[Ultrastructure and hormone secretion of a tissue culture from an adrenal gland tumor simulating Cushing's syndrome]. Tissue cultures of a surgically removed adrenocortical tumour causing Cushing's syndrome, and tissue cultures from the attached, tumour-free adrenal were studied. There were two cell types characteristic of tumour tissue. The cell type occurring most frequently had pronounced hypertrophied agranular endoplasmic reticulum. A fewer number of lipidrich cells containing many electron-dense granules could also be found. The ratio of cells changed during cultivation. In the 17 days tumour culture, a higher percentage of lipid-rich cells could be observed. In spite of continuous ACTH treatment, the initially high hydrocortisone level decreased, gradually. It may be assumed that the lipid-rich cells are of reduced ability as regards hydrocortisone production.


[Clinical aspects of intrauterine infections (author's transl)]. Examples of typical embryopathy (rubella, cytomegaly) and fetopathy (toxoplasmosis, listeriosis) demonstrate the disastrous effects intrauterine infections may have on the fetus. Even if they play a relatively small role numerically, they deserve our particular attention because we feel and hope that, by extending maternity care to environmental hygiene and prophylactic vaccination, they can be partly prevented and, by improving prenatal serological diagnosis and intensifying the cooperation of specialists, introduced early to an effective prenatal therapy, thus preventing severe damage to the child.


[Diagnostic possibilities for the prevention and identification of intrauterine infections (author's transl)]. The demonstration of antibody titer movements in the hemagglutination inhibition test, in the complement fixation test and a differentiation between IgM and IgG antibodies allow information to be obtained on the age of an intra-uterine rubella infection. Specific IgM antibodies in neonates are reliable evidence, because the maternal IgM antibodies cannot cross the placenta. A rise in total IgM level in cord blood does not, on the other hand, indicate an embryopathy in every case. In contradistinction to rubella, cytomegaly and herpes simplex virus can be comparatively easily and quickly cultivated, so that identification of the virus is of use for clinical purposes. Of 2360 children investigated in our laboratory from 1969-1974, 120 certainly and 57 extremely probably showed rubella embryopathies.


Basis for recurring ventricular fibrillation in the absence of coronary heart disease and its management. A 39-year-old man twice experienced ventricular fibrillation and exhibited numerous ventricular premature beats. Coronary arteries were normal, and no impaired cardiac function was found upon catheterization. Evidence was adduced that the ventricular premature beats were related to higher nervous activity. The patient had serious psychiatric problems; the ventricular premature beats were provoked by psychophysiologic stress, increased during REM sleep, were reduced by meditation, and were controlled by beta-adrenergic blockade, phenytoin and digitalization. We conclude that psychologic and neurophysiologic factors may predispose to life-threatening cardiac arrhythmia in the absence of organic heart disease. Effective management of the recurrent ventricular arrhythmia involved; acute drug testing for assessing antiarrhythmic efficacy; use of programmed trendscription to provide on-line information on drug action; a treatment program involving more than one agent; and use of measures to reduce sympathetic nervous activity.


Serum lipoproteins and coronary heart disease in a population study of Hawaii Japanese men. To ascertain the frequency of defined hyperlipoproteinemia and to investigate the relation between lipoprotein fractions and coronary heart disease, we measured serum lipoprotein cholesterol levels in a population-based sample of Hawaii Japanese men 50 to 72 years old. Type II hyperlipoproteinemia was present in 3 per cent of 1859 men, and Type IV in 26 per cent. Relative risks for coronary heart disease, based on 264 prevalence cases, were found to be 1.8, 1.8 and 0.46, between the upper and lower quartiles of total, beta, and alpha cholesterol, respectively. We found no significant relation between triglyceride and coronary heart disease. The inverse relation of alpha cholesterol of prevalence of coronary heart disease was independent of beta cholesterol, obesity, and other factors. The data suggest the need for further evaluation of the protective effect of the alpha lipoprotein fraction on the development of coronary heart disease.


Respiratory syncytial virus infections within families. To examine intrafamily spread of respiratory syncytial virus infections and their associated illnesses, 36 families with 188 members were studied during an outbreak of such infections. Nurses visited every three to four days to obtain specimens for viral isolation and interview household members. The virus infected 44.4 per cent of families, and 21.9 per cent of all members. All age groups had appreciable attack rates (with a range of 16.8 per cent in adults to 29.4 per cent in infants). In infected families, 45.9 per cent of members became infected, including 10 of 16 infants. Secondary attack rate for all ages was 27 per cent, and that for infants 45.4 per cent. An infant's older sibling appeared most likely to introduce the virus into the family. Associated acute respiratory illnesses occurred in 94.9 per cent of cases, and appeared more severe than those not associated with respiratory syncytial virus. When the virus was introduced into a family the high attack rate produced an illness of age-related severity.


Liver-cell adenomas associated with use of oral contraceptives. During the past six years an increasing number of liver-cell adenomas in women were diagnosed, and we undertook a study of possible etiologic factors, including use of oral contraceptives, in 42 women. There was a significant difference between cases and matched controls in mean months of oral-contraceptive use: 73.4 as compared to 36.2 (P less than 0.001). The women with adenomas took mestranol-containing pills much more commonly than the controls (P less than 0.0001). Hemorrhage into the tumor was often associated with the onset of menstruation in women taking oral contraceptives. Long-term use of oral contraceptives, especially those containing a high total dose of steroids or mestranol as the synthetic estrogen, should be balanced against risks of side effects of the drugs.


[Lacrimators as acceptors for NADH].

[Lacrimators as acceptors for NADH]. Lachrymators of varied structure are reduced either by hydrogen addition or halogen substitution using NADH model compounds as donors. Similar compounds without lachrymatory activity were reduced either very slowly or not at all. CS (o-Chlorobenzalmalonitril) is reduced by NADH, the reaction being catalyzed by an enzyme present in erythrocytes. Thus the lachrymatory action follows a general scheme for the activity of sensory transduction. This scheme consists of a reception in the nerve cell membrane and a fast or simultaneous chemical transformation in an enzymic reaction.


Outbreaks of swine vesicular disease in japan: virus isolation and epizootiological survey. Outbreaks of a vesicular disease occurred among pigs in Kanagawa and Ibaraki Prefectures in Japan in November, 1973. Another outbreak was observed in Aichi Prefecture in December. The clinical signs of the disease observed included fever and vesicular lesions on the coronary bands, bulbs of the heel and in the interdigital spaces. In some pigs, vesicular lesions were observed on the snout, tongue and skin overlying the legs and abdomen. All the vesicular samples produced cytopathic changes on cultures of primary swine kidney cells of PK-15 cells. Three isolates of cytopathic agents tested were identified as swine vesicular disease virus from their physicochemical properties and antigenicity. The virus strains isolated from vesicular epithelial samples obtained from Ibaraki, Kanagawa and Aichi Prefectures were designated as Japan/Ibaraki/1/73, Japan/Kanagawa/1/73 and Japan/Aichi/1/73 strain, respectively. An outbreak of the disease among pigs due to swine vesicular disease virus was confirmed by the serum neutralization test with serum samples collected from pigs on affected farms. Approximately 80% of the pigs housed in affected shed showed high levels of neutralizing antibody titers. This is the first to report an occurrence of swine vesicular disease among pigs in Japan.


Diagnosis of bovine respiratory syncytial virus infection by complement fixation test. The complement fixation test by the microtiter method was applied to the serological diagnosis of bovine respiratory syncytial (RS) virus infection. When used as complement fixing antigens, untreated infected cell culture fluid, fluorocarbon-treated, and ether-treated materials showed no differences in antigenicity among them. The complement fixing antigenicity of bovine RS virus appeared in bovine kidney and Vero cell cultures for the first time 4 days after inoculation. Both the infectivity and complement fixing antigenicity reached a maximum 6 days after inoculation. In detecting complement fixing antibody from infected cattle, the most outstanding specific reaction was obtained when 5% fresh normal calf serum had been added to the diluent of complement. Neutralizing and complement fixing antibodies were examined in serum samples from two cattle in the course of experimental infection. It was found that both antibodies turned to be positive 2 weeks after inoculation. There was a linear correlation between neutralizing and complement fixing antibody titers, when serum samples from 40 natural cases were tested in the acute and convalescent stages. In addition, common antigenicity was demonstrated between the virus of bovine origin and the Long strain of human RS virus by complement fixation test.


[Cholinergic mechanisms of interneuronal transmission in the retina]. Acetylcholine applied to the isolated perfused retina (frog and man) elicits a slow corneapositive potential. Such an effect of ACh besides the data known from the literature confirms an assumption on cholinergic synaptic transmission within the retina. The retinal potential elicited by ACh depends in its size and time course on definite parameters of the ACh application as well as on the illumination state of the retina: light stimulation diminishes the electrogenic action of ACh, flickering illumination being more effective than steady one. On the other hand electrical responsiveness of the retina to light lowered during the perfusion of the preparation with ACh. The b-wave seems to be the most sensitive part of the ERG and can be abolished completely, thus isolating the component PIII. The mentioned results as well as the data from the literature indicate the synapses between the first and second retinal neurons as the points of ACh action. Experimental results can be interpreted as the consequence of a desensitization of the cholinergic receptors within the retina.


[Synaptic processes in neurons of the cat pericruciate cortex evoked by pyramidal tract stimulation]. The influences of pyramidal tract stimulation on the activity of neurons in the pericruciate cortex were investigated on 423 neurons (81 neurons were studied intracellularly and 342--extracellularly), 78 of them having background activity. Pyramidal stimulation is shown to evoke not only antidromic spikes (0.5-16.0 ms latency) in the pyramidal cells, but also lateral and recurrent PSPs in the pyramidal and unidentified units of all cortical layers. IPSPs were observed in 46.7% of the investigated neurons, EPSPs--in 21.0%, mixed responces--in 26.0%. The latency of IPSPs was 1.5-14.0 ms, their amplitude ranged from 1.3 to 17.0 mV, the duration of the rising phase varied from 4 to 18 ms and the whole duration was 18-120 ms reaching sometimes 250-500 ms. In 30% of cases it was possible to divide the IPSPs into two phases: a fast one with a duration of 10-20 ms and a slow one. The latency of IPSPs was 2.6-19.0 ms, their amplitude--1.0--7.8 mV and duration--from 10.0 to 50.0 ms. The antidromic discharge in the pyramidal tract inhibited the background activity for 200-400 ms in 51.2% of spontaneously active units; acceleration was observed in 19.5% and mixed effect in 7.4% of units. The participation of pyramidal axonal collaterals and cortical interneurons in generation of the described processes is discussed.


[Generation and propagation of direct responses of intact and chronically isolated strips of cortex]. Generation and transmission mechanisms of direct cortical responses were studied in the acute experiment on unanesthetized curarized cats ant intact cortex or a chronically isolated cortical strip. All studied properties of the direct response at the intact cortex and isolated strip of gyrus suprasilvius are identical. The blockage of cortical muscarinic cholinoreceptors between the stimulating and recording electrodes prevents or significantly decreases excitation transmission throught his part of the cortex. This can be explained by a polysynaptic mechanism of the response transmission. Neuronal nets providing the propagation of direct responses are anisotropic. Both normal and inverted (by GABA, nembutal or coagulation of the cortical surface) direct responses are increased aiter anticholinesterase drug (galanthamine) application. Amizil abolishes all these potential. A suggestion is made that both deep and superficial components of the direct cortical response are generated by endogenous cortical cholinergic elements.


[Postsynaptic membrane sensitivity to acetylcholine during blockade of excitatory transmission from nerve to skeletal muscle during long-term low-frequency transmission]. Blocking of myoneural transmission resulting from indirect low-frequency stimulation (fatigue) was not followed by amplitude changes in miniature end-plate potentials and acetylcholine-potentials. A conclusion was drawn that in performed experiments the acetylcholine sensitivity of the postsynaptic membrane was not changed and the decrease of endplate potentials amplitude during fatigue was due to the lowering of EPPs quantum content. A reduction in the quantum content of EPP results from negative antidromic influence of the muscle on the motor nerve endings caused by chemical agents formed in muscle during its contractile appartus activity.


Incorporation of [3H] leucine into brain stem protein fractions: the effect of a behaviorally active, N-terminal fragment of ACTH in hypophysectomized rats. In hypophysectomized rats, the incorporation of [3H] leucine into rat brain stem protein, measured 5 min after injection of the precursor into the diencephalon, was decreased. Chronic treatment of these rats with the N-terminal fragment of ACTH, ACTH1-10 increased the incorporation rate. Brain stem proteins were sequentially extracted with a hypotonic buffer, Triton X-100 and SDS. Analysis of these protein fractions on polyacrylamide gels revealed that hypophysectomy caused a general decrease in leucine incorporation into all protein species studied, whereas treatment of hypophysectomized rats with ACTH1-10 enhanced this incororation into all proteins. Superimposed on these general effects were minor differences found in two protein bands. Thus, removal of the pituitary and subsequent, chronic treatment of rats with ACTH1-10 interferes with overall protein metabolism rather than with certain protein species in particular.


[Clinical value of magnification cerebral angiography (author's transl)]. Magnification carotid and vertebral angiography was performed via femoral catheter with  magnification factor of 2.5 to 3.0, utilizing 0.1 X 0.1 ultra fine focal spot, manufactured by Shimadzu Seisakusho, Ltd. The standard angiograms were compared with magnification angiograms in a group of 67 patients with brain tumors and vascular disorders. The magnification angiography was more valuable  than conventional angiography in 12 of 17 vascular tumors, while it was rarely valuable for diagnosis of vascular tumors. Aneurysms, occlusive diseases, collateral vessels, and capillary blush were also visualized to better advantage on magnification angiograms. Magnification cerebral angiography is a useful tool for elucidating small vascular branches, but further refinement in the design of focal spots and angiographic techinques is required for further improvement of magnification cerebral angiography.


[The rapid microscopical diagnosis of brain and spinal cord tumors by cryostat-cut frozen section (author's transl)]. The rapid histological diagnosis of neurosurgical biopsy material is of prime importance for the neurosurgeon. Cryostat-cut sections of fresh frozen tissues have been used in our department. The surgical materials are rapidly frozen at - 150 degrees C, and serial sections, 10 microns thick,  are cut in a cryostat at -18 degrees to -20 degrees C. The sections are  adhered to cover glasses and stained with Metachrome solution (a mixture of 1% Azure A and 0.5% Erie Garnet B). The rapid and accurate diagnosis can be obtained within 5 minutes. The advantage of cryostat-cut frozen section is the preservation of cell density and tissue architecture, which is most important in the diagnosis of neurosurgical materials, especially in case of diffusely infiltrating glioma. The previously reported methods, such as smear technique and frozen sections by using freezing microtome are also reviewed and discussed.


[The effect of radiotherapy for glioblastoma: a review of 516 cases (author's transl)]. 516 cases of supratentorial glioblastoma, treated in the Department of Surgical Neurology, University of Edinburgh from 1950 through 1970, were analysed with a particular attention to the effect of radiotherapy and the factors contributing to better prognosis. The length of postoperative survival was known in 349 cases and it was more than one month in 238 cases. A megavoltage linear accelerator was introduced in 1955 for radiotherapy of glioblastoma and steroids were started to be used almost routinely in 1966 for pre- and postoperative peritumoral cerebral edema. The policies of the treatment of glioblastoma in this series were; 1) to establish the histological diagnosis and 2) to prolong "useful" postoperative survival of the patient. Among the 516 cases, 271 cases (52.5%) were treated by biopsy alone. More radical procedures and/or radiotherapy were indicated only when survival of the patient was expected to be "useful" for himself and his family. "Useful" life was defined as the condition where the patient was conscious and orientated, and would, on the whole, be glad that he was still alive. Patients with disturbed consciousness, profound aphasia, bedridden or mentally disorganized, were regarded as having useless life. Radiotherapy, if indicated, was given by a 4-megavoltage linear accelerator to the whole brain with a total dose of 4500 rads in a period of 4 weeks. In order to evaluate the effect of radiotherapy, 238 cases who survived more than one month postoperatively were selected, because it took at least one month to complete the course of radiotherapy, because most cases with a biopsy alone survived less than one month, and because unexpected early death due to postoperative complications occurred in one month. The average survival for the irradiated 138 cases was 13.8 months, as compared to 5.2 months for the non-irradiated 100 cases. This difference of 8.6 months was highly significant as confirmed statistically by U-test. Although patients with profound aphasia or severe dimentia were not irradiated, aphasia and dimentia would not affect the length of biological survival of the patient. Therefore, the difference of 8.6 months could be considered as the biological effect of radiotherapy. Among the factors considered, young age, early epilepsy and relatively benign histology (astrocytoma, grade 3) appeared to be related to better prognosis. There was no evidence that a macroscopic circumscribed appearance would contribute to better prognosis. In conclusion, radiotherapy should be indicated for the cases whose survival is expected to be "useful", although its effect is limited.


[Morphological differentiation in vitro of human continuous and functional neuroblastoma cell line, NB-I under treatment of (But)2cAMP (author's transl)]. Human neuroblastoma cell line (NB-I), obtained from metastatic neck lymph node of 2.3 y-o, boy, is established and maintained in vitro for more than 3 years in our department. The cells have been proliferated in 10% calf serum in EAGLE's medium, in closed stational culture system. The cell line has characteristic activities of rather rapid growth (Tg = 40 or 50h, 1974) catecholamine metabolism and chromosomal abnormality, bearing two marker chromosomes. NB-I cells are uncloned because of co-adhesiveness but biochemically identified as adrenergic line. Morphologically, they are round or tear-drop like in shape with delicate short protoplasmic processes. NB-I cells exhibit remarkable morphological differentiation and maturation under treatment of (But)2cAMP (final concentration, 1mM/ml). A longer time (1-2 weeks) for differentiation, however, is needed in comparison with that of mouse neuroblastoma cell lines. The NB-I cells exhibit hypertrophy of cytoplasma and nuclei in size, elongation of cytoplasmic processes, as well as the tendency of flattness and multipolarity of cells. Electronmicroscopically, the cells that showed scant organella but plentiful polysomes in the specimen, obtained during autopsy, reveal an increase of cell organellae after implantation in vitro: Golgi apparatus, neurotubules and neural dense bodies appeared in cytoplasma. These organellae characteristic of sympathetic neuron are much more increased under treatment of (But)2cAMP in 3 weeks. Core-vesicle like dense bodies are extremely rich in periphery of perikaryon and elongated processes (100 - 150 mu in diameter). Microtubules are also increased, running in bundles in processes. The NB-I cells are considered to differentiate and approach to matured neurons in vitro.


'Glioblastoma'. Induction of a reproducible autochonous tumor in rats with murine sarcoma virus. Inoculation of 0.02 ml of high-titer Kirsten strain Murine Sarcoma Virus into the brains of 10-day-old Wistar/Furth rats yields, with 100 percent incidence, a uniform glioblastoma-like tumor within 16 days. Light and electronmicroscopy confirmed the neuroectodermal origin of the parenchymal cells. The remarkable vascular component was studied with extracellular tracers. The permeability of the abnormal endothelium to constituents of the blood vascular compartment was confirmed. Accessory vascular channels, and blood channels devoid of endothelium entirely, were observed. This reporducible system should provide a useful model for further studies of the biology of brain tumors.


Jaw and blind reflexes in trigeminal nerve lesions. An electrodiagnostic study. Eighteen patients with intracranial trigeminal nerve lesions were investigated electrodiagnostically. The trigeminal motor root function was studied on the basis of the jaw reflex and masseter myograms and was disturbed in all cases. The jaw reflexes were abnormal in 16 and the masseter myograms in eight cases. An impressive trigeminal sensory root function was obtained from blink reflex, which was abnormal in 12 cases. The results show the diagnostic value of bilateral recording of the jaw reflex in such patients.


Type V hyperlipoproteinaemia: a family study. Family screening for hyperlipoproteinaemia was undertaken when a patient presented with myocardial infarction in association with Type V hyperlipoproteinaemia. The clinical features of this unusual form of hyperlipoproteinaemia are discussed together with its frequency in the general population and in relatives of affected patients.


Paget's disease of the vulva. Recurrence in skin grafts. This report is directed toward problems with recurrent Paget's disease of the vulva. In addition, the behavior of a skin graft within the anal canal and recurrent disease within two separate grafts warrants recognition.


Herpesvirus infection in the newborn. Its treatment by exchange transfusion and adenosine arabinoside. A case is reported of herpesvirus infection in the newborn following maternal genital herpesvirus infection. The rationale for treating a newborn infant with signs of disseminated herpesvirus infection by exchange transfusion in addition to adenosine arabinoside is discussed. Because of the lack of data concerning therapy for a disease with a 50 to 100% mortality rate, it is important to present material such as this in order to provide information about possible new therapeutic modalities.


Trial of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU; NSC-79037) in advanced bronchogenic carcinoma 1,2,3. A total of 103 patients with lung cancer was treated with CCNU 130 mg/m2 orally every 6 weeks; 65 patients survived at least 6 weeks. Partial responses occurred in 7 patients. Leukopenia, thrombocytopenia, and nausea were frequent toxic effects. CCNU has slight efficacy in advanced lung cancer. No increase in survival was attributed to therapy.


A phase III study in lung carcinoma comparing hexamethyl-melamine (NSC 13875) to dibromodulcitol (NSC 104800) 1,2. A phase III study was designed comparing the effectiveness of Hexamethyl-melamine (NSC 13875) to Dibromodulcitol (NSC 104800) in lung carcinoma. 250 of the 316 patients entered on the study were stratified into groups according to stage of disease and cell type. The results showsed Hexamethylmelamine to be more effective in patients with squamous cell carcinoma and slightly superior to Dibromodulcitol in patients with anaplastic/undifferentiated cell carcinoma, whereas Dibromodulcitol proved to be more effective in patients with adenocarcinoma.


Solubilized nuclear DNA-dependent RNA polymerases from normal rat mammary glands and from transplantable R-35 rat mammary tumors. The three major nuclear DNA-dependent RNA polymerases (enzymes I, II and III) were present in nuclear extracts from transplantable R-35 rat mammary tumors. Except for somewhat less enzyme III, their relative distribution resembled that of nuclear extracts from late-pregnant rats. When enzyme II from normal tissue extracts was incubated for RNA synthesis with cyclic AMP, inhibition was frequently observed, but this occurred less often with nuclear extracts from the R-35 tumor. In some experiments with both normal and tumor tissue, cyclic AMP and cyclic GMP increased the apparent activity of nucleolar enzyme Ib and nucleoplasmic enzyme II, respectively. Nuclear extracts from both normal and tumor tissue contain proteins which bind radioactive cyclic AMP and cyclic GMP. Their patterns of binding were not identical. These results are consistent with the following hypothesis: altered binding by the tumor of cyclic nucleotides to putative nuclear 'r-gulatory' proteins (e.g. protein kinase subunits, or possibly other high affinity cyclic nucleotide-binding proteins unrelated to protein kinases) contributes to atative nuclear 'regularory' proteins (e.g. protein kinase subunits, or possibly other high affinity cyclic nucleotide-binding proteins unrelated to protein kinases) contributes to and may be responsible for some of the differences in response to cyclic nucleotides that were observed. It is possible that such defects occur in other tumors, or even represent a fundamental defect in all cancer cells. Several explanations for these results are discussed.


Stimulatory effect of a serum factor on DNA synthesis in isolated hepatoma nuclei. A serum protein present in normal rat serum and absent from the serum of hepatoma-bearing animals at advanced stages has a stimulatory effect on 3H-thymidine incorporation into hepatoma cells in suspension. Liver cells maintained in a similar suspension are not affected by the factor. The stimulation appears to be at the level of chromatin or DNA. Isolated membrane-denuded nuclei from Morris hepatoma 7777 incorporate more 3H-TTP when the factor is present in the incubation mixture. Nuclei from host liver are not stimulated. The factor also stimulates incorporation of 3H-TTP in a system using calf thymus DNA as primer and an extracted DNA polymerase. In this system incorporation is stimulated with DNA polymerase from both tissues, host liver and hepatoma 7777. It is concluded that the factor does not act on the DNA polymerase but on chromatin or DNA.


Study on the photochemical reaction of HCN and its polymer products relating to primary chemical evolution. The photochemical reaction of HCN at 184.9 nm is studied in the gas phase. (CN)2, H2, CH4, NH3, N2H4, C2H6, and CH3NH2 are identified as gas phase products, and a reaction mechanism is proposed. HCN polymers are also obtained as solid reaction products, and their structure is investigated by Infrared Spectorscopy, UV-Visible Spectroscopy, Mass Spectrometry, and Amino Acid Analysis. The process and nature of the formation of the polymers are discussed.


The influence of pinealectomy and of pinealectomy combined with thymectomy oncogenesis caused by polyoma virus in rats. The inoculation of Polyoma virus suspension to neonatal pinealectomized rats did not provoke a growth of neoplasia. However, if the pinealectomy was combined with neonatal thymectomy, renal tumors occured in a rate of 50 of animals. An incidence of renal tumors was observed also in 57.6% of thymectomized rats, but not in non operated control group. The stimulating effect of pinealectomy on the growth of transplantable tumors has already been described several times (2, 3, 6, 8). The question of a possible change in the viral oncogenesis after pinealectomy was raised. For this reason Polyoma virus was used and its oncogenic effect was studied on the neonatally pinealectomized rats, on rats neonatally thymectomized and on neonatally pinealectomized rats which were simultaneously thymectomized.


[Corticosterone, a synchronizing factor for liver cells]. The clone Fu 5 a of the Reuber Hepatoma cell line was incubated with corticosterone for 48 hours. The incorporation of labelled thymidine into DNA was inhibited to 10%. After this time corticosterone was removed from the medium. The DNA-synthesis increased for ten hours, the maximum was sixteen times higher than in the controls. The conclusion drawn was that corticosterone is able to block liver cells in a part of G1 phase of the cell cycle, so that the cells become synchronous.


A study of the immuno-suppressive activity of Herpes simplex virus type 2 and the tumor enhancing potential of the virus on Yoshida sarcoma. The relative delays of Yoshida sarcoma (YS) tumor induction were used as indicator for the immunosuppressive potential caused by the subcutaneous infection of Sprague-Dawley rats with approximately 10(7) TCD50 HSV-2. The tumor formation is clearly accelerated and the number of tumors is increased as compared to sham injected rats. The impairment of immunity is at its maximum when the virus and the YS cells are applied simultaneously, whereas virus given after the tumor is of no consequence. The adoptive neutralization test of Winn showed that the neutralization potential of spleen cells originating from HSV-2 infected donors is decreased from day 8 to day 11 post infection but rturns back to normal a week later. The stimulation of spleen cells with PHA is reduced up to 50% of control values on day 6 to 8 post virus infection but PFC and RFC values are not noticeably affected and neither are the hemagglutination titres. The implications of the immuno-suppressive faculty of HSV-2 for human genital malignancies are discuss.ed.


Effects of the diazafluoranthen derivative AC-3579 (NSC-170561), a new experimental antitumour drug, on rat hepatoma. The effects of AC-3579 (NSC-170561), a new experimental antitumour drug which depresses phospholipid catabolism in liver, were compared in rat aflatoxin-induced hepatoma and in non-neoplastic hepatocytes surrounding the tumour. Ultrastructural lesions, characterized by the hypertrophy of the smooth endoplasmic reticulum and the presence of lamellate cytosomes appeared in both tissues. They were less marked in hepatoma than in non-neoplastic cells. As in control livers, they were related to an invrease in pohspholipid concentration due to the decrease of phospholipid breakdown. In addition, chemical analysis demonstrated differences between hepatoma, non-neoplastic aflatoxin-treated liver and control liver: total and free cholesterol were decreased, relative concentration of sphingomyelin increased and that of phosphatidylethanolamine decreased by about 50%, in both hepatoma and non-neoplastic liver. Phospholipid concentration was decreased by 50% in tumour cells. After AC-3579 treatment total cholesterol was increased 3.2 fold in non-neoplastic liver and 2.5 fold in hepatoma but not in control liver.


Fat accumulation in tha adrenal gland: effect of oestradiol benzoate on the zona reticularis of the Mongolian gerbil adrenal gland. Oestrogen promotes accumulation of abundant lipid droplets in the zona reticularis of the Mongolian gerbil. Lipid was observed in zona reticularis parenchymal cells but droplets were especially numerous in macrophages. Enlargement of lipid droplets was facilitated by an envelopment with lysosomal membranes. Focal areas or entire parenchymal cells may then undergo degeneration and necrosis and be incorporated into large macrophages recognizable by distinct areas of lipid-containing material as well as by small mitochondria with short cristae. Oestrogen may well act directly on zona reticularis cells inasmuch as nafoxidine, an antioestrogen, prevents these changes. Zona fasciculata cells were identical to those in controls and an intermediate region between the zona fasciculata and zona reticularis persists, although concentric whorls of rough endoplasmic reticulum seen in controls were replaced by disorganized, randomly dispersed cisternae of rough endoplasmic reticulum. Oestrogen does not elevate cortisol in the plasma.


Early effects of inorganic lead on immature rat brain mitochondrial respiration. Inorganic lead, added to the diet of suckling rat in high doses, produces an encephalopathy similar to that seen in the immature human. Pathologic changes of edema and hemorrhage are seen earliest and are most prominent in the cerebellum. In this study, we measured respiration in cerebral hemisphere and cerebellar mitochondria isolated from led-fed and age-matched normal rat pups. Lactating mothers were begun on ad libitum feedins containing 4% lead carbonate when their pups were 2 weeks old. Mitochondria were isolated by differential centrifugation. Oxygen consumption was measured polarographically, NAD-linked respiration was measured with oxidation of the substrate pair, glutamate and malate. Cytochrome oxidase (cytochrome c oxidase, EC. 1.9.3.1) activity was measured in the presence of tetramethyl-p-phenylenediamine dihydrochloride (TMPD) and ascorbate. Within 2 days of starting lead feedings, rat pups showed a significant loss in body weight (P less than 0.02) and, after 1 week, a significant loss in cerebral hemisphere wet weight (P less than 0.01) compared with controls. Overt encephalopathy appeared in pups from two of nine litters receiving lead feedings for 1 week and in half of the litters after 2 weeks of feedings. None of the lead-fed mothers developed encephalopathic signs. With oxidation of the NAD-linked substrate pair, there was a progressive decrease, relative to controls, in ADP/O ratios in both cerebellar and cerebral mitochondria from lead-fed animals. After 2 weeks these differences were significant in mitochondria from both regions (cerebellum, P less than 0.02; cerebrum, P less than 0.005). Respiratory control ratios were significantly lower in cerebellar mitochondria from lead-fed rats within 2 days of beginning feedings (P less than 0.02) and in mitochondria from both regions after 2 weeks of lead feedings (cerebellum, P less than 0.01; cerebrum, P less than 0.05). The decrease in control ratios in cerebellar mitochondria from animals receivint lead feedings for 1 week or less was due to a small decrease in state 3 respiration and a large, but inconsistent, increase in state 4 respiration. The decrease in control ratios in both cerebellar and cerebral hemisphere mitochondria after 2 weeks of lead feedings was due to a marked inhibition of state 3 respiration, relative to controls (cerebellum, P less than 0.01; cerebral hemisphers, P less than 0.05). In cerebellar mitochondria from lead-fed animals, cytochrome oxidase activity showed similar changes compared with controls: a highly significant (P less than 0.001) increase within 2 days of beginning feedings and a significant (P less than 0.01) decrease after 2 weeks of feedings.


Apneic spells and sleep states in preterm infants. The incidence of apneic spells during different sleep states active sleep, quiet sleep, and undifferentiated sleep was determined in eight preterm infants of 30 to 35 weeks' conceptional age, by means of a polygraphic recording technique. They were free of perinatal and postnatal complications other than apnea. During their active or rapid eye movement (REM) sleep they showed significantly more apneic episodes which were also longer lasting and they were accompanied by bradycardia of a greater severity. The organization of the immature nervous system with a preponderance of inhibitory synaptic connections and the additional inhibition of spinal motoneurons during REM sleep are likely to be the cause of apneic spells in otherwise "normal" preterm infants.


Effect of breast-feeding on seroresponse of infants to oral poliovirus vaccination. Three hundred Indian infants between 6 and 51 weeks of age were divided into six groups and given three doses of trivalent oral polio vaccine (OPV) of known adequate potency. One group was on unrestricted breast-feeding with mandatory breast-feed during the interval between 30 minutes before and 15 minutes after each dose of OPV. In four groups of infants breast-feeding was withheld for three, four, five, and six hours both before and after each dose of OPV. The sixth group was bottle-fed. Samples of blood were collected from all infants before vaccination and from 227 infants further samples were collected four weeks after the first and/or third doses of OPV. Antibody responses to poliovirus types 1, 2, and 3 were determined following one dose and three doses of OPV, and the rates of response were found to be approximately equal in all groups of breast-fed infants irrespective of their feeding schedules, as well as in bottle-fed infants. Thus breast-feeding is shown to have no inhibitory effect on antibody response of infants beyond the newborn period to OPV.


Pediatric familial type II hyperlipoproteinemia: therapy with diet and colestipol resin. Effects of a low-cholesterol, polyunsaturate-rich diet and a synthetic organic bile sequestrant polymer (U26,597A, colestipol) were studied in 21 children, heterozygous for familial hypercholesterolemia. Total cholesterol, beta-lipoprotein cholesterol, and triglyceride were measured twice on habitual diet, monthly for six months on a low-cholesterol diet, and monthly for six months on low-cholesterol diet plus 10 gm of colestipol per day. Total cholesterol (mean +/- 1 SD) was 295 +/- 37 on habitual diet, 278 +/- 29 on low-cholesterol diet, and fell significantly to 242 +/- 29 mg/100 ml on diet plus colestipol. Low-density lipoprotein (LDL) cholesterol was 234 +/- 37 on habitual diet, 220 +/- 28 on low-cholesterol diet, and fell significantly to 179 +/- 26 mg/100 ml on diet plus drug. Plasma triglyceride levels on habitual diet were 79 +/- 31, remained unchanged on low-cholesterol diet, 86 +/- 22, and were unaffected by low-cholesterol diet plus drug, 85 +/- 17 mg/100 ml. On diet alone, plasma LDL was not normalized (less than 170 mg/100 ml) in any of the 21 children, and cholesterol fell to within normal limits (less than 230 mg/100 ml) in only one child. The combination of diet plus colestipol resin normalized total and LDL cholesterol in 52% of the children. Cholesterol was lowered to a "moderately elevated" range of 230 to 250 mg/100 ml in an additional 14% of the children and LDL was lowered to a range of 170 to 190 mg/100 ml in an additional 29%. In 33% of the children, cholesterol remained greater than 250 mg/100 ml despite diet plus colestipol,  while LDL was greater than 190 mg/100 ml in 19%. Colestipol is an effective and well-tolerated cholesterol lowering compound which, in conjunction with diet, may prove to be very useful in the treatment of children heterozygous for familial hypercholesterolemia.


Studies on chromoproteins of cardiac and skeletal muscle of caimans (Caiman sclerops). In a number of caimans (Caiman sclerops) we were unable to find traces of myoglobin in any of the histologically clearly cross-striated cardiac and skeletal muscles using spectrometric methods. The red colour of the heart is due to a high concentration of cytochrome c. This substance was also found in the muscles of the neck, back, tail, and the anterior extremities.


Potential use of ascorbic acid as a pituitary and adrenal scintigraphic agent. The distribution of intravenously administered 14C-ascorbic acid (AA) into various tissues of the dog, and the effect of dexamethasone and ACTH administration on the AA distribution was studied. As a function of time, the differences of 14C-AA distribution between adrenal and surrounding organs become greater, and 24 hours after the administration of the dose, the adrenal to liver ratio, as well as adrenal to kidney, is about 10:1. Also concentration of AA in the pituitary showed a gradual increase and reached its peak between 24 and 48 hours. Dexamethasone administration lowered significantly the uptake of 14C-AA by the adrenals, but did not affect the uptake by the pituitary. Three consecutive days administration of ACTH did not affect the uptake of the drug by either glands, but one single dose of ACTH administered one hour prior to the 14C-AA increased the uptake of AA by both glands. These results indicate that properly labeled AA may be an effective pituitary and adrenal scanning agent. Possible ways of labeling AA without changing its pharmacological properties are suggested.


Hydrolysis in 2'-chloro-2'-deoxy nucleosides, -nucleotides and -polynucleotides to arabinose-derivatives. The rate of hydrolysis of 2'-chloro-2'-deoxycytidine and -uridine, and their 5'-phosphates, -diphosphates and polymers to the corresponding arabinocytidine and arabinouridine derivatives in the presence of Tris-HCL pH 8.9 is determined. This rate is highest for the nucleosides, less for the 5'-phosphates and 5'-diphosphates and even less for the polynucleotides. In the the 2'-chloro-2'-deoxyuridine series the O2, 2'-cyclonucleoside was formed in addition to the arabinonucleoside.


The influence of fluphenazine on the interaction of noradrenaline and serotonin. The neurotransmitter catecholamines control metabolism 5-HT in pineal gland by stimulating pineal serotonin-N-acetyltransferase (EC 2.3.1.5) activity [1, 2]. To prove the influence of FP on the metabolism 5-HT provoked by norepinephrine was tested. Rat pineal gland were incubated in media containing 5-HT- 14C or 5-HT- 14C and FP (1-10(-6)-1-10(-4) M). Norepinephrine (1-10(-6)-1-10(-4)M) was added at the start of the experiment. The level of cyclic 3',5'-AMP, activity of HIOMT and metabolites of 5-HT-14C was examined. The results have suggested that the FP inhibit the provoked by norepinephrine metabolism of 5-HT in the rat pineal gland by decrease the formation of c-3',5'-AMP.


Viral hepatitis--a primer: 1. Epidem.iology. Hepatitis A is most often transmitted by the fecal-oral route under conditions of crowding, poor hygiene, and prolonged exposure to infected persons. Direct spread of hepatitis B via person-to person contact has only recently been proved. Hepatitis B is usually transmitted indirectly proved. Hepatitis B is usually transmitted indirectly by percutaneous routes, and the increase in drug abuse has brought about a change in its epidemiologic pattern.


[Preparation of highly puried pepsin by aminosilichrome chromatography]. The procedure for preparing highly purfied pepsin by chromatography on silicon dioxide with attached aminogroups (aminosilochrome) has been devised. Pepsin is eluated step=by-step: 0.0025 M and 0.05 M HCl. Results of disc-electrophoresis of purified pepsin bive evidence that the resultant preparation contains no admixtures. The activity of purified pepsin is 69 units per mg.


Cellular levels of feedback regulator of adenylate cyclase and the effect of epinephrine and insulin. We have obtained direct evidence that shows the cellular formation and subsequent release of a potent inhibitor (feedback regulator) of adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] by adipocytes, upon stimulation with epinephrine. The appearance of such a feedback regulator in adipocytes preceded its release into the medium. During a 30 min incubation, intracellular regulator levels rose rapidly and reached 39-61 units/g of adipocyte at 10 min. Release of inhibitor into the medium increased slowly and was 11-16 units/g of adipocyte at 10 min. Upon continued incubation, the cells at 30 min contained 30-41 units/g of ingibitor, slightly less than the content at 30 min; meanwhile, the medium content rose more than 3-fold. The inhibitor from both locations appeared to have the same characteristics, judging from the purification procedures and the biological activities on hormone-stimulated adenylate cyclase. Adenylate cyclase was inhibited by the feedback regulator in vitro when either epinephrine, corticotropin (ACTH), or glucagon was used as activator. The site of action of this inhibitor is therefore most likely beyond the specific hormone receptors. A new in vitro action of insulin has been found. Insulin, 50-500 microunits/ml, inhibited the formation and release of this factor from isolated rat or hamster adipocytes by 29-81% after these cells were stimulated by hormones that raise intracellular adenosine 3':5'-cyclic monophosphate. This factor enhaced the effect of insulin in lowering the adenosine 3':5'-cyclic monophosphate levels in fresh rat adipocytes. A reduced formation of such a factor may modify the metabolic events in adipocytes, and some as yet unexplained effects of insulin could therefore be linked to the metabolic effects of this factor.


The effect of nerve growth factor on cycle AMP levels in superior cerival ganglia of the rat. Nerve growth factor (NGF) produces a several-fold increase in the cyclic AMP concentration in rat superior cervical ganglia in organ culture within 5 min. An increase can be seen with as little as 40 ng/ml of NGF. Oxidized NGF is without effect. The increase in the cAMP concentration produced by NGF is prevented by the addition of antiserum to NGF.


Mapping of biological functions on RNA of avian tumor viruses: location of regions required for transformation and determination of host range. A map of the large T1 oligonucleotides of the RNA of Prague Rous sarcoma virus, strain B (Pr RSVb) has recently been established (Coffin and Billeter, submitted for publication). Since the RNA of Rous associated virus, type 1 (RAV-1) lacks many of the large 1 oligonucleotides of Pr RSV-B and contains others not present in the latter, the RNA of recombinants between RAV-1 and Pr RSV-B could be analyzed with regard to the origin of its sequences. Recombinants were selected for transforming capacity (characteristic for Pr RSV-B) and ability to grow on C/B chicken fibroblasts (characteristic for RAV-1). Four out of five recombinants examined had undergone at least two crossovers. The set of Pr RSV-B-specific oligonucleotides present in all recombinants defined an RNA region near the poly(A) segment; this must contain genetic information required for transformation required for transformation (the onc function). All recombinants lost a set of contiguous Pr RSV-B-specific oligonucleotides and concomitantly acquired a set of RAV-1-specific oligonucleotides. These define a region in the middle section of the oligonucleotide map, all or some of which must be required for determining growth capacity on C/B cells (the env function).


On the iron-sulfur cluster in hydrogenase from Clostridium pasteurianum W5. Hydrogenase, purified to an average specific activity of 328 mumol of H2 evolved/(min X mg of protein) from Clostridium pasteurianum W5, was found to have 4-5 Fe and 4-5 labile sulfur atoms per molecule of 60,000 molecular weight, in contrast with earlier reports of 12 Fe per molecule. Displacement of the iron-sulfur cluster from hydrogenase by thiophenol in 80% hexamethyl phosphoramide:20% H2O yielded the Fe4S4 (thiophenyl)4 dianion according to absorption spectroscopy. Electron paramagnetic resonance spectroscopy at 12 K showed that the iron-sulfur cluster in the enzyme could be reduced by the H2 to a state (g-values of 2.098, 1.970, and 1.898) similar to that in reduced ferredoxin and could be oxidized by dichlorophenolindophenol or H+ to a state (g-values at 2.099, 2.041, and 2.001) similar to that in high potential iron-sulfur proteins. These oxidations and reductions appeared to occur within the turnover time of the enzyme. Deuterium failed to narrow the electron paramagnetic resonance signal in either state, but the competitive inhibitor carbon monoxide reversibly formed a compound with either state and substantially altered the electron paramagnetic resonance. 13CO produced a broadening of these signals, suggesting the formation of a direct CO complex with the iron-sulfur cluster. These data are consistent with a model of the active site of the enzyme in which a four-iron four-sulfur cluster is a component that can accept one or two electrons from and donate either one or two electrons to substrates, and in which the iron-sulfur cluster serves as the site of binding of gaseous ligands.


Human papillomavirus DNA: physical map.

Human papillomavirus DNA: physical map. Human papillomavirus (HPV) DNA form I (supercoiled) was prepared from plantar warts. HPV DNA was cleaved with restriction enzymes obtained from the following sources: escherichia coli (EcoRI), Hemophilus influenzae strain Rd (both unfractionated Hind and aeparated HindII and HindIII enzymes) and Hemophilus parainfluenzae (HpaI). The cleavage products were analyzed by polyacrylamide gradient slab gel electrophoresis and electron microscopy. HPV DNA was cleaved into two fragments by EcoRI (87% and 13% of the genome) and into six fragments, ranging in size from 33.5 to 1.2% of the genome, by Hind endonucleases. The six Hind fragments result from the cleavage of three sequences recognized by HindII, two of which are also cleaved by HpaI, and of three sequence recognized by HindIII. The order of these fragments was determined by comparing their size with that of the fragments obtained with HindII, HindIII, HpaI, and the mixture of HindIII + Hpal. The two EcoRI cleavage sites were located on two adjacent Hind fragments and one of these sites has been taken for the zero point to construct a physical map. The treatment of superhelical HPV DNA with bacteriophage T4 gene 32 protein yields circular structures with a denaturation loop. The cleavage of these complexes with EcoRI and HindIII has shown two easily denatured regions which were located on the cleavage map.


Association of a cyclic AMP-dependent protein kinase with a purified translational inhibitor isolated from hemin-deficient rabbit reticulocyte lysates. In the absence of added hemin, protein synthesis in rabbit reticulocyte lysates proceeds at maximal linear rates for several minutes and then ceases abruptly. Inhibition involves the action of a translational inhibitor whose formation is regulated by hemin. Addition of the isolated inhibitor to hemin-supplemented lysates produces an inhibition of protein chain initiation similar to that observed in heme-deficiency. The inhibitor has been purified over 300-fold and contains a protein kinase activity that copurifies with the inhibitory function. With calf thymus histone II as the phosphate receptor, the inhibitor-associated protein kinase requires ATP as the phosphorylating agent. Cycle AMP stimulates kinase activity 5- to 8-fold; the concentration of cycle AMP required for halfmaximal activity is 4 X 10-8 M. Preincubation of the inhibitor in the presence of cyclic AMP significantly reduces cyclic AMP-dependent phosphorylation and inhibitory activity. The corresponding protein kinase activity from hemin-supplemented lysates displays reduced cyclic AMP-dependency and little or no inhibitory activity. These findings suggest that the protein kinase activity associated with the purified translational inhibitor is involved in the mechanism of inhibition of initiation observed in hemedeficient reticulocyte lysates.


Determination of the number of superhelical turns in simian virus 40 DNA by gel electrophoresis. Closed-circular, superhelical DNA from simian virus 40 (SV40 DNA I) was treated with an excess of DNA-relaxing enzyme in the presence of increasing amounts of ethidium bromide (EtdBr). After removal of the ethidium, each sample consisted of a group of close-circular DNA molecules differing in their number of superhelical turns (tau) around a mean value of tau in a Gaussian-like distribution. The DNA samples were analyzed by electrophoresis in agarose gels under conditions where the electrophoretic mobility was a function of the number of superhelical turns. Since the distributions around tau of DNA molecules of different samples overlappped, the difference in the mean number of superhelical turns from sample to sample, delta tau, could be determined and used to measure the mean number (tau) for native SV40 DNA I. By this criterion, SV40 DNA I contains a Gaussian-like distribution of molecules differing by integral numbers around a mean value of tau = -24 +/- 2 at 37 degree C [in 0.2 M NaC1, 10 mM Tris-HC1 (pH 7.9), and 0.2 mM EDTA]. The heterogeneity in tau is probably a consequence of thermal fluctuations in the DNA helix at the time when the last phospholiester bond is closed in vivo. When correlated to the buoyant shift of completely relaxed SV40 DNA in a CsC1-propidium diiodide gradient, the number of delta tau = 24 +/- 2 of superhelical versus relaxed DNA implies an unwinding of the DNA helix by 26-28 degree upon intercalation of one molecule of EtdBr.


Partial amino-acid sequence of NAD-specific glutamate dehydrogenase of Neurospora crassa. Parts of the primary structure of the NAD-specific glutamate dehydrogenase [L-glutamate:NAD oxidoreductase (deaminating), EC 1.4.1.2] from Neurospora crassa are presented. Segments of the sequence representing 886 unique amino-acid residues have been determined; the largest contains 267 residues. There are only short regions of possible homology between this enzyme and the glutamate dehydrogenases of bovine liver or the NADP-specific enzyme of Neurospora. The large size of the subunit (116,000 molecular weight) of the NAD-specific glutamate dehydrogenase is unusual when compared to other known dehydrogenases.


In vitro synthesis of full-length DNA transcripts of Rous sarcoma virus RNA by viral DNA polymerase. Varying the concentration of Triton X-100, a nonionic detergent used to promote the DNA polymerase activity of Rous sarcoma virus in an endogenous reaction, showed a very sharp peak at about 0.02% (vol/vol) for optimal DNA synthesis. The yield of DNA at this concentration of Triton exceeded yields obtained at concentrations above the optimum by a factor of 2-5 for the 90-min reaction. At optimal Triton concentration, about 1-7% of the DNA made in the absence of actinomycin and about 4-10% of the DNA made in the presence of actinomycin was 2.5 X 10(6) daltons or greater, as estimated by formamide polyacrylamide gel electrophoresis and by alkaline sucrose gradient sedimentation. No large DNA was obtained at higher than optimal Triton concentrations. The large DNA molecules were rendered totally resistant to single-strand specific nuclease S1 after hybridization to an excess of viral RNA. It was concluded that at optimal detergent concentration, the viral DNA polymerase can synthesize full-size DNA transcripts of viral RNA.


Apoprotein stability and lipid-protein interactions in human plasma high density lipoproteins. Temperature-dependent conformational changes of the principal apoprotein of human plasma high density lipoprotein (HDL), apoA-I, have been studied in the isolated apoprotein, in complexes of apoprotein with phospholipid, and in intact HDL. Differential scanning calorimetry shows that in solution apoA-I undergoes a reversible, two-state thermal denaturation (midpoint temperature 54 degrees). The enthalpy (2.4 cal/g)(10.0 J/g) and specific heat change (0.08 cal/degrees C per g)(0.33 J/degrees C per g) associated with the denaturation were used to calculate the free energy difference (deltaG) between native and unfolded apoA-I at 37 degrees. DeltaG (2.4 kcal/mol)(10.0 kJ/mol) is less than that of other globular proteins (typically 8-14 kcal/mol)(33-59 kJ/mol), indicating that at 37 degrees native apoA-I has a loosely folded conformation. Turbidity studies show that apoA-I is able to solubilize phospholipid in its native but not in its denatured form. Mixtures of apo-HDL (the total apoprotein of HDL) or apoA-I with dimyristoyl lecithin show a thermal transition at about 85 degrees not present in the lecithin or the apoprotein alone, which indicates that the native conformation of the apoprotein is stabilized by phospholipid. Scanning calorimetry of intact HDL shows a high-temperature endotherm associated with disruption of the HDL particle, suggesting that in HDL the conformation of apoA-I is also stabilized by interaction with lipid. The loosely folded conformation of native, uncomplexed apoA-I may be especially adapted to the binding of lipid, since this process may involve both hydrophobic sites on the surface of the protein and concealed apolar amino acid residues that are exposed by a cooperative, low energy unfolding process.


Chemically induced dynamic electron polarization in chloroplasts at room temperature: evidence for triplet state participation in photosynthesis. A transient electron paramagnetic resonance emission is observed after flash excitation of chloroplasts at room temperature. The spectrum of the emission signal is centered at g = 2.0037 and has a linewidth deltaHpp = 4G (4 X 10(-4) tesla). Inhibitor studies and chemical oxidation indicate that the signal is associated with Photosystem I, but the spectrum and kinetics indicate that it is neither P700 nor an iron-sulfur protein. The emission signal rises with the 2-musec time response of the instrument, and decays during the actinic flash. The emission signal is produced on only the first of a pair of strong flashes separated by 100 musec, indicating that the precursor has not been regenerated in that time. The results are discussed with reference to the two currently accepted mechanisms for chemically induced dynamic electron polarization: the radical pair and the photochemical triplet. For several reasons the photochemical triplet mechanism is the more attractive of the two. It is suggested that at room temperature the primary photochemistry of photosystem I proceeds via a triplet state of chlorophyll, and that the species giving rise to the emission signal is the primary electron acceptor of Photosystem I.


Primary charge separation in bacterial photosynthesis:  oxidized chlorophylls and reduced pheophytin. Bacteriopheophytin, the magnesium-free base of bacteriochlorophyll, undergoes reversible one-electron reduction in organic solvents to yield an anionic free radical with characteristic optical and electron spin resonance spectra. The reduction potential of bacteriopheophytin, E1/2 approximately --0.55 V against a normal hydrogen electrode, compared to E1/2 approximately --0.85 V for bacteriochlorophyll, renders it a likely electron acceptor in the primary charge separation of photosynthesis. Comparison of these data with picosecond optical changes recently observed upon pulsed laser excitation of bacterial reaction centers leads us to propose that bacteriopheophytin is indeed a transient electron acceptor and that the primary charge separation of bacterial photosynthesis occurs between the bacteriochlorophyll complex P870 and bacteriopheophytin to yield the radicals of the oxidized chlorophyll dimer cation and reduced pheophytin anion.


Failure of human cells transformed by simian virus 40 to form tumors in athymic nude mice. Four individual lines and one subline of human cells, permanently established in tissue culture after infection with simian virus 40, failed to form tumors when inoculated into athymic nude mice.  Under identical conditions, three established human cell lines of neoplastic origin and a spontaneously established human lymphocyte line formed tumors. Nude mice that failed to grow tumors from inocula of simian virus 40-transformed human cells, grew tumors from subsequent injections of authentic human cancer cells. Further efforts to demonstrate an immunologic basis for the growth suppression of human simian virus 40 transformants were also negative. The data suggest that the changes in morphology and in vitro growth behavior induced by the viral information are not sufficient for, or are only coincidentally related to, the neoplastic state.


Cytoplasmic microtubules in normal and transformed cells in culture: analysis by tubulin antibody immunofluorescence. Monospecific antibody directed against bovine brain tubulin was used as an immunofluorescent probe to evaluate the distribution of microtubules in normal and transformed cells grown in tissue culture. The fluorescent staining pattern of transformed and nontransformed cells is significantly different and may be used in conjunction with other morphological features to identify transformants in mixed cell populations. Normal cells are flattened, elongated, and fibroblastic; they display numerous Colcemid-sensitive fluorescent cytoplasmic filaments, presumably microtubules. Transformed cells, however, are smaller, more polygonal in shape, and contain very few cytoplasmic tubules. During mistosis the cytoplasmic microtubule complex of normal cells completely disappears, but reappears after cell division. Treatment of transformed cells with dibutyry-adenosine 3':5'-cyclic monophosphate plus testosterone or theophylline restores the normal fibroblastic appearance of the cells and stimulates the assembly of numerous cytoplasmic microtubules. This study provides further evidence for two separate microtubule entities in cycling nontransformed cells: a cytoplasmic microtubule complex and the microtubules of the mitotic spindle. Although an interchange of tubulin dimers seems to exist between microtubules in the two systems, control of tubule assembly may be under separate constraints. Stimulation of cytoplasmic microtuble assembly in transformed cells by derivatives of adenosine 3':5'-cycle monophosphate suggests that impairment of the cytoplasmic microtubule complex in these cells may be due to suboptimal levels of adenosine 3':5'-cyclic monophosphate.


Influence of a novel hypophyseal factor on steroid metabolism in cultured hepatoma cells. A rat hepatoma cell line in tissue culture (HTC cells) was treated with hypophyseal extracts from adult male and female rats. Cell homogenates were then assayed for steroid metabolizing enzymes using 4-androsten-3,17-dione as substrate. The major products were the 5 alpha-reduced derivatives (5 alpha-androstane-3,17-dione, androsterone, and epiandrosterone). When the cells were grown in the presence of female hypophyseal extract the apparent activity of the 5 alpha-reductase increased markedly, whereas treatment with male hypophyseal extract was without effect. Treatment with female hypophyseal extract resulted in a marked decrease in the apparent Km for 5 alpha-reductase from 667 +/- 102 to 99 +/- 4 muM in addition to a decrease in the apparent Vmax from 67 +/- 12 to 46 +/- 2 pmol of product/min per mg of protein. A logarithmic dose-response was obtained with female hypophyseal extract. Treatment of the HTC cells with purified rat hypophyseal follicle-stimulating hormone, luteinizing hormone, growth hormone, thyrotropic hormone, and prolactin had only marginal effects on 5 alpha-reductase activity. Crude female hypophyseal extracts were at least 6-fold more potent than any of the standard hormone preparations and at least 250-fold more potent than male hypophyseal extracts when based on activity per mg of pituitary tissue.  Chromatography of crude female hypophyseal extracts on Sephadex G-25 indicated that the factor was of high molecular weight. The identity of this activity with a hypophyseal "feminizing" factor is postulated.


Signal input for a chemotactic response in the cellular slime mold Dictyostelium discoideum. Drops with different concentrations of 3':5'-cyclic AMP were deposited at various distances from small populations of Dictyostelium discoideum amoebae, and the distances over which 50% of the amoebae drops reacted positively were determined. The linear regression analysis of a bouble logarithmic plot of distance against concentration gives a straight line with a slope of 1/4.25, which value suggests that the amoebae respond to a spatial gradient of cyclic AMP concentration. The threshold value for the signal is 3.6 X 10(-9) M/mm with a sensitivity of measurement of about 1%. These findings are discussed in relation to our present knowledge of cyclic AMP receptors.


Selective killing of transformed baby hamster kidney (BHK) cells. We report here that certain drugs can protect Syrian baby hamster kidney cells (BHK) in culture against the lethal agents cytosine arabinonucleoside, hydroxyurea, and colcemid. Polyoma virus-transformed BHK cells (PyBHK) are killed under the same conditions. The protective drugs include caffeine and streptovitacin A. Kinetic studies show that these drugs act specifically in G1, and that they shift BHK cells from G1 into the G0 state at the restriction point, similar to the effects of high cell density or serum deprivation. These drugs do not block the growth of PyBHK cells nearly as effectively, consistent with a reduced effectiveness of restriction point control in virus-transformed cells. Consequently, the transformed cells around their cycle and are killed by the cell cycle phase-specific toxic agents, in contrast to the arrested BHK cells. These findings provide a model for studies on differential killing of tumor versus normal cells in vivo.


Chicken neutrophils: oxidative metabolism in phagocytic cells devoid of myeloperoxidase. Polymorphonuclear leukocytes isolated from chicken peritoneal exudates have been found to catalyze cyanide-insensitive stimulation of respiration and the hexose monophosphate shunt upon exposure to heat-inactivated Staphylococcus aureus. However, there was no demonstrable formate oxidation concomitant with phagocytosis in either the presence or absence of exogenous catalase. Moreover, chicken polymorphonuclear leukocytes failed to oxidize scopoletin concomitant with phagocytosis in the presence of horseradish peroxidase. While oxygen uptake was increased 2- to 3-fold by the stimulus of phagocytosis, the oxidation of [1-(14)C]glucose was increased approximately 20-fold. The cells contain two mechanisms, a glutathione reductase-glutathione peroxidase system and an NADPH-NAD+ transhydrogenase, each of which is present in sufficient capacity to accommodate the enhanced shunt activity. Although chicken polymorphonuclear leukocytes were found to possess a substantial capacity to catalyze the cyanide-insensitive oxidation of either NADH or NADPH, the total or specific activities of such processes were not demonstrably affected by phagocytosis.


A structural gene mutation affecting the regulatory subunit of cyclic AMP-dependent protein kinase in mouse lymphoma cells. Compared to the wild-type parental line of S49 mouse lymphoma cells, intact cells of a mutant line (kin.A) are 10-fold less sensititive to biologic effects of exogenous cyclic adenosine 3':5'-monophophosphate (cAMP), such as induction of cAMP phosphodiesterase, cell cycle-specific growth inhibition, and cytolysis. The cAMP-dependent protein kinase (ATP:protein phosphotransferase; EC 2.7.1.37) activity of kin.A cells exhibits an apparent Ka for activation by cAMP 10-fold greater than that of wild type, and is much more resistant to inactivation by heat. These differences between the wild-type and mutant enzymes persist through a high degree of purification, suggesting a structural alteration in the kin.A holoenzyme. Heterologous reconstitution experiments, using separated R and C subunits of the wild-type and kin.A cAMP-dependent kinases, show that the altered cAMP affinity and thermolability are conferred by the R component of the kin.A enzyme. These results are most consistent with a structural mutation in the kin.A gene coding for the R subunit of cAMP-dependent protein kinase. Evidence for a structural mutation helps to define one mechanism of heritable variation in cultured somatic cells. The phenotype produced by the kin.A structural mutation also greatly strengthens the conslusion that cAMP-dependent protein  kinase is essential for cAMP regulation of growth and enzyme induction in intact S49 cells.


Virus-specific neutralization by a soluble non-immunoglobulin factor found naturally in normal mouse sera. A low-molecular-weight substance found naturally in mouse serum neutralizes mouse xenotropic C-type virus. It has no effect on endogenous ecotropic viruses. This neutralizing factor does not belong to the known immunogloblin classes, and its activity is not associated with the antivirus immunoglobulins that can be detected by radioimmunoprecipitation. Preparations of xenotropic virus absorb out this neutralizing activity in mouse sera. The specificity of this factor for X-tropic virus suggests that it represents a newly recognized type of response of the host to an endogenous virus. Its possible role in the regulation of endogenous C-type viruses is considered.


Translation of messenger ribonucleic acid from isolated pancreatic islets and human insulinomas. RNA preparations from isolated rat pancreatic islets and from human insulinomas were injected into oocytes of Xenopus laevis which were then incubated with [3H]leucine. Acid-ethanol extracts of the oocytes were immunoprecipitated with anti-insulin serum using the double antibody technique. Sodium dodecyl sulfate disc gel electrophoresis of the immunoprecipitates showed the presence of an insulin-displaceable immunoreactive material with a molecular weight of about 18,000 in extracts from oocytes injected with RNA of 9-11 S. This immunoreactive product was not detected in extracts from oocytes injected with buffer or 4-8S RNA or RNA heavier than 11 S. These observations suggest the involvement of a precursor larger than proinsulin in the biosynthesis of insulin.


Energy-dependent binding of dansylgalactoside to the lac carrier protein: direct binding measurements. High specific activity 6'-N-[3H]dansyl)aminohexyl 1-thio-beta-D-galactopyranoside (Dns6-Gal) has been synthesized, and its binding to Escherichia coli membrane vesicles measured directly by flow dialysis. With ML 308-225 vesicles containing the lac carrier protein, specific binding is not detected in the absence of D-lactate or reduced phenazine methosulfate. In the presence of these electron donors, binding is observed, and the binding constant and number of binding sites are approximately 4 muM and 1.5 nmol/mg of membrane protein, respectively. These values are in excellent agreement with those obtained by fluorescence titration. p-Chloromercuribenzenesulfonate, which directly inactivates the lac carrier protein, and carbonylcyanide m-chlorophenylhydrazone, which collapses the membrane potential, cause release of bound Dns6-Gal. Moreover, significant binding is not observed with membrane vesicles that are devoid of the lac carrier protein. The results provide qualitative and quantitative confirmation of previous studies which indicate that changes in dansylgalactoside fluorescence observed on "energization" of membrane vesicles reflect binding of the probe to the lac carrier protein.


Oxidation of ferrocytochrome c by mitochondrial cytochrome c oxidase. Attempts to rationalize the kinetics of cytochrome c oxidation catalyzed by solubilized mitochondrial cytochrome c oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1.9.3.1) have been based on assumptions of productive complex formation (Michaelis-Menten approach). However, the range of substrate concentrations used has not, in general, been sufficient to establish a general rate equation. Data adequate to derive such a rate expression are presented, as well as a method for estimation of constants which appear in the rate law deduced and reported herein. It is shown that either of two types of mechanisms, one assuming productive complex formation, as opposed to the other postulating dead-end complex formation, accurately predict the rate equation as deduced from experiment.


Binding, internalization, and degradation of low density lipoprotein by normal human fibroblasts and by fibroblasts from a case of homozygous familial hypercholesterolemia. Skin fibroblasts from a patient with homozygous familial hypercholesterolemia (HFH) were compared with normal skin fibroblasts with regard to binding, internalization, and degradation of iodinated human low density lipoprotein (LDL). Like other cell lines from HFH patients, the mutant cells showed no suppression of sterol synthesis by LDL. Surface binding, measured at 0 degrees to eliminate the appreciable internalization that was shown to occur at 37 degrees, was on the average slightly less for HFH cells than normal cells at low LDL concentrations but comparable or even greater at high LDL concentrations (greater than 60 mug of LDL protein per ml). A major defect observed was in the rate of internalization of LDL at 37 degrees, which was only 1-10% of that in normal cells. LDL degradation was also markedly reduced but not to the same extent. Thus, a larger fraction of the LDL taken up appeared to be degraded by the mutant cells. The most striking defect observed, then, was not in surface binding of LDL but in rate of LDL internalization. While this might be secondary to a defect in specific binding sites of LDL, the magnitude of the observed differences in binding at low temperature seems too small to account for the huge differences in internalization (13- to 115-fold).


Cellular cyclic nucleotides and enzyme secretion in the pancreatic acinar cell. Cellular levels of cAMP and cGMP were measured in guinea pig pancreatic lobules incubated in vitro, during basal or stimulated secretion. Stimulation with optimal concentrations of carbamylcholine (carbachol) (10(-5) M), pancreozymin (0.1 unit/ml), and caerulein (10(-9) M) resulted within seconds in a sharp rise in cGMP levels, from five to more than 20 times that of basal levels. cAMP levels did not change significantly. cGMP increases were maximal at 2 min then subsided by 4-7 min to a plateau about two to three times that of basal level. This plateau was maintained for the duration of the secretagogue stimulus. Removal of the carbachol stimulus resulted in a rapid decrease in cGMP levels to that of the basal state. The cellular cGMP levels observed within the first 2 min of stimulation correlated closely with the dose of carbachol and the secretory response. Atropine at 10(-4) M blocked the cGMP elevation due to carbachol but not that due to pancreozymin, while carbonyl cyanide m-chlorophenyl hydrazone, an uncoupler of oxidative phosphorylation, blocked the response to both secretagogues. Similar though less extensive findings were observed using rabbit pancreatic lobules incubated in vitro. High concentrations  (10(-2)-10(-3) M) of the dibutyryl and 8-bromo analogues of both nucleotides were effective, though suboptimal, secretagogues. In the case of the cAMP analogues, the secretory response was associated with a rise in endogenous cGMP levels, similar to that observed during suboptimal carbachol stimulation. These findings suggest that cGMP may be an intracellular mediator in the process of stimulus secretion coupling in the acinar cell of the exocrine pancreas.


Surface marker characteristics and Epstein-Barr virus studies of two established North American Burkitt's lymphoma cell lines. Tumor cell lines have been established in continuous culture from two North American Burkitt's lymphomas. The SU-AmB-1 line, derived from a patient with low serum antibody titers to Epstein-Barr virus (EBV), was devoid of EBV genomes by the reaction for EBV-associated nuclear antigen (EBNA), could not be induced to express EBV antigens, and was highly refractory to EBV superinfection. Conversely, the SU-AmB-2 cell line, derived from a patient with "African type" serology, yielded a positive EBNA reaction and was readily inducible and superinfectable. Although both cell lines possessed B (bone-marrow-derived) cell characteristics, they had different surface marker patterns. It is postulated that two different classes of undifferentiated B cell lymphomas exist, one of which is positive for the presence of EBV genomes and occurs endemically in Africa and New Guinea and sporadically in other parts of the world, the other of which is EBV-negative and occurs sporadically throughout the world, including the endemic areas.


Antigen related to mammalian type-C RNA viral p30 proteins is located in renal glomeruli in human systemic lupus erythematosus. An antigen related to mammalian type-C RNA viral p30 proteins was shown by the use of anti-p30 sera and the indirect immunofluorescence method to be present in the kidneys and spleen in a fulminant fatal case of human systemic lupus erythematosus and to be located in the glomeruli, the site of active lupus diffuse glomerulonephritis.


A ribosome-dependent GTPase from yeast distinct from elongation factor 2. Three proteins required for poly(U)-directed polyphenylalanine synthesis have been separated from yeast. Two of the factors correspond to the elongation factors 1 and 2 described for other eukaryotic systems, according to the criteria of phenylalanyl-tRNA binding and diphtheria toxin-catalyzed ADP-ribosylation. The third protein, while absolutely required for polyphenylalanine synthesis, was a more active ribosome-dependent GTPase than elongation factor 2.


Fluorescence-detected magnetic circular dichroism of fluorescent and nonfluorescent molecules. We have modified a spectrometer for the measurement of fluorescence-detected magnetic (and natural) circular dichroism (FDMCD). The instrument can be operated either in a direct mode in which the average and polarization-induced differential fluorescent intensities are recorded separately or in a mode which records their ratio. We have measured the FDMCD of tryptophan, which is itself fluorescent. With the aid of an added fluorescent molecule we also measured the FDMCD of Fe(III) cytochrome c, which is nonfluorescent. In both cases, the FDMCD agreed with the conventional transmission-detected magnetic circular dichroism within experimental uncertainty. The FDMCD of both fluorescent and nonfluorescent molecules is potentially a useful technique for investigating optically dense materials and biological molecules in their native environments and systems in which there is inter- or intramolecular energy transfer.


5'-terminal structure of poliovirus polyribosomal RNA is pUp. Poliovirus RNA purified from virus-specific polyribosomes does not contain m7G in a 5'-5'-pyrophosphate  linkage at its 5'-end. The only potential 5'-end found in ribonuclease digests of this RNA is pUp, which is present in a yield of 1 mole/mole of poliovirus RNA. We conclude that a 5'-terminal m7G is not required for translation of at least one RNA species in animal cells.


Regulation of transsynaptically elicited increase of 3':5'-cyclic AMP by endogenous phosphodiesterase activator. Experiments with cold exposure confirmed previous studies indicating that the endogenous protein acitvator of phosphodiesterase (PDEA) isolated by Cheung participates in the in vivo regulation of 3':5'-cyclic adenosine monophosphate (cAMP) in adrenal medulla. This activator of cAMP phosphodiesterase (PDE) (3':5'-cyclic-AMP 5'-nucleotidohydrolase, EC 3.1.4.17) is present in the particulate as well as the soluble fractions of rat brain. It was found that a purified cAMP-dependent protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37), in the presence of ATP and cAMP, stimulates 3-fold the release of PDEA from the particulate fraction of rat brain and adrenal medulla. The substrate for this phosphorylation could be either a membrane protein that binds PDEA or PDEA itself. In vivo evidence, however, obtained by injecting rats intraventricularly with [gamma-32P]ATP, indicates that the PDEA does not contain radioactive phosphate in its structure. Also, PDEA could not be phosphorylated by protein kinase in vitro. The following mechanism is postulated: when the intracellular content of cAMP increases it activates a protein kinase which phosphorylates a PDEA-binding membrane protein and releases PDEA. In turn this binds to activator-deficient high Km PDE and decreases its Km to facilitate the hydrolysis of the increased concentration of cAMP.


Binding of Cibacron blue F3GA to proteins containing the dinucleotide fold. A simple, convenient, and sensitive spectrophotometric procedure is described for quantitative measurement of nucleoside phosphate binding sites constructed by the dinucleotide fold. The procedure involves difference spectral titration of such enzymes with the dye Cibacron blue F3GA in a spectral region remote from the intrinsic absorbance of proteins or natural ligands. The titration curves can be analyzed to determine the affinity of nucleoside phosphate binding sites for both the dye and the natural ligand over a potentially wide range of experimental conditions. The interaction of the dye with two proteins which contain the dinucleotide fold, lactate dehydrogenase (L-lactate:NAD+ oxidoreductase, EC 1.1.1.27) and phosphoglycerate kinase (ATP:3-phospho-D-glycerate 1-phosphotransferase, EC 2.7.2.3), is illustrated.


"Early" simian-virus-40-specific RNA contains information for tumor antigen formation and chromatin replication. Simian virus 40 (SV40) induces tumor (T)-antigen formation, chromatin replication, and mitosis in primary mouse kidney cells arrested in G0 phase of the mitotic cycle. The temporal and quantitative relation between these early virus-specific reactions led to the hypothesis that the early SV40 mRNA contains information necessary for T-antigen formation and induction of cellular DNA synthesis. To get direct experimental evidence for this hypothesis, the early strand of SV40 DNA was transcribed in vitro by Escherichia coli DNA-dependent RNA polymerase and the SV40-specific cRNA was transferred by microinjection into epitheloid cells of confluent primary mouse kidney cultures. T-antigen formation and stimulation of DNA synthesis were investigated in the recipient cells. The experimental results obtained agree with the hypothesis that T-antigen is a virus-coded protein and that the early virus-specific mRNA contains information necessary for stimulation of cellular DNA replication in the arrested cells.


The 5' end of poliovirus mRNA is not capped with m7G(5')ppp(5')Np. Poliovirus was grown in HeLa cells in the presence of phosphorus-32 and actinomycin D. Three to four hours after infection, viral mRNA was recovered from polyribosomes and its identity verified by two-dimensional gel electrophoresis of RNase T1 digests. Digestion of the viral [32P]mRNA with RNase T2 and separation of the products by ion exchange chromatography at pH 5 yielded pUp as possible 5' terminus but no "capping group" of the structure m7G(5')ppp(5')Np. Total cytoplasmic [32P]RNA of HeLa cells, on the other hand, was found to contain capping groups. Neither the capping group nor ppNp or pppNp was found in an RNase T2 digest of poliovirion [32P]RNA, in agreement with previous results [Wimmer, E. (1972) J. Mol. Biol. 68, 537-540]. The data indicate that 5'-terminal m7G(5')ppp(5')Np is absent from poliovirus RNAs and, therefore, is not involved in poliovirus protein synthesis.


Order of transcription of genes of vesicular stomatitis virus. The effect of ultraviolet radiation on the expression of the genes of vesicular stomatitis virus (VSV) was studied in a cell-free system which executed coupled transcription and translation of the viral genes. In this system, which contained detergent-activated virus and a cytoplasmic extract of mouse L-cells, three of the five viral proteins (N, NS, and M) were synthesized accurately and efficiently; a putative precursor to the viral glycoprotein (G) was also made, but synthesis of the L protein was not detected. In response to irradiation of VSV, the synthesis of each protein in the coupled system displayed a characteristic single-hit inhibition. This permitted calculation of the apparent target-sizes for expression of the different viral genes relative to the entire genome (3.6 to 4.0 X 10(6) daltons) as measured by loss of infectivity. These are: N, 0.55 X 10(6); NS, 0.83 X 10(6); M, 1.12 X 10(6); G, 1.76 X 10(6) daltons of RNA. Only the N protein gene has a target-size which corresponds to that predicted from the molecular weight of its messenger RNA (molecular weight: 0.55 X 10(6)). The target-sizes for the other three genes are two to four times larger than expected, and are not proportional to the molecular weights of their corresponding messenger  RNAs (molecular weights: NS, 0.28 X 10(6); M, 0.28 X 10(6); G, 0.7 X 10(6)). The polar effect of UV irradiation is inconsistent with independent transcription of each of the genes of VSV. Rather, the target-sizes appear to be cumulative, suggesting that trnascription initiates at a single point on VSV RNA and proceeds in the order 3'-N-NS-M-G-(L)-5'.


Location of envelope-specific and sarcoma-specific oligonucleotides on RNA of Schmidt-Ruppin Rous sarcoma virus. Envelope-specific and sarcoma-specific nucleotide sequences have been located within the 10,000 nucleotides of the RNA of nondefective Schmidt-Ruppin Rous sarcoma virus (nd SR). For this purpose, about 30 RNase-T1-resistant oligonucleotides were ordered relative to the 3'-poly(A) terminus of the RNA, to construct an oligonucleotide map of the nd SR RNA. A cluster of seven envelope-specific oligonucleotides, identified by their absence from an otherwise very similar oligonucleotide map of an envelop-defective deletion mutant (which lacks the major viral glycoprotein), mapped at a distance of 2800-5000 nucleotides from the poly(A) end of nd SR RNA. A cluster of two sarcoma-specific oligonucleotides, identified by their absence from an otherwise nearly identical oligonucleotide map of a transformation-defective deletion mutant, mapped at a distance of 1000-2000 nucleotides from the poly(A) end of nd SR RNA. The oligonucleotide maps of nd SR and of the two deletion mutants were the same from the poly(A) end up to 650 nucleotides and included one terminal oligonucleotid, termed C, which is found in all avian tumor viruses tested so far. A possible gene order consistent with our data suggests that sarcoma-specific nucleotide sequences map between envelope-specific nucleotide sequences and the poly(A) end of the RNA.


Primary structure determination of two cytochromes c2: close similarity to functionally unrelated mitochondrial cytochrome C. The amino-acid sequences of the  cytochromes c2 from the photosynthetic non-sulfur purple bacteria Rhodomicrobium vannielii and Rhodopseudomonas viridis have been determined. Only a single residue deletion (at position 11 in horse cytochrome c) is necessary to align the sequences with those of mitochondrial cytochromes c. The overall sequence similarity between these cytochromes c2 and mitochondrial cytochromes c is closer than that between mitochondrial cytochromes c and the other cytochromes c2 of known sequence, and in the latter multiple insertions and deletions must be postulated before a match can be obtained. Nevertheless, these two cytochromes c2 show no better reactivity with the mitochondrial cytochrome c oxidase than do the less well-matched cytochromes c2. The bearing of these findings on possible evolutionary relationship between mitochondria and prokaryotes is discussed.


Mutants of Balb/c 3T3 fibroblasts defective in adhesiveness to substratum: evidence for alteration in cell surface proteins. Two mutants of Balb/c 3T3 cells defective in adhesiveness to substratum have been isolated. After exposure to a mutagen a cell population was treated with prostaglandin E1 and methylisobutylxanthine to elevate the cells 3':5'-cyclic AMP levels and adhesion to the substratum; then, loosley adherent cells were harvested by gentle detachment. After four cycles of selection two clones were isolated. Both are characterized by a round morphology and a decreased adhesion to substratum. The defect in adhesion appears to be responsible for the change in cell shape, since the mutant cells become flattened and look similar to wild-type cells when treated with dibutyryl cyclic AMP even though their adhesiveness remains decreased. Further, the surface of both mutants is chemically altered. Lactoperoxidase-catalyzed iodination of the cell surface shows that two iodinated polypeptides  (molecular weight, Mr, 90,000 and 135-140,000) present in wild-type cells are not detected on the surface of the mutants, and another iodinated band (110-120,000 Mr) is markedly decreased. In addition, one mutant is also missing the high-molecular-weight (230,000) cell surface protein that is sensitive to transformation. No changes have been found in the content of cyclic AMP or free or polymerized tubulin. The exponential growth rate and saturation density are not altered in these adhesion--eficient cells. This last result suggests that modulation of adhesion to substratum per se does not play an important role in the growth control of Balb/c 3T3 cells in vitro.


Effect of Xenopus laevis oocyte extract on supercoiled simian virus 40 DNA: formation of complex DNA. A soluble cell-free extract derived from stage 6 Xenopus laevis oocytes is described. From supercoiled simian virus 40 DNA the extract produces nicked circles (having a single-strand scission), linear molecules of full unit size, shorter length fragments, and various forms of complex DNA.


Association of cell surface receptors for melanotropin with the Golgi region in mouse melanoma cells. Binding of beta-melanotropin (betal-melanocyte stimulating hormone) to mouse melanoma cells occurs in a region on the cell surface overlying the Golgi complex. This association was demonstrated by labeling cells with fluorescein isothiocyanate hormone and by locating the Golgi complex with a histochemical test for thiamine pyrophosphatase activity. The biologically active fluorescent hormone appears on the surface and later in vesicles in the malanized cells, as judged by fluorescence microscopy. It is conceivable that internalization of the hormone is instrumental in the process of hormonally induced melanization. Because initial and late events of hormonally induced pigmentation are related to the Golgi complex, it is likely that instructions that follow the attachment of melanotropin to receptors are carried out in a compartmentalized manner.


Regulation by steroid hormones of phosphorylation of specific protein common to several target organs. The effect of in vivo administration of steroid hormones on the endogenous phosphorylation of individual proteins in cell sap from several target tissues has been studied using the technique of discontinuous sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The hormones studied (and their respective target organs) were: 17 beta-estradiol [1,3,5(10)-estratriene-3, 17beta-diol] (uterus); testosterone (17 beta-hydroxy-4-androsten-3-one) (ventral prostate and seminal vesicle)' cortisol (11beta, 17alpha, 21-trihydroxy-4-pregnene-3,20-dione) (liver); aldosterone (the 18.11-hemiacetal of 11beta,21-dihydroxy-3,20-dioxo-4-pregnen-18-al) (toad bladder). In each of the five target organs studied, pretreatment with the appropriate hormone reduced the amount of 32P incorporated from [gamma-32P]ATP into an apparently common protein band present in the cytosol fraction. The endogenous phosphorylation and dephosphorylation of this protein was also regulated by cAMP. This protein, designated SCARP (steroid and cyclic adenosine 3':5' monophosphate regulated phosphoprotein), was estimated to have an apparent molecular phoprotein), was estimated to have an apparent molecular weight of 54,000 in the gel electrophoresis system used. The effect of the steroid hormones in decreasing the phosphorylation of SCARP was specific for their respective target tissues. The effect of 17beta-estradiol and of testosterone on SCARP could be observed as early as two hours after a single dose of the steroid. A protein synthesis inhibitor, cycloheximide, abolished the effect of the steroid hormones, but not that of cAMP, on the endogenous phosphorylation of SCARP. The results suggest that steroid hormones regulate either the amount of SCARP or its ability to become phosphorylated. This regulation of a single species of protein by several types of steroid hormones in different target organs raises the possibility that this common biochemical action may be a component of the mechanism by which these steroids achieve some of their biological effects.


Inhibition of tumor cell proliferation by dimerized ribonuclease. A cross-linked dimer of pancreatic ribonuclease A (ribonucleate 3'-pyrimidino-olitonucleotidohydrolase, EC 3.1.4.22), at a 10 mg/liter concentration, blocks proliferation of tumor cells. The protein retains this ability after inactivation by iodoacetate. The cytostatic effect of ribonuclease preparations on various cell lines correlates well with their rate of uptake: for example, monomeric ribonuclease A is much less effective and is taken up into the cells 10 t0 15 times more slowly. Cell fractionation studies on hepatoma cells indicate accumulation of the dimer in the lysosomal system. Ribonuclease dimer induces a labilization of the lysosomes when added to cell homogenates, raising the possibility that its antitumoral effect may be mediated by endocytosis and lysosomes.


New gene locus modifying susceptibility to certain B-tropic murine leukemia viruses. A new gene, SRV, the dominant allele of which occurs in mice of strain C57BL, independently modifies the slope ("hitness") and level of sensitivity of the titration curves obtained when one subclass of B-tropic murine leukemia viruses is propagated on mouse embryo cells of nonpermissive Fv-1 genotypes. Replication of another subclass of B-tropic viruses is not modified with respect to hitness, and that of N-tropic viruses is not affected.


Suppression of murine type-C RNA virogenes by type-specific oncornavirus vaccines: prospects for prevention of cancer. Immunization of crossbred and F1 mice with combined killed and live Gross leukemia virus AKR type-C viral vaccines suppressed endogeneous N-type AKR virus up to 10,000-fold for significant periods during early life. Since several previous studies in the same and similar crossbred systems revealed direct correlations between low and high levels of type-C virus early in life with low and high incidences of leukemia and other cancers later in life, we believe that prospects for suppression of spontaneous neoplasms are good; however, 8-14 months will be required to achieve the final results. Should cancers be prevented by serotype-specific vaccines, such evidence would provide conclusive proof of endogenous viral etiology.


Activation of an endogenous mouse type C virus by ultraviolet-irradiated herpes simplex virus types 1 and 2. Infection of BALB/c mouse cells with UV-irradiated herpes simplex virus (HSV) types 1 and 2 resulted in activation of a xenotropic type C virus detected by infectious center formation in permissive rat cells. The levels of type C virus activated by HSV were related to the UV dose and the multiplicity of infection used. The ability of HSV to activate type C virus was eliminated by heat-inactivation and by neutralization with specific antiserum against HSV, but was not affected by purification or treatment with DNase and RNase. Maximum levels of type C virus in the cells and medium were observed within 1 day after HSV infection, and the levels returned to control cell values within 3-4 days. The possible significance of these findings with respect to the putative oncogenic potential of HSV is discussed.


The pathogenesis of simian varicella virus in cynomolgus monkeys. The MLM herpesvirus is infectious for cynomolgus monkeys. The disease in this species, possibly modulated by preinoculation antibody resembles human varicella. Virus has been recovered from blood during the early incubation period, and from liver, lymph nodes, kidney, bladder and urine during the eruptive period of infection. The major target organs were skin and liver; specific pathological changes developed in both. Appropriate antibody responses, including those to Herpesvirus varicellae followed infections mounted by parenteral inoculation of cynomolgus monkeys.


Effects of hyperlipoproteinemic serum and exogenous proline concentration on collagen synthesis by isolated rabbit aortas. Collagen synthesis was measured in segments of normal rabbit aorta, incubated in vitro, by monitoring the formation of peptidyl-14C-hydroxyproline from [U-14C]-L-proline added to the incubation medium. The effect of hyperlipoproteinemic rabbit serum on the rate of collagen synthesis was compared with the effect of normal rabbit serum. No differences in the rates of synthesis were detected between the two batches of serum, despite a 60-fold difference in serum cholesterol concentration. Increases in free proline concentration in the incubation medium resulted in changes in proline flux between medium and tissue pools of free proline, but medium proline concentration had no effect on the rate of collagen synthesis.