[Antiproliferative activity of cimetidine and a copper-cimetidine complex on rat fibroblasts cultured in vitro]. Cimetidine is an active H2-receptor antagonist which, combined with other drugs, has antitumor effect. It has been recently shown that some drugs complexed with copper increase their inhibitory effect on tumor growth and promote cell differentiation. In the present investigation we analysed the modifications of some cell proliferation parameters (mitotic, labelling and apoptotic indices and DNA synthesis) by microfluorometry and autoradiography in 2-day low and high cell density fibroblast cultures in presence of 20 microM cimetidine or copper-cimetidine complex. In low density cultures, a more intense proliferation activity was found while in the high density ones there was accumulation of 4c and 8c polyploid cells. The highest antiproliferative effect was found in low density cultures in presence of copper cimetidine complex, which caused mitotic anomalies and increased chromocenters. Simultaneous evaluation of nuclear DNA content and 3H-T labelling indicated the extension of G1 and G2 phases, followed by a lower cell proliferation and degeneration.


Changes in fibronectin production in rat liver during cirrhotic evolution due to treatment with CCl4 and steroid hormones: correlation with plasmatic fibronectin. Previous our studies showed that some steroid hormones, as pure crystalline Progesterone (pPc) and 17-alpha-hydroxyprogesterone capronate (17 alpha HPC) heightened the cirrhogenic action produced in rat liver by carbon tetrachloride. Medroxyprogesterone (MPA), however, did not appear to promote cirrhosis, but increased just steatosis. In the present paper, we have studied the above mentioned steroid hormones for their possible capability of inducing changes in plasma fibronectin concentration. For this purpose, the soluble plasma fibronectin level was measured in female rats 45 days after CCl4-induced cirrhosis, and it was compared with the insoluble fibronectin of liver (detected by immunostaining) and the collagen content in the organ. The results obtained show that, after treatment with CCl4 and MPA, both plasma and liver fibronectin content strongly increases, whereas liver collagen content lowers. However, after treatment with CCl4 alone or in association with the other two steroid hormones, any changes in fibronectin content is not observable, but, on the contrary, is evident a heightened collagen production associated with a cirrhotic change of liver.


[Non-radioactive in situ hybridization of alpha-satellite sequences in cytogenetic diagnosis]. Non isotopic in situ hybridization with alpha-satellite DNA probes in the cytogenetic diagnosis. Standard banding cytogenetic techniques do not always allow to define the structure and the origin of chromosome rearrangements involving the centromere region. Non-isotopic in situ hybridization of alphoid sequences has allowed to determine the origin of the centromeres in the metaphases of 5 patients referred to us for: 2 structural rearrangements involving chromosome 21, 2 structural rearrangements involving chromosome Y and 1 reciprocal translocation involving on chromosome 20 and one chromosome 15.


[Adenomatoid tumor of the epididymis: cytologic diagnosis by fine-needle aspiration (FNA)]. Adenomatoid tumor of the epididymis diagnosed using fine needle aspiration (FNA). The adenomatoid tumor of the male genital system is a relatively uncommon, well recognized neoplasm with benign histologic appearance, and mostly asymptomatic clinical features. We describe a case of adenomatoid tumor of the epididymis diagnosed before surgery by fine needle aspiration cytology (FNA). This diagnostic procedure has allowed a rapid, reliable, and conclusive surgical approach.


[Comparison between the use of anti-secretory-component (sc) antibodies and anti-mucin-like-cancer-antigen (MCA) antibodies in the identification of cancer cells in serous effusions]. Evaluation between Antibodies SC and MCA in identification of carcinomatous cells in serous effusions. Authors have tested with MAbs MCA and SC twenty-seven cell-blocks from malignant effusions (epithelial neoplasms) in order to investigate the capacity of each antibody to discriminate neoplastic cells from mesothelial cells; moreover the two antibodies have been tested in relation to PAS-diastase test to verify their sensitivity to mucin detection. The "Chi square" statistical test demonstrates that MCA is much better than SC for sensitivity towards neoplastic cells, but that the SC is the best between two antibodies as markers of mucous producing features, and preferable to PAS-diastase test for better standardisation and interpretation.


[A case of desmoplastic malignant melanoma]. Desmoplastic malignant melanoma: report of a case. Authors report a case of desmoplastic malignant melanoma, a variant of the common primary cutaneous malignant melanoma in a 42 years old man, who was admitted into Hospital for metastatic node disease. Differential diagnoses on the primary, eventually discovered on the right knee, are discussed.


[Image processing of early gastric cancer cases]. Computer image processing was used to enhance gastric lesions in order to improve the detection of stomach cancer. Digitization was performed in 25 cases of early gastric cancer that had been confirmed surgically and pathologically. The image processing consisted of grey scale transformation, edge enhancement (Sobel operator), and high-pass filtering (unsharp masking). Gery scale transformation improved image quality for the detection of gastric lesions. The Sobel operator enhanced linear and curved margins, and consequently, suppressed the rest. High-pass filtering with unsharp masking was superior to visualization of the texture pattern on the mucosa. Eight of 10 small lesions (less than 2.0 cm) were successfully demonstrated. However, the detection of two lesions in the antrum, was difficult even with the aid of image enhancement. In the other 15 lesions (more than 2.0 cm), the tumor surface pattern and margin between the tumor and non-pathological mucosa were clearly visualized. Image processing was considered to contribute to the detection of small early gastric cancer lesions by enhancing the pathological lesions.


Monocytoid B-cell lymphoma of the prostate. A case of a primary malignant non-Hodgkin's lymphoma of the prostate, with the histological and immunohistochemical features of monocytoid B-cell lymphoma, is presented. The tumor histology was identical to that described in the forms of node-based monocytoid B-cell lymphoma being composed of a dense, monomorphous lymphoid infiltrate with ovoid nuclei and rather abundant, pale cytoplasm. Phenotypic analysis revealed high expression of B markers 4KB5 and L26, and negativity for T-associated antigens. This unusual localization broadens the spectrum of extra-nodal sites of monocytoid B-cell lymphoma.


[Two-dimensional time-of-flight MR angiography of mediastinum and pulmonary hilar vessels: initial clinical experiences]. Two-dimensional time-of-flight magnetic resonance angiography (2D TOF MRA) of mediastinal and pulmonary hilar vessels was performed in 10 patients, seven men and three women with a mean age (range) of 65.7 (48-88) years. The rate of visualization of the vessels and the diagnostic ability of 2D TOF MRA were assessed in comparison with contrast-enhanced CT. A radiofrequency-spoiled gradient echo sequence (SPGR) was used during repeated breath-holding (8-27 seconds) in coronal (8 patients) and axial (2 patients) imaging planes on a 1.5 Tesla superconducting scanner under the following conditions: repetition time/echo time/flip angle/excitation: 25-33/7-8 ms/45 degrees/1, field-of-view: 30 x 30 cm, slice thickness: 2.5 mm, 32 slices, 256 (frequency) x 192 (phase) matrix, with gradient moment nulling technique. Visualization sufficient to enable diagnosis of the vascular lesion was obtained in 95 (52%) vessels, mere visualization in 63 (35%), and non-visualization in 24 (13%) of the 182 evaluable vessels. The rates of good visualization of pulmonary hilar vessels (26/86, 30%) and veins (26/48, 54%) were significantly lower than that of arteries (43/48, 90%, p < 0.05). The sensitivity and specificity of 2D TOF MRA were 77% (10/13) and 100% (83/83), respectively, in 96 evaluable vessels of nine patients. 2D TOF MRA of mediastium and pulmonary hili is clinically feasible, and may be useful because of its high specificity.


[Changes in mammographic features of breast cancer--comparison with previous films]. Mammographic features of 87 breast cancer patients were studied in comparison with their previous survey films. Changes in the mammographic features included microcalicification (28 cases), tumor shadow (35 cases) and intratumorous microcalicifications (6 cases). Seven cases had several extremely faint calcifications on the previous films, and three of six cases with clustered and scattered microcalcifications that extended over an entire breast quadrant had increased in number, density and extent. Eight cases in which clustered microcalcifications had increased in number, density and extent suggested a relationship between the increase in the extent of microcalcifications and length of time between visits. In most cases with tumor shadow, a slight localized increase in mammary gland density, irregular margins and straightened trabeculae were overlooked because of breast density.


[Fundamental study of helical scanning CT--evaluation of spatial resolution in the longitudinal axis]. We evaluated spatial resolution in the longitudinal axis with helical scanning CT using a fourth-generation fast CT scanner. We made a phantom by stringing acrylic balls (65 mm phi x 8 and 9 mm phi x 6). The acquired images were processed by MPR and assessed visually to evaluate axis resolution. With the conventional scanning method, the partial volume effect varied with the starting position, but helical scanning was able to reconstruct high-resolution images using continuous raw data. During helical scanning, axis resolution varied depending on the slice width and sliding speed of the couch top. Even if the sliding speed was kept constant at 4 mm/sec, axis resolution was superior with a slice width of 2 mm than with one of 5 mm.


[Dental abnormalities in cleidocranial dysplasia. Presentation of 4 cases]. Four patients, three in the same family, with cleidocranial dysplasia, are reported. The birth of a male newborn with cleidocranial dysplasia has allowed to investigate his mother and grandmother with the same condition. An other 30-year-old patient with cleidocranial dysplasia is described. All the adult patients showed the main clinical manifestations of the disease with the peculiar abnormalities of the dentition.


[CT features of chronic maxillary osteomyelitis in adults]. CT findings were reviewed in four adult patients with chronic maxillary osteomyelitis (CMO) that was histologically proved. The CT features of CMO included bone destruction and soft tissue mass, predominantly in the inferior portion of the maxillary antrum (all 4 cases), thickening of the antral wall (3 cases) and abnormal soft tissue around the antrum associated with or without bony wall destruction (3 cases). CMO could not be distinguished from cancer of the maxillary antrum on CT because of the similar findings. However, abnormal soft tissue around the antrum together with an undestructed bony antral wall may be useful for differentiating the two diseases.


[MR imaging of bone metastasis].

[MR imaging of bone metastasis]. One hundred and three patients with known malignancy and suspected bone metastasis underwent magnetic resonance imaging. In 93%, the metastatic lesions, detected both on plain films and radionuclide bone scans, showed decreased signal intensity on T1-weighted images and increased signal intensity on T2-weighted images. All lesions, except one lesion showing much blastic change on plain films, showed contrast enhancement with Gd-DTPA administration. Although further investigation is needed, a correlation between the type of primary malignancy and the signal intensity of the metastatic lesion is suggested. Among 312 metastatic lesions detected by MR imaging, 272 lesions (87%) and 134 lesions (40%) were also detected by radionuclide bone scans and plain films, respectively. In five cases, MR imaging clearly revealed the lesion compressing the spinal cord, which helped us formulate a therapeutic plan. We conclude that MR imaging is the most sensitive examination for detecting bone metastasis and is necessary for planning treatment.


[Single maxillary central incisor and holoprosencephaly]. A holoprosencephalic female was born to a mother with a single central maxillary incisor. The newborn had microcephaly, hypotelorism, cebocephaly, palatoschisis, micrognathia, and normal chromosomes. Her brain computed tomography showed alobar holoprosencephaly. The mother was of normal intelligence and stature, and her brain computed tomography was normal. Other relatives did not have single central maxillary incisors, hypotelorism, or oral clefts. We show a further evidence of a single central maxillary incisor as an indicator of potential holoprosencephaly in the next generation, confirming an autosomal dominant trait with wide variety in penetrance and expressivity.


[MR imaging of stereotactic thalamotomy using radiofrequency methods]. Sixty-eight stereotactic radiofrequency (rf) thalamic lesions in 57 patients with movement disorders were evaluated by magnetic resonance (MR) imaging. Postoperative periods ranged from 5 days to 4 years and 9 months. All 68 rf lesions were clearly detected on T2-weighted images (T2WI). Changes in signal intensity on T2WI were classified into five patterns, as follows: Pattern I: lesions with three concentric zones consisting of an inner hypointense, middle hyperintense and outer hypointense zone (31 lesions); Pattern II: lesions consisting of an inner hypointense and outer hyperintense zone (4 lesions); Pattern III: lesions consisting of an inner hyperintense and outer hypointense zone (27 lesion); Pattern IV: lesions of a hyperintense area alone (2 lesions); Pattern V: lesions of a hypointense area alone (4 lesions). The outer hypointense rim in Patterns I and III is thought to represent hemosiderin deposition. The abnormal signal intensity on T2WI caused by rf ranged from 2 to 12 mm in diameter, and lesions in the late phase were smaller than lesions in the early phase.


A retrospective survey of delayed adverse reactions to ionic and nonionic contrast media. A comparative survey of delayed adverse reactions (DARs) to ionic and nonionic contrast media (CM) was carried out. In Japan high osmolar CM have mostly been replaced by new low osmolar CM. Therefore, a retrospective survey was performed limited to inpatients who had received an enhanced CT examination in our hospital. The material consists of 715 consecutive cases given ionic CM (iothalamate and diatrizoate) and 902 given nonionic CM (iopamidol and iohexol). DARs were picked up based on descriptions in the medical record up to 5 days after the examination. Most DARs had occurred within two days after the examination. Overall DARs were noted in 36 cases (5.0%) of the ionic group, and 44 cases (4.9%) of the nonionic group. Skin rash as well as other symptoms revealed no significant difference in incidence between the ionic and the nonionic group. There were no significant associations with immediate reactions, allergy, previous adverse reactions to CM. One patient given an ionic CM developed hypotension (60 mmHg) after return to the ward. It should be emphasized that a delayed shock had been recorded.


[Hydralazine-induced enhancement of hyperthermia treatment in vivo]. Hydralazine (Hyd) is a vaso-active drug that significantly affects the nature of blood flow in tumors. As a result, Hyd reduces blood flow and oxygen tension in tumors, causing an increase in the toxic effect of hyperthermia treatment. We investigated enhancement of the anti-tumor effect of hyperthermia by Hyd on SCC-VII tumors in C3H mice. Hyd was administered by intraperitoneal injection, and tumors were heated by water bath. We measured the tumor temperature in animals receiving Hyd by thermocouple. We found no significant change in tumor temperature with Hyd treatment. The effect of Hyd (2.5 mg/kg, 5.0 mg/kg, 7.5 mg/kg) on tumors was evaluated in terms of a growth delay value at which tumor volume reached four-fold. The growth delay values obtained were 6.25 +/- 0.82, 7.14 +/- 0.90, 8.50 +/- 0.98, 9.72 +/- 0.92, and 9.84 +/- 1.3 days for: hyperthermia alone, Hyd of 1.0 mg/kg, 2.5 mg/kg, 5.0 mg/kg, respectively. This effect was independent of the time course of administration of Hyd. These results indicate that Hyd can increase the therapeutic efficacy of hyperthermia treatment. Changes in the microenvironment, such as low pH and tumor hypoxia, induced by arterial embolization may have increased the sensitivity of tumors to heat.


[MR angiography of the lung using 3-dimensional imaging method]. Three-Dimensional MR angiography (3D-MRA) of the lung was performed in 5 healthy volunteers and 14 patients with primary pulmonary cancer in the hilum, and compared with 2D-MRA. As 3D-MRA could be obtained with single breath holding after intravenous bolus injection of Gd-DTPA, pulmonary vessels of the hilum and peripheral zone were demonstrated more definitely than those of 2D-MRA. In all patients except one case, the relation between the tumor and pulmonary vessels was visualized more clearly by 3D-MRA than 2D-MRA. This technique is considered a non-invasive and effective method for evaluation of pulmonary vessels in the patient with hilar tumor.


[MR angiography enhanced by sodium acetazolamide]. We tried to use sodium acetazolamide to enhance MR angiography (MRA). Sodium acetazolamide, one of carbonic anhydrase inhibitors, has an effect to increase regional cerebral blood flow. We made a direct comparison between pre and post enhanced MRA (both 3D-TOF and 3D-PC techniques). MRA after administration of sodium acetazolamide could depict more details of cortical branches without enhancement of back-ground. So we recommend this simple method to be used widely in any examination of MR angiography.


[How is simplex glaucoma diagnosed?].

[How is simplex glaucoma diagnosed?]. Although glaucoma has traditionally been regarded as a disease affecting people over 60 years of age, improved diagnostic methods now enable glaucoma-related changes to be detected already in the 35-40-year-old age group, long before any functional visual changes are present. Since there is no treatment capable of restoring lost visual capacity, the diagnosis should be made and treatment initiated at an early stage before visual deterioration has become manifest. Measurements of intra-ocular pressure and visual acuity provide insufficient guidance, whereas periodic photography of the nerve fibre layer yields early diagnostic information and an indication of the appropriate individual level of intra-ocular pressure to prevent progression of the disease.


[Laser treatment in diabetes-related eye complications]. Argon laser photocoagulation for diabetic retinopathy predominantly involves scatter photocoagulation of either the macula (for diabetic macular edema) or a large portion of the retina (panretinal photocoagulation). In both cases the laser treatment destroys a part of the photoreceptor layer, allowing the diffusion of oxygen and nutrients from the choroid to the inner retina, thus improving the metabolic state of the inner retina. Metabolic imbalance (hypoxia) can be linked with diabetic macular edema through hemodynamic principles according to Starlings law. The connection between the metabolic imbalances and retinal neovascularization is less clear and may involve chemical mitogens, hemodynamic considerations and other unknown factors.


[Current diagnostic and therapeutic possibilities in tapetoretinal degeneration]. Recent advances in research have markedly improved the possibility of differentiating between the different kinds of retinitis pigmentosa, and of elucidating prognosis, heredity and in certain cases the precise pathogenesis. Although treatment trials have begun, as yet no adequate treatment is available for the majority of patients with diseases belonging to this category. One approach under discussion is transplantation of fetal retinal cells.


[Alcohol during pregnancy damages eye and vision development]. Maternal alcohol abuse during pregnancy causes malformations of the eyes with serious consequences to the vision of the affected children. A high percentage (up to 90 per cent) of children suffering from the fetal alcohol syndrome have eye abnormalities of different kinds. The most typical are hypoplasia of the optic nervehead and increased tortuosity of the retinal vessels, especially of the arteries, both occurring in up to half of the cases. Visual acuity is often reduced. Considering the developmental timing of different tissues of the eye, one can presume that there is a risk for deleterious effects of alcohol on eye structures at any point of time from early gestation until development is completed.


[Contrast sensitivity and glare].

[Contrast sensitivity and glare]. The ability of the visual system to detect the information presented to the eye in form of spatial or temporal contrast can be characterized by the contrast sensitivity function (CSF). CSF measurements can be used to visualize loss in visual capacity in patients with different diseases and to monitor recovery of vision. In the clinic CSF is most commonly used to evaluate disability glare in patients with cataract.


The crack cocaine problem. A warning from North America. This article describes crack cocaine and its adverse clinical and public health effects. Although crack cocaine does not yet constitute a major drug problem in the Nordic countries, heed should be paid to North America where crack cocaine has developed into a serious and widespread problem only a few years after its introduction. In spite of highly publicized efforts to limit its spread, after a small decline in 1990, the use of crack cocaine is once again increasing. Strong efforts should therefore be made to keep this substance away from the Nordic countries.


[Diagnosis in acute narcotics poisoning]. The article presents a brief summary of symptoms in acute narcotic drug intoxication. It emphasizes the need for correct diagnosis by using drugs of abuse testing, and when these tests may be applicable. With three case reports the authors show the need for correct diagnosis from both medical and legal aspects.


[Sexual abuse of children--a public health problem?]. Owing to variation in the reported prevalences of sexual abuse of children, international publications were reviewed to analyse the definitions and other criteria used. Reported prevalences varied from 4 to 67 per cent, the variation being due, among other things, to inconsistency in the definitions used. By some of the investigators sexual acts without actual physical contact between the offender and the victim had been included in the category, exhibitionism. Approximately half of the reported experiences were isolated events. Future research in this field will require greater conformity in the definitions and criteria used.


[Effort-induced myocardial wall motion abnormalities in the magnetic resonance tomogram: a comparison with effort MIBI SPECT]. In 20 patients with an angiographically documented coronary artery stenosis of > or = 70% and normal LV function at rest MRI was performed before and after dipyridamole infusion (0.75 mg/kg BW). In all patients MIBI-SPECT was obtained at rest and after dynamic symptom-limited exercise. In 18 patients MIBI-SPECT showed ischemia and in 18 patients dipyridamole MRI showed a wall motion impairment. In the segments representing the 29 stenosed vessels ischemia in MIBI-SPECT was diagnosed correctly in 24 instances (sensitivity 83%, specificity 90%) and a wall motion abnormality was present in MRI in 23 instances (sensitivity 79%, specificity 90%). Dipyridamole GE-MRI is not superior to MIBI-SPECT in the diagnosis of ischemia.


[A phosphine-substituted diphosphonic acid (HMPD) for improved scintigraphic detection of bone lesions]. Although the radiopharmaceuticals used for bone scintigraphy are of very high quality, the search for an "ideal" agent continues. To optimise the detectability of bone lesions, we analysed 244 different 99mTc-labeled phosphonates in animal experiments. In osteosarcoma-carrying rats 99mTc-labeled 1-Hydroxy-3-methyl-phosphinic-1, 1-propanediphosphonic acid (HMPD) was shown to produce the best lesion/normal bone ratio. 99mTc-MDP was used as reference. The ratio was found to be 1.28 for 99mTc-HMPD. The transferability of our results in animals to the situation in man was studied in 10 patients with bone metastases. There was for 99mTc-HMPD an improvement of the lesion/normal bone ratio by more than 60% but also an additional reduction of the soft tissue contrast by about 40%. 15% of the metastases were detected by scintigraphy using 99mTc-HMPD only and not with 99mTc-MDP. The new agent should make possible a better and earlier discrimination of bone lesions in the scintigram.


[The value of a new IRMA for determining thyroglobulin]. A total of 277 patients suffering from differentiated thyroid carcinoma with individual follow-up periods of up to five years had been investigated. More than 1,000 sera were collected. The present paper reports on the results of the parallel serum Tg determinations by means of a recently introduced Tg-IRMA system in comparison with the previously used, well established Tg-RIA method. The intra- and the interassay variation of the IRMA were found at 37% and 46%, respectively, for 4 ng Tg/ml, and 3% and 6%, respectively, for Tg values above 40 ng/ml. Several effects that could interfere with the serum Tg determination (freezing or repeated freezing and thawing of sera, hemolysis or lipemia or dilution of sera, preanalytical use of serum separating tubes) were examined. Although statistically significant in some instances, at least, all of the above mentioned effects were without any practical relevance for the clinical routine use of the IRMA. In patients being in complete remission and on complete TSH suppressive thyroid hormone treatment but having no residual thyroid tissue (n = 70), the level of clinical significance was 1 ng Tg/ml for the IRMA system, resulting in a sensitivity of 99% and a specificity of 90%, respectively. For the RIA system, however, the level of clinical significance was found to be 10 ng Tg/ml with a sensitivity of 89% and a specificity of 95%. We could additionally define the grey-zone for the practical use of the IRMA (1-3 ng Tg/ml). The IRMA system is significantly more sensitive and detects more sera correctly positive than the Tg-RIA.(ABSTRACT TRUNCATED AT 250 WORDS)


Quantitative bone scintigraphy and 24-hour whole-body counting of 99mTc-methylene diphosphonate in patients with prostatic carcinoma. Thirty-four patients with prostatic carcinoma were studied with quantitative bone scintigraphy and whole-body counting (WBC) 1 and 24 h after injection of 99mTc-MDP before as well as two weeks and two months after orchiectomy. Thirteen of the patients had normal bone scintigrams and WBR at the three different investigations; 21 had skeletal metastases. The latter showed throughout the study higher local gamma camera count rates as well as WBR values than the patients with normal scintigrams. In these patients a "flare phenomenon", with an increase in count rate two weeks after orchiectomy followed by a decrease two months post-operatively, was seen with quantitative bone scintigraphy but not with WBC. However, WBC may be a valuable method indicating the total extent of skeletal metastases in the body, while quantitative bone scintigraphy is more accurate in the interpretation of individual skeletal metastases.


[Immunoscintigraphy using 111In-antimyosin-antibodies in the clinical diagnosis of myocarditis]. Thirty patients suspected of having acute myocarditis underwent examination with 111In-labeled antimyosin antibodies. The heart/lung ratio was used for scintigraphic evaluation, with a value of > 1.5 being regarded as positive. The values were correlated with a score based on typical clinical parameters, separating myocarditis into categories "unlikely", "possible" and "highly probable". There was complete correlation in the category "myocarditis highly probable"--with a heart/lung ratio > 1.5 (11 patients)--and in the category of "myocarditis unlikely"--with a heart/lung ratio of < or = 1.5 (5 patients). The category "myocarditis possible" included 2 cases with a scintigraphic vote for the presence of myocarditis and 12 cases against. Immunoscintigraphy with antimyosin antibodies has shown itself to be a valuable non-invasive tool in the investigation of suspected myocarditis.


[MR tomography of the lower limbs of children with diseases of the hematopoietic system]. In 41 children with disorders of the hematopoetic system 119 MR examinations of both tibiae and knees were performed (T1 and T2 spin-echo sequences and a T2 gradient-echo sequence). Before therapy bone marrow changes in leukemia were diffuse, and patchy during and after therapy. Signals in T2-weighted images were different for infiltrations, fibrosis, necrosis and siderosis. In 3 children suspected of suffering from osteomyelitis, Ewing-sarcoma or coxitis, MR examination was the first to show the correct diagnosis. Therefore in children indications for MR tomography should be handled more generously to win time for therapy. Using only a T1 spin-echo sequence and a T2 gradient-echo sequence screening of more than one region is capable of controlling bone marrow diseases.


[Tuberous sclerosis: broadening of the diagnosis using MR tomography and computed tomography]. Ten children and a 22-y old women with tuberous sclerosis were studied. All children were epileptic but only 8 had skin lesions (white spots, sebaceous adenoma), 4 had rhabdomyoma of the heart and 4 children had astrocytoma of the retina; only in 4 children was the entire triad of tuberous sclerosis seen. The young woman with a hemorrhage of an angiolipoma of the kidney had no other symptoms of tuberous sclerosis. All patients had pathologic CT and MRT findings; 4 different types of lesion were seen in MRT. In order to elucidate human genetic aspects and to demonstrate the underlying disease all children with epileptic disorders and also all patients without the full triad of symptoms should be examined by MRT.


[99mTc-MAB bone marrow scintigraphy and probe-guided biopsy in aplastic anemia]. The diagnosis of systemic haematological disorders is often confirmed by bone marrow biopsy which is usually performed "blind". False-negative results may be obtained because this method is restricted to a small part of the blood-forming organ. Bone marrow scintigraphy has the potential of detecting the distribution of active haematopoietic bone marrow and to localize infiltrative lesions. This case report of a patient with pancytopenia demonstrates that the diagnosis of aplastic anaemia can be confirmed by bone marrow scintigraphy in combination with probe-guided bone marrow biopsy.


[Electrolyte abnormalities in elderly people. Prevalence and relation to drug treatment. Multicenter study of 631 subjects aged 70 years and over]. In order to assess the responsibility of medicinal drugs for the disorders in electrolytes observed in elderly people, the prevalence of abnormal natraemia and kalaemia was prospectively established in 631 subjects aged 70 or more on the first day of hospitalization. Among 337 subjects not taking drugs likely to create problems (diuretics, angiotensin-converting enzyme inhibitors, anti-inflammatory agents) 3.6 percent had hyponatraemia (below 130 mmol/l) and 12.2 percent had hypokalaemia (below 3.5 mmol/l). Subjects with normal renal function who were taking angiotensin-converting enzyme inhibitors or non-steroidal anti-inflammatory agents showed no more electrolytic abnormalities than those who did not take them. Conversely, subjects on thiazides were more frequently hyponatraemic (11.8 percent) or hypokalaemic (16.2 percent; 20 percent in the absence of associated distal diuretics). These abnormalities were more frequent in women than in men (21.7 and 13 percent). This, together with the fact that 68 percent of subjects taking thiazides were women accounts for the marked female predominance of electrolytic abnormalities reported in the literature.


[Survival after gastrectomy for cancer. 209 cases]. The long-term results of 209 gastrectomies performed for adenocarcinoma, including 117 which were prospectively collected, are presented. Resection was curative in 154 cases (73.6 percent). The TNM distribution of the tumours was: stage I (TxNOMO) 75 cases, stage II (TxN1MO) 46 cases, stage III (TxN2MO) 33 cases and stage IV (TxNxM1) 55 cases. Lymph node involvement was more frequent in the prospective than in the retrospective study. With a more than 5 years' follow-up of 80 percent of the patients operated upon, the actuarial survival rate at 5 years (operative mortality included) was 38 percent for all lesions, 52 percent for curative resection and 2 percent for palliative resection. Following curative resection, the survival rates for tumours of the upper, middle and lower thirds of the stomach were 40, 60 and 55 percent respectively. These rates were 60 percent for stage I tumours, 54 percent for stage II tumours and 25 percent for stage III tumours. The results obtained in this series, where most of the curative gastrectomies included excision of N1 and N2 lymph nodes, show that lymph node involvement has no significant importance for the prognosis when it is proximal (N1) and is not incompatible with prolonged survival when it is pedicular (N2).


[Efficacy of danazol in autoimmune hemolytic anemia with cold agglutinins. 4 cases]. The treatment of haemolytic anaemia related to primary cold agglutinin disease is often unsatisfactory. Corticosteroids are not useful in most cases and, beside protection against cold, treatment is frequently limited to transfusions. We report the good results obtained with long-term danazol therapy (600 mg per day initially, than 400 mg per day) in 3 cases of chronic cold agglutinin disease and 1 case of idiopathic haemolytic anaemia with cold agglutinin in serum. The only side-effect was a transient increase of transaminase level requiring reduction of a maintenance dose which could be given during 3 to 7 years.


[Infectious respiratory complications of AIDS]. Respiratory infections are particularly frequent in HIV infection. They depend upon the degree of immunodeficiency, the geographical region and a possible prophylaxis. Bronchopneumopathies caused by pyogenic organisms (notably pneumococci) appear when the number of T4 lymphocytes is little reduced. Pulmonary tuberculosis, particularly frequent in Africans and Haitians, occurs in patients with moderate immunodeficiency (T4 between 200 and 300/mm3). HIV infections modify the epidemiology of tuberculosis in Africa, but also in the USA and probably in Europe. Despite a well-established prophylaxis, pneumocystosis, which develops when the number of T4 cells falls below 200/mm3, is the opportunistic pathology which in most cases points to AIDS in the USA and in France. Atypical mycobacterial infections (Mycobacterium avium complex) and CMV infections occur at a late stage of the disease in patients with severe immunodeficiency. Noticeable advances have recently been made in the treatment of these complications.


[Bronchial cancer associated with atheromatous arteriopathy. Prognostic and therapeutic consequences in pulmonary excision]. Between January 1980 and January 1985, 462 consecutive patients underwent lung resection for bronchogenic carcinoma. Eighty of these patients (17.3 percent) had atherosclerosis involving mainly the coronary arteries (40 percent). The mortality rate was significantly higher in patients with atherosclerosis (10 percent) than in those without it (4.7 percent), but the long-term survival was not affected with the presence of atherosclerosis. The lung carcinoma-atherosclerosis association raises two problems: what investigations should be carried out in this group of patients at risk, and what treatment should be given priority when vascular lesions are present and likely to become complicated by pulmonary surgery? However, the presence of atherosclerosis should not lead to a re-evaluation of the need for lung resection.


[Systematic parasitic coprologic testing. Value in hospital kitchen staff]. The results of annual parasitological coprology in 56 members of the kitchen staff of a hospital over a 6-year period have been studied. Among these subjects, 27 had at least one positive examination. The results were compared with those from other hospitals. Among the parasites found were 2 Strongyloides stercoralis, 4 Schistosoma mansoni and 31 protozoa with direct transmission. The value of this examination is discussed, taking into account the increasing number of immunodepressed patients.


[Treatment of wounds with honey. 40 cases]. Honey has been used long ago to treat wounds and cutaneous ulcers, and its healing properties have recently been rediscovered. Its applications on wounds or infected burns give satisfactory results. In a study of 40 patients with wounds of various origin honey provided healing in 88 percent of the cases. At the end of the healing process a few microorganisms were isolated from the wounds, but they did not prevent consolidation. This treatment, therefore, seems to be effective; moreover, as it is simple and inexpensive it should be better known and added to the list of commonly used antiseptic products.


[Tubular gastrostomy using celioscopy].

[Tubular gastrostomy using celioscopy]. Surgical feeding gastrostomy is attended by a significant morbidity and mortality. Today, the percutaneous endoscopic route is regarded as the best method, but it is not feasible in case of pharyngo-oesophageal stenosis and has its own drawbacks. A new method using laparoscopy to perform a Depage-Janeway gastrostomy is presented here. The theoretical advantages of this method are clear: the limited parietal damage should avoid cicatricial complications, and the limited ventilatory aggression should reduce the amplitude of respiratory complications. Moreover, in surgical practice this is a simple, rapid and efficient operation.


[Cardiovascular consequences of obesity associated with arterial hypertension]. Hypertensive obese subjects run an increased cardiovascular risk. Their predominantly abdominal obesity is often associated with hypertriglyceridaemia and insulin-resistant diabetes, and their cardiovascular status is characterized by cardiac hyperdynamics and hypervolaemia responsible for left ventricular hypertrophy and dilatation. Insulin resistance and subsequent hyperinsulinaemia are thought to explain the obesity-hypertension association, the cardiovascular effects observed and the metabolic and cardiovascular complications which might result from this situation. Successful control of both arterial pressure and overweight should contribute to regression of the left ventricular hypertrophy. Simultaneous treatment of abnormalities in carbohydrate and lipid metabolism is also necessary to prevent cardiovascular complications.


Population genetics in the forensic DNA debate. The use of matching variable number of tandem repeat (VNTR) profiles to link suspects with crimes is potentially very powerful, but it has been quite controversial. Initial debate over laboratory procedures has largely given way to debate over the statistical and population genetic issues involved in calculating the frequency of a profile for a random member of a population. This frequency is used to weight the evidence of a match between suspect and crime scene material when the suspect denies responsibility for that material. A recent report from the National Research Council, intended to put to rest some of the issues, has instead raised further debate by advocating a procedure based on maximum frequencies of profile components over several different populations.


Androgen-specific gene activation via a consensus glucocorticoid response element is determined by interaction with nonreceptor factors. A fundamental issue in steroid hormone regulation is the question of how specific transcription is attained in vivo when several receptors can bind the same DNA sequence in vitro. We report an enhancer of the mouse sex-limited protein (Slp) gene that, unlike previously characterized enhancers, is activated by androgens but not by glucocorticoids or progestins. Potent androgen induction requires both a consensus glucocorticoid (hormone) response element and auxiliary elements also present within a 120-base-pair DNA fragment. Cotransfection assays with wild-type and mutant receptors reveal that glucocorticoid receptor can bind, but not transactivate from, the hormone response element within the enhancer. The positive effect of androgen and the null effect of glucocorticoid appear to require the amino-terminal domains of the respective receptors. Thus, exclusive transcriptional response to androgens, and lack of response to glucocorticoids, derives from factor interactions that are determined by the context of the receptor binding site rather than by its distinct sequence.


Transcription factor IIA is inactivated during terminal differentiation of avian erythroid cells. Avian histone H5 and alpha A-globin genes are transcribed much more efficiently in whole cell extracts derived from immature polychromatic erythrocytes than in extracts from mature duck erythrocytes. We found that these differential activities are detectable only if assayed with promoters containing a functional TATA box. The addition of either highly purified human or recombinant yeast transcription factor IIA (TFIIA) to extracts from mature erythrocytes resulted in a significant increase in transcription from TATA-containing promoters, whereas transcription from TATA-less promoters remained unaffected. Moreover, the activity of TFIIA was found to be reduced in extracts from mature erythrocytes. These data support the proposition that inactivation of TFIIA may contribute to a general repression of gene activity in avian erythrocytes, and only those genes with alternative mechanisms of initiation complex formation continue to be expressed in these cells. In the case of the histone H5 gene, such an alternative mechanism could be mediated via the interaction between duck erythrocyte upstream stimulating factor and TFIID.


The PR264/c-myb connection: expression of a splicing factor modulated by a nuclear protooncogene. We have previously reported that expression of the c-myb gene in normal avian thymic cells proceeds through the intermolecular recombination of ET (thymus-specific) and c-myb coding sequences, thereby generating a novel type of c-myb product. Antisense transcripts expressed from the ET locus encode the extremely well-conserved splicing factor PR264/SC35. We now show that the human PR264 promoter sequences contain several myb-recognition elements that efficiently interact in vitro with the c-myb DNA-binding domain. Moreover, expression from the PR264 promoter is transactivated, both in vitro and in cultured cells, by different c-myb products. Thus, the PR264 gene is most likely a physiological target for the c-myb family of transcription factors.


A different sort of Mott cell.

A different sort of Mott cell. NYC is a B lymphoma cell line derived from B/W mice. Upon fusion of NYC cells with a plasmacytoma, which itself produces no immunoglobulin, the resulting NYCH hybridoma cells are Mott cells; i.e., they contain large intracellular vesicles filled with immunoglobulin, the so-called Russell bodies. When NYCH.kappa, a variant of NYCH that had lost the ability to produce heavy chain, was transfected with a heavy-chain construct, this concentration of immunoglobulin in the intracellular vesicles occurred only when the transfected immunoglobulin heavy chain had the same variable region as NYC. Moreover, unlike conventional Mott cells, the hybrid cells secrete immunoglobulin at a normal rate.


Ascaris hemoglobin gene: plant-like structure reflects the ancestral globin gene. Animal globin genes have two introns at strictly conserved positions, while plant globin genes have both of these as well as an additional, central intron. It has been proposed that a common ancestor gene had three introns, one of which was subsequently lost from animal but not plant globin genes. We have elucidated the cDNA sequence and gene structure of a hemoglobin from the parasitic nematode Ascaris suum and found a plant-like central intron, providing strong evidence for a three-intron ancestor of modern globin genes.


Identification of a functionally important sequence in the C terminus of the interferon-gamma receptor. We have previously shown that the intracellular domain of the interferon-gamma (IFN-gamma) receptor plays an obligate role in receptor-mediated signal transduction. Moreover, we have specifically identified two regions within the human IFN-gamma receptor's intracellular domain required for functional activity: the membrane-proximal 48 amino acids required for both functional activity and receptor-mediated ligand internalization and the C-terminal 39 amino acids required exclusively for biologic response induction. Herein we report the identification of the 3 amino acids within the C-terminal region of the receptor that are obligatorily required for receptor function. By using a set of overlapping truncation mutants, the minimal functional sequence within the C-terminal region was localized to residues 434-444 (APTSFGYD-KPH). By mutating each individual residue within this sequence to alanine, three residues (Tyr-440, Asp-441, and His-444) were identified as being critical for IFN-gamma-dependent (i) upregulation of major histocompatibility complex class I proteins, (ii) activation of the IFN regulatory factor 1 gene, and (iii) stimulation of cells to produce nitric oxide. The more conservative Tyr-440-->Phe substitution also resulted in a nonfunctional receptor. Subsequent mutational analysis of all five of the IFN-gamma receptor's intracellular tyrosine residues revealed that Tyr-440 was the sole tyrosine required for receptor activity. These results thus identify a unique sequence in the IFN-gamma receptor that is required for initiation of IFN-gamma-dependent biologic responses and highlight the importance of the hydroxyl side chain of Tyr-440 in this process.


Circadian oscillations in period gene mRNA levels are transcriptionally regulated. The period (per) gene is involved in regulating circadian rhythms in Drosophila melanogaster. The per gene is expressed in a circadian manner, where fluctuations in per mRNA abundance are influenced by its own translation product, which also cycles in abundance. Since per gene expression is necessary for circadian rhythmicity, we sought to determine how certain features of this feedback loop operate. The results of this study reveal that fluctuations in per mRNA are primarily controlled by fluctuations in per gene transcription, that per mRNA has a relatively short half-life, and that sequences sufficient to drive per mRNA cycling are present in 1.3 kilobases of 5' flanking sequences. These and other results indicate that the per feedback loop has all of the basic properties necessary to be a component of a circadian oscillator.


Insulin-like growth factor II stimulates motor nerve regeneration. Injury to mammalian motor nerves can lead to paralysis, but relatively successful regeneration may occur when conditions are favorable. Elucidation of the mechanism upholding successful regeneration is of theoretical and clinical interest. In this study, the hypothesis that insulin-like growth factor II (IGF-II) can stimulate motor nerve regeneration was tested. When IGF-II was infused continuously near a site of crush on the sciatic nerve, the distance of motor axon regeneration was increased significantly in rats. In contrast, spontaneous regeneration was inhibited when an anti-IGF-II antiserum was infused through a "window" in the epineurium. Thus, infused IGF-II can increase, and endogenous IGFs can support, the regeneration of motor axons in lesioned nerves.


Carbonic anhydrase in the membrane of the endoplasmic reticulum of male rat liver. We have prepared subcellular fractions of male rat liver homogenate by the method of Lewis and Tata [Lewis, J. A. & Tata, J. R. (1973) J. Cell Sci. 23, 447-459], further purifying the membranes of the microsomal fraction by exposure to 0.01% Triton X-100 and centrifugation. We determined the purity of the fractions with marker enzymes and measured carbonic anhydrase (CA; EC 4.2.1.1) activity in intact and solubilized particulates with 18O exchange between CO2/HCO3- and water. We measured the concentration of CA by titration with a sulfonamide inhibitor, ethoxzolamide, obtaining an average value of 3.8 mumol/mg of microsomal membrane protein. The equilibrium constant for binding ethoxzolamide was 0.49 x 10(-9) M. The Km for CO2 was 1.7 mM and the turnover number was 560,000 sec-1, characterizing this as a membrane-bound, high-activity isozyme of type IV. By electron microscopy of tissue sections after staining with a cobalt precipitation technique, CA was seen in small cytoplasmic vesicles in hepatocytes and in microsomal particles and membranes. There was a sulfonamide-resistant (isozyme type III) and a sulfonamide-sensitive (isozyme type II) CA in the cytosol but none in the rapidly sedimenting endoplasmic reticulum. We conclude that there is no CA normally within the matrix of the cell endoplasmic reticulum but that the CA type III found in the microsome may have been captured from the cytosol during resealing. Thus the adult male rat hepatocyte contains CA type IV in the membrane of the endoplasmic reticulum and CA type II and CA type III in the cytoplasm.


Honeybee colony integration: worker-worker interactions mediate hormonally regulated plasticity in division of labor. Adult workers in honeybee (Apis mellifera) colonies exhibit plasticity in hormonally regulated, age-based division of labor by altering their pattern of behavioral development in response to changes in colony conditions. One form of this plasticity is precocious development: levels of juvenile hormone increase prematurely and bees begin foraging as much as 2 weeks earlier than average. We used two experimental paradigms inspired by developmental biology to study how bees obtain information on changing colony needs that results in precocious foraging. An analog of "cell culture," with bees reared outside of colonies in different sized groups, revealed that worker-worker interactions exert quantitative effects on endocrine and behavioral development. "Transplants" of older bees to colonies otherwise lacking foragers demonstrated that worker-worker interactions also affect behavioral development in whole colonies. These results provide insights to a long-standing problem in the biology of social insects and further highlight similarities in the integration of activity that exist between individuals in insect colonies and cells in metazoans.


Expression of hepatitis B surface antigen in transgenic plants. Tobacco plants were genetically transformed with the gene encoding hepatitis B surface antigen (HBsAg) linked to a nominally constitutive promoter. Enzyme-linked immunoassays using a monoclonal antibody directed against human serum-derived HBsAg revealed the presence of HBsAg in extracts of transformed leaves at levels that correlated with mRNA abundance. This suggests that there were no major inherent limitations of transcription or translation of this foreign gene in plants. Recombinant HBsAg was purified from transgenic plants by immunoaffinity chromatography and examined by electron microscopy. Spherical particles with an average diameter of 22 nm were observed in negatively stained preparations. Sedimentation of transgenic plant extracts in sucrose and cesium chloride density gradients showed that the recombinant HBsAg and human serum-derived HBsAg had similar physical properties. Because the HBsAg produced in transgenic plants is antigenically and physically similar to the HBsAg particles derived from human serum and recombinant yeast, which are used as vaccines, we conclude that transgenic plants hold promise as low-cost vaccine production systems.


Ligand-dependent conformational changes in the progesterone receptor are necessary for events that follow DNA binding. Hormones and antihormones induce related, but distinct, conformational changes in the progesterone receptor [Allan, G. F., Leng, X., Tsai, S. Y., Weigel, N. L., Edwards, D. P., Tsai, M.-J. & O'Malley, B. W. (1992) J. Biol. Chem. 267, 19513-19520]. In both cases the conformational change precedes the dissociation of heat shock proteins and binding to DNA. We have now investigated the steps in hormone action which are dependent upon this conformational change. We show that in the absence of ligand, monoclonal antibodies directed against different regions of the progesterone receptor can induce high-affinity binding to its response element in vitro. This antibody-induced DNA binding is presumably facilitated by enhanced dimerization of receptor monomers. However, antibodies do not induce the hormone-specific conformational change in the progesterone receptor and do not induce in vitro transcription by the receptor. In contrast, the antiprogestin ZK98299, which inhibits receptor binding to DNA, fully induces the antihormone-specific conformational change. Thus, our data imply that steroids induce a conformational change in their receptors which is necessary for events subsequent to DNA binding, most likely for transactivation.


Modulation of synaptic gain by light.

Modulation of synaptic gain by light. Although synaptic transmission in the retina has been assumed to be static, it appears that the voltage gains of the synapses between photoreceptors and second-order cells can be enhanced by light. Voltage gains of the synapses between rods and bipolar (or horizontal) cells are about 10 times higher in the presence of dim background light than in darkness. This increase in synaptic gain may compensate for the loss of rod light responsiveness caused by weak background light so that the animal can maintain good rod sensitivity under moonlight or starlight, the natural lighting condition for mating and food catching.


Poly(ADP-ribose) polymerase activity in mononuclear leukocytes of 13 mammalian species correlates with species-specific life span. Poly(ADP-ribosyl)ation is a eukaryotic posttranslational modification of proteins that is strongly induced by the presence of DNA strand breaks and plays a role in DNA repair and the recovery of cells from DNA damage. We compared poly(ADP-ribose) polymerase (PARP; EC 2.4.2.30) activities in Percoll gradient-purified, permeabilized mononuclear leukocytes from mammalian species of different maximal life span. Saturating concentrations of a double-stranded octameric oligonucleotide were applied to provide a direct and maximal stimulation of PARP. Our results on 132 individuals from 13 different species yield a strong positive correlation between PARP activity and life span (r = 0.84; P << 0.001), with human cells displaying approximately 5 times the activity of rat cells. Intraspecies comparisons with both rat and human cells from donors of all age groups revealed some decline of PARP activity with advancing age, but it was only weakly correlated. No significant polymer degradation was detectable under our assay conditions, ruling out any interference by poly(ADP-ribose) glycohydrolase activity. By Western blot analysis of mononuclear leukocytes from 11 species, using a crossreactive antiserum directed against the extremely well-conserved NAD-binding domain, no correlation between the amount of PARP protein and the species' life spans was found, suggesting a greater specific enzyme activity in longer-lived species. We propose that a higher poly(ADP-ribosyl)ation capacity in cells from long-lived species might contribute to the efficient maintenance of genome integrity and stability over their longer life span.


Domain motions in phosphoglycerate kinase: determination of interdomain distance distributions by site-specific labeling and time-resolved fluorescence energy transfer. 3-Phosphoglycerate kinase is composed of two globular domains separated by a wide cleft. The substrate binding sites are situated on the inner surfaces of the two domains. By analogy to other kinases, it has been postulated that the catalytic mechanism of phosphoglycerate kinase involves a hinge bending domain motion that brings the substrates together to allow phosphoryl transfer. To characterize this large-scale conformational change, as well as the dynamics of the unliganded enzyme in solution, we have applied site-directed mutagenesis and time-resolved nonradiative energy transfer techniques. Two genetically engineered cysteines (Cys-135 and Cys-290), one in each of the two domains, were covalently labeled with a donor and acceptor pair of fluorescent probes. Analysis of subnanosecond fluorescence decay curves yielded the equilibrium distribution of interdomain distances. In the absence of substrates, the distribution of distances between the two labeled sites was very broad, with a full width at half maximum estimated as 20 A or broader, indicative of a large number of conformational substates in solution. The mean distance, 31.5 +/- 1 A, was 8 A smaller than in the crystal structure. Upon addition of ATP alone or of ATP and 3-phosphoglycerate, the average distance increased to 38 +/- 1 A and the width of the distribution decreased. Addition of 3-phosphoglycerate alone induced a similar but smaller change. The rate of conformational state fluctuations (interconversion between states) was found to be slow on the nanosecond time scale, as expected for a protein with a relatively large interdomain contact area.


Amplifiable messenger RNA.

Amplifiable messenger RNA. RNA molecules were prepared that consisted of an mRNA encoding chloramphenicol acetyltransferase embedded within the sequence of midivariant RNA, which is a template for the RNA-directed RNA polymerase Q beta replicase. These recombinant RNAs were shown to be bifunctional: they are amplified exponentially by incubation with Q beta replicase, and the replicated RNA serves as template for the cell-free synthesis of enzymatically active chloramphenicol acetyltransferase. The availability of amplifiable mRNAs will enable relatively large amounts of protein to be synthesized in vitro.


Molecular cloning of a gene encoding a seed-specific protein with nutritionally balanced amino acid composition from Amaranthus. An albumin with a well-balanced amino acid composition and high levels of the essential amino acids was purified to homogeneity from the mature seeds of Amaranthus hypochondriacus. The amino acid composition of this protein is comparable to the World Health Organization recommended values for a highly nutritional protein. The protein is a 35-kDa monomer with four isoforms that can be separated by chromatofocusing. Antibodies raised against one of the isoforms, AmA1, cross-reacted with the other three isoforms. Affinity-purified AmA1 antibodies were used to isolate cDNA clones from a developing-seed expression library. The six immunopositive recombinants obtained were found to be related. The cDNA of the largest clone (1.2 kilobases) has a single major open reading frame corresponding to a 304-amino acid polypeptide. The clone was confirmed by hybrid-selected translation and immunoprecipitation. The size of the immunoprecipitated product was identical to the mature protein. Analysis of RNA and protein in developing seeds showed that AmA1 is synthesized during early embryogenesis, reaching a maximum by midmaturation. No RNA was detected in 1-day-old seedlings although the protein showed delayed breakdown on germination. Expression of the AmA1 gene was found to be seed-specific, as no protein or RNA was detected in other plant tissues.


Myc/Max and other helix-loop-helix/leucine zipper proteins bend DNA toward the minor groove. A distinct family of DNA-binding proteins is characterized by the presence of adjacent "basic," helix-loop-helix, and leucine zipper domains. Members of this family include the Myc oncoproteins, their binding partner Max, and the mammalian transcription factors USF, TFE3, and TFEB. Consistent with their homologous domains, these proteins bind to DNA containing the same core hexanucleotide sequence CACGTG. Analysis of the conformation of DNA in protein-DNA complexes has been undertaken with a circular permutation assay. Large mobility anomalies were detected for all basic/helix-loop-helix/leucine zipper proteins tested, suggesting that each protein induced a similar degree of bending. Phasing analysis revealed that basic/helix-loop-helix/leucine zipper proteins orient the DNA bend toward the minor groove. The presence of in-phase spacing between adjacent binding sites for this family of proteins in the immunoglobulin heavy-chain enhancer suggests the possible formation of an unusual triple-bended structure and may have implications for the activities of Myc.


Human T-cell leukemia virus type I infection of monocytes and microglial cells in primary human cultures. The pathogenesis of progressive spastic paraparesis [HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP)], a serious consequence of human T-cell leukemia virus type I (HTLV-I) infection, is unclear. T and B lymphocytes can be naturally infected by HTLV-I, but the susceptibility to HTLV-I infection of other cell types that could contribute to the pathogenesis of HAM/TSP has not been determined. We found that a human monocyte cell line (THP-1), primary human peripheral blood monocytes, and isolated microglial cells but not astrocytes or oligodendroglial cells derived from adult human brain were infected by HTLV-I in vitro. Infection with HTLV-I enhanced the secretion of interleukin 6 in human microglial cell-enriched cultures but did not stimulate the release of interleukin 1 from monocytes or microglial cells. Tumor necrosis factor alpha production was stimulated by HTLV-I infection of monocytes and microglial cells and could be enhanced by suboptimal amounts of lipopolysaccharide. Since both tumor necrosis factor alpha and interleukin 6 have been implicated in inflammatory demyelination and gliosis, our findings suggest that human microglial cells and monocytes infected with and activated by HTLV-I could play a role in the pathogenesis of HAM/TSP.


Progesterone regulates the activity of collagenase and related gelatinases A and B in human endometrial explants. Explants of human endometrium were cultured to study the release of matrix metalloproteinases (MMPs). Analysis of conditioned media by zymography revealed latent and active forms of collagenase (MMP-1, EC 3.4.24.7), 72-kDa gelatinase A (MMP-2, EC 3.4.24.24), and 92-kDa gelatinase B (MMP-9, EC 3.4.24.35). These proteinases were identified by their M(r), their inhibition by tissue inhibitor of metalloproteinases, and the activation of their zymogens by trypsin or aminophenylmercuric acetate. In the absence of sex hormone, explants released large amounts of enzyme activities, as measured by densitometry of zymograms or in soluble assays. Physiological concentrations of progesterone (10-200 nM) almost totally abolished the release of collagenase, of total gelatinase activity, and of the active form of gelatinase B and largely inhibited the release of the active form of gelatinase A. These effects, which were antagonized by mifepristone (RU 38486), suggest that progesterone restrains endometrial tissue breakdown by blocking the secretion and activation of MMPs.


Cloning of cDNAs of the MLL gene that detect DNA rearrangements and altered RNA transcripts in human leukemic cells with 11q23 translocations. Recurring chromosomal abnormalities involving translocations at chromosome 11 band q23 are associated with human myeloid and lymphoid leukemia as well as lymphoma. We have identified the gene located at this break-point and have named it MLL (for myeloid-lymphoid, or mixed-lineage, leukemia). The t(4;11), t(6;11), t(9;11), and t(11;19) are among the most common reciprocal translocations in leukemia cells involving this chromosomal band. We now have evidence that the breakpoints in all of these translocations are clustered within a 9-kilobase (kb) BamHI genomic region of the MLL gene. By Southern blot hybridization using a 0.7-kb BamHI cDNA fragment of the MLL gene called MLL 0.7B, we have detected rearrangements of DNA from cell lines and patient material with an 11q23 translocation in this region. Northern blot analyses indicate that this gene has multiple transcripts, some of which appear to be lineage-specific. In normal pre-B cells, four transcripts of 12.5, 12.0, 11.5, and 2.0 kb are detected. These transcripts are also present in monocytoid cell lines with additional hybridization to a 5.0-kb transcript, indicating that expression of different-sized MLL transcripts may be associated with normal hematopoietic lineage development. In a cell line with a t(4;11), the expression of the 12.5-, 12.0-, and 11.5-kb transcripts is reduced, and there is evidence of three other altered transcripts of 11.5, 11.25, and 11.0 kb. Thus, these 11q23 translocations result in rearrangements of the MLL gene and may lead to altered function(s) of MLL and of other gene(s) involved in the translocation.


Metabolic engineering of medicinal plants: transgenic Atropa belladonna with an improved alkaloid composition. The tropane alkaloid scopolamine is a medicinally important anticholinergic drug present in several solanaceous plants. Hyoscyamine 6 beta-hydroxylase (EC 1.14.11.11) catalyzes the oxidative reactions in the biosynthetic pathway leading from hyoscyamine to scopolamine. We introduced the hydroxylase gene from Hyoscyamus niger under the control of the cauliflower mosaic virus 35S promoter into hyoscyamine-rich Atropa belladonna by the use of an Agrobacterium-mediated transformation system. A transgenic plant that constitutively and strongly expressed the transgene was selected, first by screening for kanamycin resistance and then by immunoscreening leaf samples with an antibody specific for the hydroxylase. In the primary transformant and its selfed progeny that inherited the transgene, the alkaloid contents of the leaf and stem were almost exclusively scopolamine. Such metabolically engineered plants should prove useful as breeding materials for obtaining improved medicinal components.


Calcium ions are involved in Escherichia coli chemotaxis. Escherichia coli regulates intracellular free Ca2+ at about 90 nM [Gangola, P. & Rosen, B. P. (1987) J. Biol. Chem. 262, 12570-12574]. To increase intracellular free Ca2+, nitr-5/Ca2+, a "caged" Ca2+ compound, was electroporated into cells and then its affinity for Ca2+ was reduced by exposure to 370-nm light. Upon release of the Ca2+ ions, the cells tumbled. Studies on mutant strains showed that the receptor proteins (methyl-accepting chemotaxis proteins, MCPs) were not required for the Ca(2+)-induced tumbling but that CheA, CheW, and CheY proteins were required. Similar results were obtained with DM-nitrophen/Ca2+, another caged calcium compound that releases Ca2+ upon illumination at 340 nm. Diazo-2, a caged Ca2+ chelator that takes up Ca2+ upon illumination at 340 nm, was used to decrease intracellular free Ca2+, and this caused smooth swimming.


Identification of human TFIID components and direct interaction between a 250-kDa polypeptide and the TATA box-binding protein (TFIID tau). Previous studies have indicated that human transcription initiation factor TFIID is a large complex that contains a TATA-binding polypeptide (TFIID tau or TBP) and other components that qualitatively alter promoter interactions and are uniquely required for activator-dependent (versus basal) transcription. TFIID tau-specific antibody columns have been employed to identify a number of human TFIID polypeptides that are tightly associated with TFIID tau. These differ in size from polypeptides in known general initiation factors, including the initiator-binding factor (TFII-I) which shares some promoter binding characteristics with TFIID. The largest component (p250) identified in TFIID was shown to interact directly and tightly with TFIID tau, suggesting that it may play a major role in the assembly of the TFIID complex.


Characterization of the Ca(2+)-triggered conformational transition in troponin C. Troponin C is the Ca(2+)-binding subunit of troponin in vertebrate striated muscle. Binding of Ca2+ to troponin C is thought to induce a conformational change that triggers subsequent events in the initiation of muscle contraction. A molecular modeling study has proposed that, when Ca2+ binds to the N-terminal triggering sites, helices B and C separate from the helices D and A, thereby exposing a crucial interaction site for troponin I, the inhibitory subunit of troponin [Herzberg, O., Moult, J., and James, M. N. G. (1986) J. Biol. Chem. 261, 2638-2644]. In the present study the question of whether this separation actually occurs is addressed directly. A mutant rabbit skeletal troponin C containing a pair of cysteines at position 12 in helix A and position 49 in the polypeptide segment linking helices B and C was created by site-directed mutagenesis. Pyrene excimer fluorescence and resonance energy transfer studies on the labeled mutant troponin C reveal a Ca(2+)-induced increase in distance between the two cysteines. Under certain assumptions, the distance increase could be estimated from the extent of energy transfer to be approximately 13 A, in good agreement with the distance increase predicted by molecular modeling. Our results provide further experimental support for the model proposed by Herzberg et al. (above).


Expression of tissue factor by melanoma cells promotes efficient hematogenous metastasis. Metastasis is a multistep process which requires highly adapted interactions of tumor cells with host target organs. Compared with nonmetastatic cells, metastatic human melanoma cells express 1000-fold higher level of tissue factor (TF), the major cellular initiator of the plasma coagulation protease cascades. To explore whether TF may contribute to metastatic tumor dissemination, we analyzed the effect of specific inhibition of TF function on human melanoma metastasis in severe combined immunodeficient (SCID) mice. Using species-specific antibodies to TF, we demonstrate that initial adherence in insufficient for successful tumor cell implantation in a target organ. Rapid arrest of human tumor cells in the lungs of mice was not diminished by inhibition of TF. However, inhibition of TF receptor function and consequent reduction in local protease generation abolished prolonged adherence of tumor cells, resulting in significantly reduced numbers of tumor cells retained in the vasculature of the lungs. The growth of pulmonary metastases was also significantly inhibited by a blocking anti-TF monoclonal antibody and Fab fragments thereof, whereas a noninhibitory antibody lacked antimetastatic effects. Cell surface expression of functional TF thus contributes to melanoma progression by allowing metastatic cells to provide requisite signals for prolonged adhesive interactions and/or transmigration of tumor cells across the endothelium, resulting in successful metastatic tumor implantation.


A nuclear localization signal and the C-terminal omega sequence in the Agrobacterium tumefaciens VirD2 endonuclease are important for tumor formation. The T-DNA portion of the Agrobacterium tumefaciens tumor-inducing (Ti) plasmid integrates into plant nuclear DNA. Direct repeats define the T-DNA ends; transfer begins when the VirD2 endonuclease produces a site-specific nick in the right-hand border repeat and attaches to the 5' end of the nicked strand. Subsequent events generate linear single-stranded VirD2-bound DNA molecules that include the entire T-DNA (T-strands). VirD2 protein contains a nuclear localization signal (NLS) near the C terminus and may direct bound T-strands to plant nuclei. We constructed mutations in virD2 and showed that the NLS was important for tumorigenesis, although T-strand production occurred normally in its absence. A tobacco etch virus NLS, substituted for the VirD2 NLS, restored tumor-inducing activity. Amino acids (the omega sequence) at the C terminus of VirD2, outside the NLS and the endonuclease domain, contributed significantly to tumorigenesis, suggesting that VirD2 may serve a third important function in T-DNA transfer.


A human interleukin 3 analog with increased biological and binding activities. Human interleukin 3 (IL-3) variants generated by site-directed mutagenesis were analyzed in multiple biological and binding assays to identify residues critical for IL-3 activity. Two mutants carrying substitutions in the predicted hydrophilic region within the first alpha-helix, [Ala21,Leu22]IL-3 and [Ala21,Leu22,Ala25]IL-3 showed loss of biological activity and high-affinity binding. Mutants in a second predicted hydrophilic region, [Ala44,Leu45,Ala46]IL-3 and [Ala44,Ala46]IL-3, however, showed similar biological and binding activities to wild-type IL-3. Mutations in a C-terminal hydrophilic region that overlaps the fourth predicted alpha-helix led to either loss or gain of function. IL-3 analogs [Glu104,Asp105]-, [Leu108]-, [Asn108]-, [Thr108]-, and [Ala101,Leu108]IL-3 were less active than wild-type IL-3, whereas [Ala101]IL-3 and [Val116]IL-3 were 2- to 3-fold more potent. Significantly, the double mutant [Ala101,Val116]IL-3 exhibited a 15-fold greater potency than native IL-3. Receptor binding studies showed that [Ala101,Val116]IL-3 exhibited increased binding to the high- and low-affinity receptors of monocytes. These results show the generation of an IL-3 analog with increased biological and binding activities and support a model where the C terminus of IL-3 interacts with the alpha chain of the IL-3 receptor, making this region a useful focus for the development of more potent IL-3 agonists or antagonists.


Axon substitution in the reorganization of developing neural connections. Insights into the mechanisms of normal and pathological neural development may be gained by studying the reorganization of developing neural connections, caused experimentally or by disease. Many reorganized connections are assumed to arise by the anomalous stabilization of transient connections that occur during normal development. We report that, although the retina projects transiently to the somatosensory system in normal developing hamsters, the permanent retinal projections to the somatosensory system that arise as a consequence of early brain lesions are not formed by the stabilization of the normally transient projection. Instead, the transient retinal axons are replaced by retinal axons that do not normally project to the somatosensory system. The distinction between anomalous stabilization and substitution is significant for determining the cellular mechanisms underlying the development of neural connectivity.


Interaction of the yeast Swi4 and Swi6 cell cycle regulatory proteins in vitro. In budding yeast, two transcription factors, Swi4 and Swi6, control the expression of important cell cycle regulatory proteins (the G1 cyclins, Cln1 and Cln2, and the cyclin-like Hcs26) as well as the HO gene, whose product initiates mating-type switching. Both Swi4 and Swi6 are components of a protein complex that forms at a repeated sequence element, SCB (SWI4, -6-dependent cell cycle box), found in the upstream regulatory sequences of target genes. We show, by using proteins synthesized in vitro, a direct association between Swi4 and Swi6. The cdc10-Swi6 or ankyrin motifs present in both Swi4 and Swi6 are dispensable for their association, which is mediated instead by a region near the C terminus of each protein. Furthermore, we show that interaction with Swi6 is not necessary for specific recognition of the SCB sequence by the Swi4 protein; we propose that Swi4 is responsible for binding to the SCB sequence while Swi6, through its association with Swi4, regulates activity of the complex.


Insulin-like growth factor I accelerates recovery from ischemic acute tubular necrosis in the rat. The effects of administering insulin-like growth factor I (IGF-I) were examined in a model of ischemic acute tubular necrosis in rats. Injury was induced by 75 min of bilateral renal artery occlusion. Compared to rats administered vehicle, rats administered IGF-I (100 micrograms/day via continuous subcutaneous infusion) had significantly lower serum creatinine and blood urea nitrogen levels over the course of 7 days postocclusion. Glomerular filtration rate as determined by inulin clearance was examined on day 2 postocclusion and was significantly increased in IGF-I-treated animals (0.16 +/- 0.02 ml per min per 100 g of body weight) compared to vehicle-treated controls (0.08 +/- 0.02 ml per min per 100 g of body weight). The weight loss that occurred during the course of acute tubular necrosis was ameliorated by IGF-I. Mortality was reduced from 36.7% in vehicle-treated rats to 7.1% in rats administered IGF-I. Histologically, there was much less renal injury evident at day 7 postocclusion in the IGF-I-treated rats compared to vehicle-treated controls. In contrast, growth hormone (200 micrograms administered subcutaneously for 4 days) did not affect recovery of renal function or reduce mortality postreperfusion. This report demonstrates a beneficial effect of IGF-I administration in the setting of acute tubular necrosis. Several properties of IGF-I render it a pharmacological agent with excellent potential for treatment of this condition in humans.


1H NMR studies on the catalytic subunit of aspartate transcarbamoylase. The 1H NMR spectrum of the catalytic subunit of Escherichia coli aspartate transcarbamoylase (EC 2.1.3.2) was simplified by using strains auxotrophic for the aromatic amino acids and a growth medium containing fully deuterated Trp, Phe, and His and partially deuterated Tyr. 1H resonances for Tyr in the catalytic trimer (M(r) = 10(5)) were partially resolved into five peaks at 27 degrees C, which above 50 degrees C were further resolved to give a distinct resonance for each of the eight Tyr residues in the polypeptide chain. Experiments on chemically modified catalytic subunits and on a mutant form in which Tyr-165 was converted to Ser-165 led to the assignment of resonances for Tyr-165, Tyr-240, and Tyr-185. Binding of the substrate, carbamoyl phosphate, caused shifts of two of the unassigned resonances, and the subsequent binding of the aspartate analog succinate perturbed the resonances corresponding to Tyr-165 and Tyr-240. The bisubstrate analog N-(phosphonacetyl)-L-aspartate produced a spectrum differing considerably from that caused by the combination of carbamoyl phosphate and succinate. The NMR spectrum for the Tyr-165-->Ser mutant trimer showed clearly that the single amino acid substitution caused conformational changes affecting the environment of residues remote from the position of the replacement. In contrast, the inactive mutant subunit in which Gly-128 was replaced by Asp exhibited a spectrum virtually identical to that of the wild-type protein. However, addition of the substrate carbamoyl phosphate caused a marked change in the spectrum of the mutant enzyme, whereas that of the wild-type trimer was altered only slightly, showing that the effect of the amino acid substitution was manifested in the NMR spectrum only with the liganded enzyme.


Major histocompatibility complex class II genes of zebrafish. Twenty cDNA clones derived from beta-chain-encoding class II genes of the zebrafish (Brachydanio rerio) major histocompatibility complex (MHC) have been sequenced. They fall into three groups identifying three loci of expressed genes. The length and organization of these genes are similar to those of their mammalian homologs. Amplification by polymerase chain reaction and sequencing of genomic DNA from zebrafish collected at different locations in India indicate the existence of a fourth group of sequences (fourth locus). A high degree of polymorphism at the B. rerio MHC loci and concentration of variability to the putative peptide-binding region of the beta 1-domain-encoding part of the gene are also indicated. Large genetic distances between alleles suggest trans-specific evolution of fish MHC polymorphism. Zebrafish genes appear to be derived from a different ancestor than the various class II gene families of other vertebrates. In spite of great sequence divergence between fish and mammalian MHC genes, there seems to be a striking conservation in their overall organization.


Identity of 4a-carbinolamine dehydratase, a component of the phenylalanine hydroxylation system, and DCoH, a transregulator of homeodomain proteins. The principal pathway for the metabolism of phenylalanine in mammals is via conversion to tyrosine in a tetrahydrobiopterin-dependent hydroxylation reaction occurring predominantly in the liver. Recently, the proposal that certain hyperphenylalaninemic children may have a deficiency of carbinolamine dehydratase, a component of the phenylalanine hydroxylation system, has widened the interest in this area of metabolism. Upon cloning and sequencing the dehydratase, we discovered that this protein is identical to DCoH, the cofactor which regulates the dimerization of hepatic nuclear factor 1 alpha, a homeodomain transcription factor. The identity of the nuclear and cytoplasmic proteins is demonstrated by size, immunoblotting, stimulation of phenylalanine hydroxylase, and dehydratase activity. The evolution of the dual functions of regulation of phenylalanine hydroxylation activity and transcription activation in a single polypeptide is unprecedented.


A 69-kDa RNA-binding protein from Xenopus oocytes recognizes a common motif in two vegetally localized maternal mRNAs. Vg1 mRNA, a maternal message encoding a member of the transforming growth factor beta superfamily, undergoes localization to the vegetal cortex of Xenopus laevis oocytes during a narrow period of oogenesis. A 340-nucleotide sequence has been identified in Vg1 RNA that directs its vegetal localization [Mowry, K. L. & Melton, D. A. (1992) Science 255, 991-994]. To understand how cis- and trans-acting factors are involved in Vg1 mRNA localization, we have looked for specific interactions in vitro between oocyte proteins and Vg1 mRNA. S100 extracts of late-stage oocytes contain a protein-binding activity that protects specific regions of labeled Vg1 mRNA from degradation by RNase T1. The use of different regions of Vg1 RNA in competition reactions reveals two binding sites, both in the first half of the 3' untranslated region of Vg1 message. UV crosslinking predominantly labels a 69-kDa protein; saturation analysis and competitor studies indicate that this protein binds with a high affinity to the down-stream site, which corresponds to the 340-nucleotide vegetal localization sequence. Binding to this region is inhibited by another vegetally localized message, transforming growth factor beta 5 but is not inhibited by an animally localized RNA, An2. These data indicate that vegetally localized mRNAs share a binding motif that helps them achieve their intracellular distribution through specific RNA-protein interactions.


Relationships between orientation-preference pinwheels, cytochrome oxidase blobs, and ocular-dominance columns in primate striate cortex. The relationships between cytochrome oxidase blobs, ocular-dominance columns, and iso-orientation domains, subsystems underlying visual perception, were explored in primary visual cortex of macaque monkey. High-resolution maps of these three subsystems were acquired. Optical imaging based on activity-dependent intrinsic signals revealed that the most prominent organizational feature of orientation preference was a radial arrangement, forming a pinwheel-like structure surrounding a singularity point. More than 80% of these pinwheels were centered along the midline of ocular-dominance columns. The iso-orientation contours of adjacent pinwheels crossed borders of ocular-dominance columns at approximately right angles. Pinwheels with the same or opposite directions of orientation-preference change were smoothly connected with each other. On the average, all orientations were equally represented. In exactly the same cortical area, the cytochrome oxidase blobs, thought to be involved in color processing, were also mapped, using cytochrome oxidase histology. Like the centers of pinwheels, the centers of blobs also lie along the midline of ocular-dominance columns. However, the centers of pinwheels did not coincide with the centers of blobs; these two subsystems are spatially independent. "Hypercolumn" modules, each including two complete pinwheels in two adjacent columns of complementary ocularity, as well as portions of a few blobs, were frequently found but did not seem to be the primary unit of cortical organization. An alternative to hypercolumns is proposed.


Deformation of DNA during site-specific recombination of bacteriophage lambda: replacement of IHF protein by HU protein or sequence-directed bends. Escherichia coli IHF protein is a prominent component of bacteriophage lambda integration and excision that binds specifically to DNA. We find that the homologous protein HU, a nonspecific DNA binding protein, can substitute for IHF during excisive recombination of a plasmid containing the prophage attachment sites attL and attR but not during integrative recombination between attP and attB. We have examined whether IHF and HU function in excisive recombination is mediated through DNA bending. Our strategy has been to construct chimeric attachment sites in which IHF binding sites are replaced by an alternative source of DNA deformation. Previously, we demonstrated that properly phased bends can substitute for the binding of IHF at one site in attP. Although this result is highly suggestive of a critical role of IHF-promoted bending in lambda integration, its interpretation is obscured by the continued need for IHF binding to the remaining IHF sites of these constructs. In the present work, we engineered a population of sequence-directed bends in the vicinity of the two essential IHF sites found in attR and attL. Even in the absence of IHF or HU, pairs of these attachment sites with properly phased bends are active for both in vitro and in vivo excision. This success, although tempered by the limited efficiency of these systems, reinforces our interpretation that IHF functions primarily as an architectural element.


Primary structure of an archaebacterial transducer, a methyl-accepting protein associated with sensory rhodopsin I. A methylated membrane protein of 97 kDa was suggested on the basis of mutant analysis to transduce signals from the phototaxis receptor sensory rhodopsin I to the flagellar motor in Halobacterium halobium. Here we report isolation of the proposed transducer protein, cloning of its gene based on partial protein sequences, the complete gene sequence, and analysis of the encoded primary structure. The 1611-base-pair gene termination codon overlaps the initiator ATG of the sopI gene, which encodes the sensory rhodopsin I apoprotein. The predicted size of 57 kDa for the methylated protein indicates an aberrant electrophoretic migration on SDS/polyacrylamide gels, as occurs with other acidic halophilic proteins. Putative promotor elements are located in an A+T-rich region upstream of the gene. Comparison of the translated nucleotide sequence with N-terminal sequence of the purified protein shows the protein is synthesized without a processed leader peptide and the N-terminal methionine is removed in the mature protein. The deduced protein sequence predicts two transmembrane helices near the N terminal that would anchor the protein to the membrane. Beyond this hydrophobic region of 46 residues, the remainder of the protein (536-amino acid residues total) is hydrophilic. The C-terminal 270 residues contain a region homologous to the signaling domains of eubacterial transducers (e.g., Escherichia coli Tsr protein), flanked by two regions homologous to the methylation domains of the transducer family. The protein differs from E. coli Tsr in that it does not have an extramembranous-receptor binding domain but instead has a more extended cytoplasmic region. Coexpression of the methyl-accepting protein gene (designated htrI) and sopI restores sensory rhodopsin I phototaxis to a mutant (Pho81) that contains a deletion in the htrI/sopI region. These results extend the eubacterial transducer family to the archaebacteria and substantiate the proposal that the methylated membrane protein functions as a signal-transducing relay between sensory rhodopsin I and cytoplasmic sensory-pathway components.


Ku autoantigen is the regulatory component of a template-associated protein kinase that phosphorylates RNA polymerase II. The carboxyl-terminal domain of RNA polymerase II contains a tandemly repeated heptapeptide sequence. Previous work has shown that this sequence is phosphorylated at multiple sites by a template-associated protein kinase, in a reaction that is closely associated with the initiation of RNA synthesis. We have purified this kinase to apparent homogeneity from human (HeLa) cells. The purified kinase phosphorylates native RNA polymerase II only in the presence of DNA and the general transcription factors TFIID (TBP), TFIIB, and TFIIF. Two kinase components are required for full activity: a catalytic component and a DNA-binding regulatory component. The regulatory component has been identified as Ku autoantigen, based on the molecular weights of its component polypeptides, its DNA-binding properties, and its reactivity with anti-Ku monoclonal antibodies. The Ku autoantigen recruits the catalytic component of the kinase to the template. Ku autoantigen has been previously proposed to interact with DNA by a characteristic bind-and-slide mechanism. This mode of interaction may provide a mechanism for targeting the kinase to the transcription complex and other DNA-bound substrates.


DNA strand-specific repair of (+-)-3 alpha,4 beta-dihydroxy-1 alpha,2 alpha-epoxy-1,2,3,4-tetrahydrobenzo[c]phenanthrene adducts in the hamster dihydrofolate reductase gene. We evaluated the formation and removal of (+-)-3 alpha,4 beta-dihydroxy-1 alpha,2 alpha-epoxy-1,2,3,4- tetrahydrobenzo[c]phenanthrene (BcPHDE)-DNA adducts in two Chinese hamster ovary (CHO) cell lines. One line of repair-proficient cells (MK42) carries a stable 150-fold amplification of the dihydrofolate reductase (DHFR) locus. The other line of repair-deficient cells (UV-5) is diploid for this gene and is defective in excision of bulky DNA lesions. Two methods were used to quantitate adduct levels in treated cells: Escherichia coli UvrABC excision nuclease cleavage and 32P-postlabeling. DNA repair was examined in the actively transcribed DHFR gene, in an inactive region located 25 kilobases downstream, and in the overall genome. Between 8 and 24 hr after BcPHDE exposure, preferential repair of the DHFR gene compared to the noncoding region was apparent in MK42 cells. This gene-specific repair was associated with adduct removal from the DHFR transcribed strand. However, UV-5 cells showed no lesion reduction from this strand of the gene. By both quantitation methods, regions accessible to repair in MK42 cells showed a 2-fold reduction in DNA adduct levels by 24 hr. That the decline in adducts reflects genomic repair was demonstrated by the constant damage level remaining in UV-5 cells. Since BcPHDE-induced mutations in DHFR apparently arise from adducted purines on the nontranscribed strand, results from the present study support the idea that a consequence of strand-specific repair is strand-biased mutations.


Identification of a 45-kDa protein at the protein import site of the yeast mitochondrial inner membrane. Import of proteins into mitochondria involves the cooperation of protein translocation systems in the outer and inner membranes. We have identified a 45-kDa protein at the protein import site of the yeast mitochondrial inner membrane. This 45-kDa protein could be crosslinked to a partly translocated precursor, which cannot be imported across the inner membrane when the matrix is depleted of ATP. In addition, an antibody against this protein strongly inhibited protein import into right-side-out inner-membrane vesicles. The 45-kDa protein accounts for only 0.1% of mitochondrial protein and appears peripherally attached to the outer face of the inner membrane. The properties of this protein suggest that it is a component of the protein import system of the mitochondrial inner membrane.


Gene-culture coevolution: toward a general theory of vertical transmission. A general formulation of cultural and genetic transmission is developed. The cultural transmission is vertical and the genetics may involve multiple loci. Each individual is represented by a phenogenotype, and conditions are given under which the evolutionary dynamics of phenogenotype frequencies are reducible to phenogametic or phenoallelic frequencies. The interaction between genes and culture is specified by an association measure, and results on the order of magnitude of this association at equilibrium are presented.


Resistance to host antimicrobial peptides is necessary for Salmonella virulence. The production of antibacterial peptides is a host defense strategy used by various species, including mammals, amphibians, and insects. Successful pathogens, such as the facultative intracellular bacterium Salmonella typhimurium, have evolved resistance mechanisms to this ubiquitous type of host defense. To identify the genes required for resistance to host peptides, we isolated a library of 20,000 MudJ transposon insertion mutants of a virulent peptide-resistant S. typhimurium strain and screened it for hypersensitivity to the antimicrobial peptide protamine. Eighteen mutants had heightened susceptibility to protamine and 12 of them were characterized in detail. Eleven mutants were attenuated for virulence in vivo when inoculated into BALB/c mice by the intragastric route, and 8 of them were also avirulent following intraperitoneal inoculation. The mutants fell into different phenotypic classes with respect to their susceptibility to rabbit defensin NP-1, frog magainin 2, pig cecropin P1, and the insect venom-derived peptides mastoparan and melittin. The resistance loci mapped to eight distinct locations in the genome. Characterization of the mutants showed that one had a defective lipopolysaccharide and another mutant harbored a mutation in phoP, a locus previously shown to control expression of Salmonella virulence genes. Our data indicate that the ability to resist the killing effect of host antimicrobial peptides is a virulence property and that several resistance mechanisms operate in S. typhimurium.


Formation of guide RNA/messenger RNA chimeric molecules in vitro, the initial step of RNA editing, is dependent on an anchor sequence. Synthetic pre-edited messenger RNA (mRNA) and guide RNA (gRNA) for the 5'-edited maxicircle-encoded ND7 cryptogene from Leishmania tarentolae formed chimeric molecules upon incubation in the presence of a mitochondrial extract. These chimeric molecules consisted of the gRNAs covalently linked to the mRNAs by short oligo(U) tails at normal editing sites in most cases. Unlike the previously reported chimeras present in steady-state kinetoplast RNA, the in vitro-synthesized chimeras showed no editing of downstream editing sites. The synthesis of chimeric RNAs required ATP and was dependent on the formation of a gRNA/mRNA anchor duplex 3' of the pre-edited region, as shown by in vitro mutagenesis of the mRNA and the gRNA. mRNA sequences 3' and 5' of the pre-edited region also affected the efficiency of the chimera-forming activity. This in vitro system may accurately represent the initial step in RNA editing.


Insertional mutagenesis of hydrophilic domains in the lactose permease of Escherichia coli. The lactose permease of Escherichia coli is a membrane transport protein postulated to contain a hydrophilic N terminus (hydrophilic domain 1), 12 hydrophobic transmembrane alpha-helices that traverse the membrane in zigzag fashion connected by hydrophilic domains, and a hydrophilic C terminus (hydrophilic domain 13). To test whether the hydrophilic domains are important for function, each domain was independently disrupted by insertion of two or six contiguous histidine residues, and the mutants were characterized with respect to initial rate of lactose transport and steady-state level of accumulation. Remarkably, histidine insertions into 10 out of 13 hydrophilic domains result in molecules that catalyze lactose accumulation effectively, although the initial rate of transport is compromised in certain cases. In contrast, insertions into hydrophilic domain 3, 9, or 10 cause a marked decrease in transport activity. As judged by immunoblots and [35S]methionine pulse-chase experiments, diminished activity is not due to decreased expression of the mutated permeases, defective insertion into the membrane, or increased rates of proteolysis after insertion. The results (i) suggest that most of the hydrophilic domains in the permease do not play an essential role in the transport mechanism and (ii) focus on the region of the permease containing putative helices IX and X as being particularly important for activity.


Induction of expression of monocyte interleukin 1 by Hageman factor (factor XII). The results reported here indicate that activated species of Hageman factor (HF, factor XII), a protein that mediates blood clotting, fibrinolysis, and activation of the complement cascade, induce elaboration of interleukin 1 (IL-1) by human monocytes. Augmentation of IL-1 production in mononuclear cell cultures was observed when HF was present along with lipopolysaccharide (LPS) but was not observed with HF alone. Furthermore, antiserum to HF abrogated the enhancement of IL-1 in cultures containing HF and LPS. Total IL-1 activity, which represents secreted and cell-associated IL-1, was enhanced in LPS-stimulated mononuclear cultures by HF. In the absence of LPS, the initial activation product of HF, HFa, which contains the serine protease enzyme activity and the surface-binding domains of the protein, induced IL-1 beta protein and mRNA. In the presence of LPS, the enzymatic moiety (HFf), which is also contained in HF and HFa, amplified IL-1 production. Induction and amplification of monocyte IL-1 by HF provides further evidence for establishing a role for HF in the acute-phase reaction and the cellular immune response.