Mechanism of pain induced by radiocontrast media. In lightly-anesthetized dogs, ionic or non-ionic RCM (Iotalamato and iohexol, respectively) when injected by intracarotid route (i.c.), elicit a pain response comparable to that caused by bradykinin (BK) or capsaicin (CAP). This response, which is characterized by vocalization, hyperpnea, bradycardia and neck muscle contraction, was dose dependent and related to the osmolarity of the RCM. In the present study we observed that indomethacin did not interfere with CAP and RCM-induced pain at dose (2 mg/kg i.c.) that reduced BK-elicited responses. In contrast, Ruthenium Red (RR), in dose (1 mg/kg i.c.) that reduced CAP and/or RCM-induced effects did not affect BK-induced phenomena. We also verified that L-NAME (50 mg/kg i.c.) reduced the BK-, but not the CAP- and/or RCM-induced pain responses which suggests that an L-arginine-derived NO or related compound is involved in BK activation of perivascular nociceptors. Indeed, we found that i.c. injection of 20 mg of S-nitrosocysteine, a putative EDRF, caused BK-like responses. On the other hand, RCM and CAP appear to activate the same RR sensitive ionic channels of primary afferent endings. Therefore, RR-analogues could constitute a novel approach to minimizing or eventually abolishing the RCM side effects.


Expression of blood group antigens on red cell microvesicles. The purpose of this study was to determine whether epitopes of the A, B, D, Fya, M, N, S, s, and K blood group antigens are present on microvesicle membranes shed by red cells during storage. Vesicles were isolated from outdated units of blood having and lacking the specified antigens. Diluted antisera were absorbed with fixed quantities of vesicles from red cells with the test antigen and red cells lacking that antigen (controls). The adsorbed and unadsorbed antisera were titrated and scored by using panel cells from persons known to be heterozygous for all the non-AB antigens. The mean titration scores following adsorption with the vesicles from A, B, D, M+N-, M-N+, S+s-, S-s+, and Fy(a+b-) units were appreciably lower than the control scores (0, 0, 3, 2, 2, 0, 4, and 4 vs. 19, 23, 34, 13, 12, 16, 18, and 29, respectively), which indicated the presence of these epitopes on the membrane of shed vesicles. The results following adsorption with K:1,2 vesicles were equivocal.


Monocyte chemotaxis and chemotactic cytokine release after exposure to hydroxyethyl starch. Hydroxyethyl starch (HES) is commonly used in leukapheresis and infused as an alternative to blood components for the treatment of hypotension due to hemorrhage and trauma. Its prolonged intravascular persistence and retention in tissue raise concerns about possible effects on humoral and cell-mediated immunity and white cell (WBC) locomotion, particularly in volunteer WBC donors or in severely burned individuals with immunologic depression and increased risk for infection. This study evaluated the effect of HES on human monocyte migration and chemotaxis and the production of antigen- and mitogen-induced WBC-derived chemotactic cytokine. A bioassay was developed to quantitate the neutrophil chemotactic activity of a cytokine generated by mononuclear WBCs stimulated in vitro by phytohemagglutinin or tuberculin protein. The time- and dose-dependent generation of the chemotactic cytokine was not affected by the presence of HES. HES by itself did not induce the generation of this cytokine, nor were human monocyte chemotaxis and spontaneous migration significantly changed by exposure to HES. These results, with those of other investigators, suggest that HES is a safe red cell-sedimenting agent for leukapheresis and an alternative to the use of blood components in shock resuscitation.


[Factors that cause a change in the antigenic structure of of the influenza virus hemagglutinin]. Crossing of norakin-resistant mutant NR1 of A/Waybridge (H7N7) strain of fowl plague virus (FPV) with human influenza virus strains produced recombinants inheriting the hemagglutinin (HA) gene of the NR1 mutant and neuraminidase (NA) genes of human influenza virus strains. The R120 recombinant produced by crossing of NR1 with A/Taiwan/1/86 (H1N1) strain, unlike other recombinants and NR1 mutant, lost the capacity of reacting in H1 test with two monoclonal antibodies (MCA) to HA7: 71/4 and 46/6. The ts mutant A/FPV/Rostok which has ts-mutation in HA-gene also had changes in the antigenic specificity of HA. The RA and RB recombinants produced by crossing R120 with the A/Krasnodar/101/59 strain and inheriting HA-gene from R120 and NA-gene from A/Krasnodar/101/59 strain recovered the initial HA antigenic structure. No changes in the antigenic properties of HA were observed in the recombinants produced by crossing the original A/FPV/Waybridge strain with A/Taiwan/1/86 strain and inheriting HA-gene from the original A/FPV/Waybridge strain and NA-gene from A/Taiwan/1/86 strain. It is concluded that ts mutations in influenza virus HA-gene may be accompanied by changes in the antigenic specificity of this virus HA. The possibilities of manifestation of phenotypic suppression at the level of influenza virus virion membrane proteins and the causes of changes in the HA antigenic structure in this virus recombinants are discussed.


[The isolation and characteristics of monoclonal antibodies to different proteins of the herpes simplex virus types 1 and 2]. The authors prepared 156 mouse hybridomas producing monoclonal (MCA) antibodies to type- and group-specific antigenic determinants of HSV-1 and HSV-2. Seven of them were studied at length by western blot and radioimmunoprecipitation methods. The cell lines 1H97 and 2H141 were shown to produce immunoglobulins of G2 beta and M class, respectively, and were directed against group-specific antigenic determinants of the major nucleocapsid protein p150. The cell lines 1H38 and 1H110 produced immunoglobulins of M and G2 beta, respectively, and were directed against type-specific antigens of HSV-1 glycoprotein gB. At the same time, the presence of group-specific antigenic determinants on glycoprotein gB molecule was indicated by MCA 1H188 belonging to immunoglobulins of G2 alpha class. Two cell lines, 2H208 and 1H225, produced immunoglobulins G2 alpha directed against type-specific antigenic determinants of HSV-2 glycoprotein gD and group-specific antigenic determinants of HSV-1 gD, respectively. The results of immunoelectron microscopy indicated that MCA 1H110 and 2H208 were directed against virus envelope proteins.


Development of a vaccine for the prevention of AIDS, a critical appraisal. The pathogenesis and clinical expression of HIV-1 infection in humans is considered in terms of classical pathogenetic studies of viral infections for which successful vaccines have been produced. The unique features of HIV pathogenesis are defined, and gaps in knowledge identified as a framework for considering designs for immune intervention. Envelope-derived candidate vaccines have been used in immunization and challenge experiments in SIV/macaque or HIV/chimpanzee models, presented either as vaccinia recombinant vectors or as subunits, singly or in sequence. These studies have been paralleled by clinical trials for safety and immunogenicity in seronegative individuals. Data generated will permit comparison of immune responses to specific antigens and delivery systems in animal models and in humans. In limited studies conducted under optimized conditions, non-human primates have been protected against virus challenge when immunized with some candidate vaccines or following passive transfer of high-titred antibody. Consideration of current information suggests that in order to prevent HIV infection it may be necessary to devise new strategies capable of inducing and maintaining high threshold titres of biologically relevant antibody as well as persistence of active cytotoxic T cells recognizing multiple epitopes.


[The therapeutic effect of aprotinin inhalations in influenza and paramyxovirus infections in mice]. Mice infected with influenza A/Aichi/2/68 (H3N2) virus or Sendai/960 paramyxovirus were treated by inhalations of aerosol aprotinin, a broad spectrum inhibitor of proteinases. A course of inhalations of finely dispersed aerosol aprotinin including 4 exposures of 35-40 min each daily for 6 days provided respiratory administration of aprotinin in a dose about 100 kallikrein-inhibiting U/mouse per day. In mice treated by aprotinin inhalations, histological examinations showed decreased pulmonary pathology, and their body weights increased as much as in uninfected animals. In the placebo group, the weight decreased until the death of the animals. The protective effect of aprotinin inhalations was 40-80% with inoculation doses 10-100 MLD50/mouse. The treated animals died 2-4 days later than those in the placebo group. The results indicate the expedience of inhalation therapy with aerosol aprotinin in influenza and paramyxovirus respiratory infections.


[Surgery of stomach cancer--an assessment of current status]. Surgical strategy for gastric carcinoma consists of total gastrectomy as a rule combined with extended lymphadenectomy. For small tumors of the intestinal type located in the distal stomach distal gastric resection can be taken into consideration. In case of a high risk patient endoscopic polypectomy of a polypoid early gastric carcinoma can be sufficient. If the operation is done for cure intraoperative radiotherapy is employed within prospective trials in order to prevent local recurrence. Extended resections prolong survival time only if curativity will be achieved. For palliative situations procedures including removal of the tumor have better results with regard to quality of life and survival than bypass methods.


[Surgical emergency interventions in acute diseases of the large intestine]. Emergency operations due to acute colonic disease between 1. 1. 1984 and 31. 12. 1991 were retrospectively analyzed with regard to causality, surgical procedure, complications and mortality. 55 of 1105 colonic operations were emergency cases requiring immediate surgical intervention. Primary continuity preserving resections were carried out whenever possible, attending not only to the acute situation but also to the primary disease. The mean age of the 26 females and 29 males was 69 [1, 9] years. 29 patients had a colonic ileus, 21 a diffuse peritonitis and 5 patients had an uncontrolled haemorrhage. Colorectal carcinomas were initially diagnosed in 20 of the 50 patients; 14 of these patients (70%) could be operated for potential cure and primary continuity preserving resections were also possible for 14 patients (70%). Continuity preserving resections were possible for 18 of 21 patients with peritonitis and 3 colonic perforations were oversutured. In the 5 patients with acute haemorrhage, 4 resections and one transanal intervention were performed. Postoperative complications were observed in 19 patients (35%). Postoperative mortality was 16% (9/55), 5% for operations due to peritonitis, 24% for operations due to colonic ileus and 20% for operations due to haemorrhage. Primary continuity preserving resections were possible for 39 of 55 patients (71%).


[The degree of virulence in Far Eastern strains of the tick-borne encephalitis virus]. Screening of immunomodulating properties of 45 tick-borne encephalitis (TBE) virus strains isolated in the southern part of the Soviet Far East was carried out. TBE virus strains were found to have different effects on the immune responsiveness of host splenocytes. Over 90% of the strains isolated from hematothermals could inhibit the immune response of the host to a heterologous antigen (sheep erythrocytes); similar properties were found in 5 strains isolated from ticks. At the same time, most strains isolated from the vectors were unable to modulate the immune response of antibody-producing cells and 3 strains even had immunostimulating properties. The existence of significant correlation (r = -0.57; p < 0.01) between the immunomodulating activity of a strain and peripheral virulence of the virus for white mice indicates that the immunomodulation parameter may be used as another pathogenetic marker. This marker and that of the peripheral activity served the basis for creation, by means of the mathematical method of discrimination analysis, of a new pathogenetic characteristic--a single marker of strain virulence.


[Simultaneous en-bloc allotransplantation of pancreas and kidney in the animal model. Comparison of separate organ and en-bloc pancreas/kidney transplantation in swine]. The high technical complication rate of pancreas transplantation requires large animal models to improve clinical transplant survival rates. The pig is a very suitable animal due to its anatomy, physiology and immunology which are similar to humans. In this study a model of en-bloc simultaneous pancreas and kidney transplantation was established which--in contrast to separate transplantation of both organs--decreases preservation time, operation time, and clamp time. Furthermore, the rates of intra- and postoperative complications were reduced compared with separate transplantation. The donor aorta (encompassing celiac axis, superior mesenteric artery, and left renal artery) is anastomosed en-bloc to the recipients aorta in a an oblique-to-side fashion. The portal vein is anastomosed end-to-side to the left common iliac vein. The exocrine pancreatic secretions are drained via duodenocystostomy to allow for monitoring of urinary amylase for rejection. The en-bloc technique is an alternative for pediatric donor organs since the risk of vascular complications is lower compared with separate implantation of the donor vessels. Based on our results in a large animal model the en-bloc technique could be used in adult uremic diabetic patients who receive a combined pancreas-kidney transplant from a pediatric cadaver donor.


Fibronectin production by chicken granulosa cells in vitro: effect of follicular development. In vitro studies were conducted to investigate the role of chicken ovarian granulosa cells in the production of fibronectin, a component of the basal lamina of ovarian follicles. Collagenase dispersed granulosa cells obtained from the first (F1; about 35 mm in diameter) and third (F3; 15-20 mm in diameter) largest preovulatory follicles, as well as from a pool of small yellow follicles (SF; 6-10 mm in diameter), were incubated in serum-free medium-199 for 24 to 96 h in the absence and presence of luteinizing hormone (LH) or forskolin. Fibronectin secreted in the medium was quantitated by enzyme linked immunosorbent assay. Basal fibronectin production (which increased with the duration of incubation) was significantly greater (p < 0.001) in granulosa cells derived from mature follicle (F1) than in F3 or SF cells. Both LH and forskolin stimulated fibronectin production in SF and F3 cells in a dose-dependent manner; however, they were without effect in F1 cells. The magnitude of increase in fibronectin production elicited by LH or forskolin was greater in SF cells than in F3 cells. The cytoplasm of cultured granulosa cells taken at all stages of follicular development stained positively for fibronectin. These findings indicate that chicken granulosa cells produce fibronectin. This ability is acquired early in follicular development and the stimulatory effect of the gonadotropin (LH) diminished as the follicle approached ovulation.


Immunocytochemistry of neurofibrillary tangles with antibodies to subregions of tau protein: identification of hidden and cleaved tau epitopes and a new phosphorylation site. Antibodies to multiple epitopes spanning the length of the tau molecule were used to study Alzheimer neurofibrillary tangles (NFT) using immunocytochemical methods and several different methods of fixation and tissue processing, including staining of vibratome sections, hydrated autoclaving of paraffin sections and immunofluorescence of NFT isolated from fresh brain tissue. Smears and sections were pretreated with trypsin and/or phosphatase to further characterize antibody binding. In tissue fixed briefly in periodate-lysine-paraformaldehyde, tau immunoreactivity was detected in astrocytes, but only a few tau epitopes were detected in NFT with this fixation method. In contrast, all tau epitopes were detected in NFT in tissue fixed in formaldehyde for prolonged periods of time. In the hippocampus, the number of NFT detected in the dentate fascia was in proportion to the duration of dementia, as we previously noted. Dentate fascia NFT were intracellular (i-NFT) and were reactive with antibodies recognizing epitopes in both the carboxy- and amino-terminal regions of tau, but not the microtubule-binding domain of tau, suggesting that microtubule-binding domain epitopes are hidden in i-NFT. In contrast, NFT in the subiculum and layer II of the parahippocampal cortex were mostly extracellular (e-NFT), especially in severe cases of long duration. e-NFT were immunoreactive with antibodies to the microtubule-binding domain, but only weakly reactive with antibodies to carboxy- or amino-terminal epitopes, suggesting that e-NFT may contain fragments of tau. In both isolated NFT and NFT in sections, amino-terminal epitopes, including the Alz-50 epitope, were sensitive to trypsin proteolysis, which suggests that the lack of staining of e-NFT by antibodies to the amino-terminal regions of tau is due to proteolysis. Antibodies reactive with amino-terminal epitopes also stained fewer NFT following hydrated autoclaving, while those reacting with the carboxy half of tau stained more NFT after hydrated autoclaving. Thus, although carboxy-terminal regions are not detected in e-NFT, they are probably masked, rather than proteolytically cleaved, since they can be revealed by hydrated autoclaving. Finally, phosphatase treatment of isolated NFT revealed enhanced immunostaining not only with Tau-1, as in previous studies demonstrating abnormal phosphorylation of tau proteins in NFT, but also with an antibody to exon 2, which reveals yet another phosphorylation site in tau of NFT.


Expression of leukocyte antigen CD34 by brain capillaries in Alzheimer's disease and neurologically normal subjects. We studied the expression of a hemopoietic progenitor cell antigen, designated CD34, in brains from subjects with Alzheimer's disease (AD) and non-neurological controls. Immunoblots of brain microvessel proteins probed with monoclonal antibody QBEND/10 to the leukocyte antigen CD34 recognized a protein band with an apparent molecular mass of 90-100 kDa. Immunocytochemical staining of brain tissue sections showed CD34 to be expressed by all microvasculature including those of the circumventricular organs. In normal control brains such specific staining exhibited by QBEND/10 was indistinguishable from that obtained with collagen IV antibodies. In AD, however, increased vascular tendrils in form of endothelial abluminal processes and intraparenchymal abnormalities were evident in cortical and hippocampal regions, predominant in cases with severe pathology. Our results demonstrate that the leukocyte antigen CD34 is localized with the vascular endothelium throughout the human brain. These results also suggest that CD34 detects endothelial abnormalities in brains of AD subjects and support previous observations on the usefulness of CD34 to label abluminal microprocesses.


Intraventricular infusion of N-methyl-D-aspartate. 2. Acute neuronal consequences. This study documents the ultrastructural features of acute neuronal injury following N-methyl-D-aspartate (NMDA) receptor activation. NMDA (100 nmol/microliters) or vehicle was infused over a 15-min period into the lateral ventricle of adult rats. After perfusion fixation, specimens demonstrating normal and abnormal patterns of vascular permeability to horseradish peroxidase were sampled for ultrastructural analysis. In NMDA-infused rats, brain regions exhibiting protein extravasation contained swollen dendritic profiles and abnormal neuronal perikarya. Although periventricular regions were most severely affected, parenchymal abnormalities were also detected in the cerebral cortex, septum, striatum, thalamus, hypothalamus and cerebellum. Mildly affected dendrites contained dark compact mitochondria, while in severely swollen dendrites mitochondria were enlarged with ruptured cristae. Focal sites of plasma membrane disruption were also detected within swollen dendrites. Swollen neurons commonly displayed peripheral pallor and increased numbers of cytoplasmic vacuoles. Other neurons appeared dark and shrunken, some containing disrupted mitochondria and pyknotic nuclei. Pretreatment with the NMDA antagonist MK-801 (2 mg/kg) attenuated the neuronal and dendritic alterations. In conditions where cerebrospinal fluid levels of glutamate are abnormally elevated, excessive NMDA receptor activation may lead to early vascular and neuronal complications which could work in concert to promote brain injury.


Brain stem serotonin-synthesizing neurons in Alzheimer's disease: a clinicopathological correlation. The location and number of brain stem serotonin-synthesizing neurons were analyzed in 11 patients with Alzheimer's disease (AD) and 5 age-matched controls using immunohistochemical techniques. In addition, the number of neuritic plaques and neurofibrillary tangles in the cortex and brain stem raphe was evaluated, as was the number of Nissl-stained raphe neurons. AD patients could be classified into two groups based on their raphe pathology; patients with such pathology (AD+) and those without (AD-). The number of large raphe neurons correlated significantly with the number of serotonin-synthesizing neurons in control material, indicating that all large neurons were serotonergic. This relationship was not apparent in AD+ patients, in whom the number of serotonin-synthesizing neurons correlated with the number of neurofibrillary tangles in the raphe of these patients. This indicates that in AD+ patients the serotonin-synthesizing neurons were selectively affected. There was no correlation between raphe and cortical pathology or raphe pathology and patient sex, age, mini-mental score or depression score, even when such scores were weighted for the interval between testing and death. There was a trend for the raphe pathology to correlate with the age of onset and duration of dementia and the Blessed dementia score in AD+ patients. Most AD+ patients with severe raphe lesions had clinical dementia only, while AD- patients had additional clinical features. The raphe lesions were more dramatic in AD+ patients with a rapid progression of symptoms.


Diagnostic criteria for human diffuse malignant mesothelioma. Diffuse malignant mesothelioma is a rare tumor in the general population, yet is unique in that it is caused almost exclusively by exposure to asbestos with long-term latency (15 years and over). Pathologists are required to provide a reliable diagnosis of the tumor for clinicians who are responsible for the treatment of affected patients. Pathological diagnosis of diffuse malignant mesothelioma is not always easy; however, it has improved over the last few decades. Currently, comprehensive analysis, including gross appearance, histology, histochemistry, immunocytochemistry and electron microscopy is recommended as the best approach to an accurate diagnosis of diffuse malignant mesothelioma.


Epitopic heterogeneity of the CD36 antigen expressed by normal and neoplastic endothelial cells. An immunohistochemical study with a novel monoclonal antibody 8C9. Phenotypic and functional heterogeneity of endothelial cells (ECs) is being recognized with increasing frequency. Here we report a novel murine monoclonal antibody (MoAb), named 8C9, that detects a unique epitope on the leukocyte differentiation antigen CD36 (platelet glycoprotein IV or IIIb) expressed by both normal and neoplastic ECs. In immunohistochemical and flow-cytometric studies, 8C9-immunoreactivity was detected on capillary ECs, adipocytes, monocytes, platelets and a human monocytoid cell line U-937, which are known to express the CD36 antigen. Blocking experiments using U-937 cells and studies on cryostat sections revealed that a murine MoAb OKM5, which detects the CD36 antigen, blocked the binding of 8C9 to its antigen. Immunoblot analysis showed that 8C9 bound to a 97-kDa membrane protein expressed by U-937 cells treated with phorbol ester. These results indicate that 8C9 detects the CD36 antigen. However, the findings that some OKM5-positive normal ECs in the liver, spleen and lymph nodes as well as neoplastic ECs in a cutaneous angiosarcoma did not react with 8C9, together with the fact that the CD36 antigen does not form a complex or associate with other proteins, suggest epitopic heterogeneity of the CD36 antigen expressed by these tissues.


Immune disorders in depression: higher T helper/T suppressor-cytotoxic cell ratio. This study investigated the leukocyte T helper and T suppressor-cytotoxic cell (sub)set profile of minor, simple major and melancholic depressives versus normal controls. Using both monoclonal antibody staining and flow cytometry, we determined the absolute numbers and percentages of the following T cell immune subsets: T helper (CD4+), T virgin (CD4+CD45+), T memory (CD4+CD45-), T suppressor/cytotoxic (CD8+), CD8+ T suppressor (CD8+CD57-) and CD8+ T cytotoxic (CD8+CD57+) cells. After computing the CD4+/CD8+ ratio, we detected a significantly increased ratio in depressed patients as compared with healthy controls. Depression per se is characterized by a higher percentage of CD4+ and a lower percentage of CD8+CD57- cells. Melancholic depressed subjects exhibit a significantly increased number of CD4+ and CD4+CD45- cells. The combined use of various percentages of CD4+ and CD8+ (sub)sets yields a high degree of marker positivity for melancholia: through cumulative evaluation of those percentages, the marker positivity increases to 68% (sensitivity) and the specificity is 95%. These results together with our previous reports may refer to a depression-related state of T cell activation.


The Swedish version of the Positive and Negative Syndrome Scale (PANSS) for schizophrenia. Construct validity and interrater reliability. The Positive and Negative Syndrome Scale (PANSS) has been translated into Swedish and tested in 88 chronic schizophrenic patients. All 4 subscales exhibited a roughly normal distribution. The overall alpha for the positive, negative and general psychopathology subscales were 0.81, 0.58 and 0.63, respectively. The correlation between the positive and negative subscales was -0.17 (NS). The interrater reliability was 0.73-0.75 for the positive, 0.65-0.74 for the negative and 0.75-0.77 for the general psychopathology subscales. The intraclass coefficients were 0.75-0.77 for the positive, 0.27-0.46 for the negative, 0.56-0.72 for the general psychopathology subscales and 0.66-0.71 for the total scale. Thus, the validity and reliability of the PANSS (Swedish version) are quite satisfactory.


Assessment of the clinical value of urinary trypsin inhibitory activity in elderly people. To assess the clinical value of urinary trypsin inhibitory activity (UTIA) in elderly people, a prospective study was carried out over 4 months in our internal medicine department. Two hundred and forty-three patients of more than 60 years of age were included. A positive correlation was observed between UTIA and serum creatinine (p < 10(-3)). In the population with serum creatinine of less than 133 mumol/l (200 patients), UTIA was independent of age, sex and serum creatinine. UTIA was compared with seven serum inflammatory proteins titrated on patient admission. The principal interest of UTIA determination appeared in bacterial infections. UTIA was significantly increased in this group (p < 10(-4)). However, a positive correlation was proved only with C-reactive protein (CRP) (p = 9 x 10(-4)). Nevertheless, CRP appeared to be the best marker of bacterial infectious diseases after receiver operating characteristic curves analysis.


Time engaged in reading. A critical factor in reading achievement. The reading achievement of deaf children may be low not only as a result of factors related to the hearing loss, such as a lag in language development. Environmental factors such as the quantity and quality of reading instruction, for example, may also cause low reading achievement. This study looked at the amount of time spent reading and the types of teacher interactions during reading instruction in classrooms at a school for deaf children and associated satellite classes in New Zealand. It was found that the deaf children spent very low levels of time engaged in reading and were subjected to teacher interactions that may inhibit the development of meaning-based reading skills. The quantity and quality of reading instruction for deaf children may differ from that experienced by most hearing children in New Zealand.


Study of the activities of Chinese herb Viscum alniformosanae Part II: The components of conditioned medium produced by Viscum alniformosanae-stimulated mononuclear cells. The human promyelocytic cell line HL-60 can be induced to monocytoid terminal differentiation by several conditioned media produced by lectin-stimulated mononuclear cells. We reported previously that a 572-conditioned medium (CM) secreted from viscum alniformosanae-stimulated mononuclear cells also had the capacity of inducing HL-60 leukemic cells into mature monocytes. In the present study, we showed that 572-CM did not contain IFN-r, TNF, IL-1 and IL-2 as determined by using ELISA tests. This CM was unable to induce granulocyte-macrophage colony formation. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was used to detect the components of this CM. After running the acrylamide gels, a wide band protein, in the 65-80 kd range was obtained and it was different from those of other mitogens.


Prediction of pregnancy outcome with single versus serial maternal serum alpha-fetoprotein tests. The purpose of our study was to determine whether the trend of three maternal serum alpha-fetoprotein samples was more predictive of pregnancy outcome than the initial sample in the evaluation of patients with unexplained alpha-fetoprotein elevations. A total of 432 patients with unexplained elevation of their first two maternal serum alpha-fetoprotein samples had a third sample drawn. Pregnancy outcomes were determined. Patients were grouped for analysis according to the level of the initial sample, the final sample, and the trend of three samples. Statistical analysis was by chi 2 and logistic regression, with p < 0.05 considered significant. The initial maternal serum alpha-fetoprotein was most predictive of preterm delivery (p < 0.001), size small for gestational age (p < 0.001), and intrauterine fetal death (p = 0.009). Neither the final value nor the trend of three values was as prognostic. The first maternal serum alpha-fetoprotein is the best predictor of pregnancy outcome. Obtaining a second sample to confirm the elevation is appropriate, but additional samples provide minimal information.


Western blot analysis of antibodies to Leishmania infantum antigens: potential of the 14-kD and 16-kD antigens for diagnosis and epidemiologic purposes. When infected with Leishmania species, patients develop specific antibodies that constitute the basis of serodiagnosis. Using Western blot analysis, we studied the specificity of anti-L. infantum antibodies in patients with visceral leishmaniasis (including patients with acquired immunodeficiency syndrome [AIDS]) and in healthy subjects living in an endemic area. Sera from patients with visceral leishmaniasis recognized numerous antigens that had a molecular mass range of 12-120 kD. The 14-, 16-, 28-30-, 46-, and 68-kD antigens were recognized by 92%, 95%, 63%, 80%, 69%, and 89% of the patients' sera, respectively. The 14-16-kD antigens had the greatest specificity for leishmaniasis. The same pattern was found with sera from AIDS patients with proven leishmaniasis, but the 14-kD band was not present in some cases; recognition of the 16-kD band was constant. In these patients, Western blotting characterized specific antibodies even when the results of classic serologic tests (indirect immunofluorescent antibody test and enzyme-linked immunosorbent assay) were negative. Western blotting was found to be more sensitive than the IFA and ELISA, and it was used to detect antibodies to the 14-, 16-, 22-, and 24-kD antigens in subjects living in an endemic area. The detection of antibodies for the 14-kD and 16-kD Leishmania antigens would be a valuable tool both in the diagnosis of visceral leishmaniasis and in epidemiologic studies.


Rapid diagnosis of Bancroftian filariasis by acridine orange staining of centrifuged parasites. A rapid diagnostic test for detection of microfilaremia using a microhematocrit tube precoated with acridine orange (the Quantitative Buffy Coat [QBC] tube) was compared with a conventional 50 microliters-thick blood film (TF) in 119 volunteers in an area of Recife, Brazil that was endemic for Wuchereria bancrofti. Both the QBC and the TF techniques were 100% specific, and thus positive predictive values were equal at 100% for each technique in all subjects studied. Both techniques had equal negative predictive values of 100% in subjects with microfilarial counts > 100 per milliliter (mf/ml). Counts < 20 mf/ml are below a cutoff equal to the calculated limit of sensitivity of each of the two techniques. For those individuals with counts between 20 and 99 mf/ml, negative predictive values were, for practical diagnostic purposes, equivalent at 97.5% for the QBC technique and 99.0% for the TF. Because the QBC technique has predictive values as high as conventional TF, the convenience and rapidity of the technique will make the QBC technique a desirable alternative diagnostic method in those clinical settings where the equipment is available. A positive result will be available in less than six min after obtaining the specimen in individuals with counts < 100 mf/ml, and individuals with lower or no microfilaremia will have a result within 6-12 min.


Diversity in the immunodominant determinants of the circumsporozoite protein of Plasmodium falciparum parasites from malaria-endemic regions of Papua New Guinea and Brazil. To determine the nature and extent of variation in the T cell sites of the Plasmodium falciparum circumsporozoite (CS) protein, a candidate antigen in the development of a malaria vaccine, we cloned and sequenced 69 recombinant clones of the CS protein gene representing 18 and 17 P. falciparum isolates from infected individuals from Madang, Papua New Guinea (PNG), a holoendemic malaria region, and Paragaminos and Jacunda, Brazil, relatively low endemic regions, respectively. As previously known, the amino acid sequence polymorphism was restricted to the three immunodominant regions of the protein, Th1R-N1, Th2R, and Th3R. While some of the observed nonsilent mutations in the T cell determinants of the CS protein were similar to those previously identified, we have found new amino acid changes in each of the polymorphic sequences in parasites from PNG and Brazil. A comparison of the CS epitope sequences of parasites from PNG and Brazil with the previously known CS epitope sequences of parasites from Brazil and The Gambia showed the following: 1) polymorphism was found in the Th1R-N1, Th2R, and Th3R region; however, while amino acid substitutions in the Th1R-N1 and Th2R region tended to be conservative, the substitutions found in the Th3R region were not, suggesting that the Th3R epitope may be rapidly evolving to allow parasites to escape host antiparasite cytotoxic T cell-enforced immune responses, and 2) the CS proteins of P. falciparum from high malaria-transmission regions (PNG and The Gambia) appear more polymorphic than the CS proteins of parasites from relatively low malaria-endemic regions in Brazil, where P. falciparum infection has been recently established.


Comparative light-microscopic and immunohistochemical study of traumatic and palisaded encapsulated neuromas of the skin. The primary hyperplastic nature of palisaded encapsulated neuromas (PENs) has been recently challenged by suggesting a traumatic origin. We studied eight cases of traumatic neuroma (TN) and 12 cases of PEN by routine light-microscopic, histochemical, and immunohistochemical methods to assess evidence of previous tissue injury. Sections from the formalin-fixed, paraffin-embedded tissue were stained with hematoxylin-eosin, trichrome, elastic, reticulin, Giemsa, colloidal iron (with and without hyaluronidase), and Bielschowsky silver stains. Antibodies were applied to collagen types I, III, and IV, MAC 387, factor XIIIa, alpha 1-antitrypsin (A1AT), epithelial membrane antigen (EMA), Leu-7, and myelin basic protein using ABC techniques. We found that (a) in TN the individual fascicles were usually surrounded by perineurial cells, whereas in PEN the perineurial cells were observed mainly in the capsular areas and only rarely within the fascicles as evidenced by EMA antibodies; (b) histochemically TN contained considerably larger amounts of collagen (types I and III), acidic mucin, and myelin products than did PEN; and (c) neither PEN nor TN contained increased inflammatory cells or cells positive for factor XIIIa, MAC 387, or A1AT. We conclude that (a) there are substantial structural and histochemical differences between TN and PEN, (b) the changes suggest that the classic form of PEN has a different histogenesis than TN, and (c) on histologic grounds, chronic minor trauma could not be excluded as an etiologic factor for PEN.


Reevaluation of host corneal tissue 1955 to 1970: Calcofluor white staining for occult acanthamoebic infection. The incidence of acanthamoebic keratitis before 1974 is not known. A retrospective study was conducted to understand better the early epidemiology of this infection. Host corneal tissue from therapeutic penetrating keratoplasties submitted to the Stanford Eye Pathology Laboratory from 1955 to 1970 were stained with Calcofluor white and observed by fluorescence microscopy. We reviewed 197 cases, and no misdiagnosed cases of acanthamoebic infection were found.


Relative sparing of nitric oxide synthase-containing neurons in the hippocampal formation in Alzheimer's disease. Nitric oxide (NO) is an endogenous neuromodulator that may mediate neurotoxic effects of glutamate. NO-synthesizing neurons are, however, resistant to NO- and glutamate-induced neurotoxicity. We now show that NO synthase neurons are selectively spared in patients with Alzheimer's disease, even in a severely affected region of the brain such as the hippocampal formation.


Delayed surgery for congenital diaphragmatic hernia: neurodevelopmental outcome in later childhood. The long term neurodevelopmental outcome was assessed in 23 survivors born with congenital diaphragmatic hernia who had been managed by an elective delay in surgical repair after a period of stabilisation. This cohort was treated in one neonatal surgical unit between 1983 and 1989 by a single team of surgeons and anaesthetists. All children underwent comprehensive neurological, developmental, and anthropometric assessment at a mean age of 56 (range 18-94) months. Two children (9%) had major disability (one with hemiplegia and one with a lower limb monoplegia) and two further children had minor disabilities (one had partial sightedness and squint, the other squint only). The mean developmental quotient (DQ) for the group was 108 (SD 10.8) and none had developmental delay (defined as DQ < 70). Infants who had spent more time in hospital, or had had a longer duration of ventilation, tended to have lower weights and lower occipitofrontal circumference centiles in later childhood. Preoperative stabilisation and delayed surgery for congenital diaphragmatic hernia is not associated with an impaired neurodevelopmental outcome.


Effect of hypothermia on pancreatic acinar cells in rats. This study was designed to evaluate the effects of direct pancreatic surface cooling on the exocrine pancreas. We measured the changes in serum amylase levels, pancreatic water, amylase and cathepsin B as a lysosomal enzyme, content, histological changes of acinar cells, and the subcellular distribution of cathepsin B after 1-2- and 3-hours of direct pancreatic cooling in rats. In addition, we evaluated the in-vivo amylase and cathepsin B output stimulated by caerulein, in-vitro lysosomal and mitochondrial fragility as well as the pancreatic adenylate energy metabolism. 2-hours cooling showed slight yet significant changes, but 3-hours cooling caused most significant changes including hyperamylasemia, increased pancreatic amylase content and very mild histological changes. Furthermore, 3-hours cooling caused a remarkable redistribution of cathepsin B activity from the lysosomal fraction to the heavier zymogen fraction, and colocalization of the lysosomal enzyme with the digestive enzyme, the impaired amylase and cathepsin B output into pancreatic juice stimulated by caerulein as well as the accelerated fragility of lysosomes and mitochondria, and impaired pancreatic adenylate energy metabolism. These results indicate the impaired exocrine pancreatic functions induced by direct pancreatic cooling injury induced by cooling as shown in the other models of experimental pancreatitis. Moreover, this cooling model of pancreatitis seems to be useful in understanding the early events in the pathogenesis of acute pancreatitis, and we must take these "cold" injuries of exocrine pancreas into considerations, particularly in the pancreas transplantation and in other major abdominal surgeries where the pancreas is exposed to cooling.


Fragility of subcellular organelles induced by pancreatic duct obstruction in rabbits. The effects of short-term (6 hours) pancreatic duct obstruction on the possible secretion of lysosomal enzyme into pancreatic juice and subcellular lysosomal and mitochondrial fragility were investigated in rabbits. Caerulein stimulated the secretion of amylase and cathepsin B into pancreatic juice in controls. Blockage of the pancreatic duct for 6 hours caused a significant decrease of amylase and cathepsin B output into pancreatic juice, and a significant rise in portal serum amylase and cathepsin B levels, and pancreatic amylase content and an accelerated leakage of cathepsin B from lysosomes and malate dehydrogenase from mitochondria in in-vitro preparations, as well as redistribution of cathepsin B in acinar cells. These changes tended to continue for up to 12 hours after removal of the pancreatic duct obstruction. These results indicate that under physiological conditions, lysosomal enzymes are secreted into the pancreatic juice in response to stimulation by gut hormones and that increased lysosomal fragility, mitochondrial fragility and impaired pancreatic energy charge levels are closely related to the pathogenesis of pancreatic injury in this model. Moreover, zymogen colocalized with lysosomal enzyme after duct obstruction was secreted into pancreatic juice in increased amount together with digestive enzymes; these findings suggest that lysosomal enzymes play important pathophysiological roles in pancreatic juice.


[Effects of duct reconstruction and estrogen treatment on endocrine and exocrine functions of fibrotic pancreas caused by ligation of the pancreatic duct in dogs]. After fibrotic pancreas was produced by ligation of the pancreatic ducts in mongrel dogs, effects of duct reconstruction and estrogen treatment on endocrine and exocrine function of the fibrotic pancreas were investigated. 1) K value in IV-GTT and sigma IRG in IV-ITT were significantly improved after duct reconstruction in group with estrogen treatment, especially K value was significantly improved as compared with that in group without estrogen treatment. 2) Total volume of pancreatic secretion, total amylase output and maximal bicarbonate concentration in P-S tests were improved significantly after duct reconstruction in the group with estrogen treatment, but not significantly improved in the group without estrogen treatment. 3) Histological changes of the islet and pancreatic fibrosis, caused by the pancreatic duct ligation, were also significantly improved after duct reconstruction in the group with estrogen treatment, as compared with those in the group without estrogen treatment.


Development and characterization of Porphyromonas gingivalis-specific rat T-cell clones. Porphyromonas gingivalis has been implicated as a major pathogen in periodontitis. To determine the role of T cells in the regulation of this disease, a method was developed for the generation and characterization of rat T-cell clones with antigen specificity to P. gingivalis whole cells. The clones studied so far demonstrated a T-helper (Th) phenotype W3/13+, W3/25+, OX8- and OX22-. These T-cell clones proliferated in vitro in response to P. gingivalis, but not to other bacteria (Prevotella intermedia, Actinobacillus actinomycetemcomitans, Wolinella recta, Fusobacterium nucleatum, Streptococcus sanguis). Limiting dilution analysis showed W3/25+, OX8- T cells preferentially respond to P. gingivalis, rather than W3/25-, OX8+ T cells. P. gingivalis-reactive W3/25+ T cells belonged to the OX22- population, suggesting that the OX22- T cells may represent memory cells. All clones tested produced interferon gamma, but not interleukin 2. The cloned T-cell F1 significantly enhanced P. gingivalis-specific antibody production (p < 0.03). The availability of these cloned T cells should bring new insight into the mechanism by which T cells regulate oral health and periodontal disease.


Purification and structure of human liver aspartylglucosaminidase. We have recently diagnosed aspartylglucosaminuria (AGU) in four members of a Canadian family. AGU is a lysosomal storage disease in which asparagine-linked glycopeptides accumulate to particularly high concentrations in liver, spleen and thyroid of affected individuals. A lesser accumulation of these glycopeptides is seen in the kidney and brain, and they are also excreted in the urine. The altered metabolism in AGU results from a deficiency of the enzyme aspartylglucosaminidase (1-aspartamido-beta-N-acetylglucosamine amidohydrolase), which hydrolyses the asparagine to N-acetylglucosamine linkages of glycoproteins and glycopeptides. We have used human liver as a source of material for the purification of aspartylglucosaminidase. The enzyme has been purified to homogeneity by using heat treatment, (NH4)2SO4 fractionation, and chromatography on concanavalin A-Sepharose, DEAE-Sepharose, sulphopropyl-Sephadex, hydroxyapatite, DEAE-cellulose and Sephadex G-100. Enzyme activity was followed by measuring colorimetrically the N-acetylglucosamine released from aspartylglucosamine at 56 degrees C. The purified enzyme protein ran at a 'native' molecular mass of 56 kDa in SDS/12.5%-PAGE gels, and the enzyme activity could be quantitatively recovered at this molecular mass by using gel slices as enzyme source in the assay. After denaturation by boiling in SDS the 56 kDa protein was lost with the corresponding appearance of polypeptides alpha,beta and beta 1, lacking enzyme activity, at 24.6, 18.4 and 17.4 kDa respectively. Treatment of heat-denatured enzyme with N-glycosidase F resulted in the following decreases in molecular mass; 24.6 to 23 kDa and 18.4 and 17.4 to 15.8 kDa. These studies indicate that human liver aspartylglucosaminidase is composed of two non-identical polypeptides, each of which is glycosylated. The N-termini of alpha,beta and beta 1 were directly accessible for sequencing, and the first 21, 26 and 22 amino acids respectively were identified.


Application of active-phase plot to the kinetic analysis of lipoxygenase in reverse micelles. A new plot for explaining the complex expression of the enzymic activity in reverse micelles has been developed as an extension of the theoretical model described by our group [Bru, Sánchez-Ferrer & García-Carmona (1990) Biochem. J. 268, 679-684]. The plot describes the changes in the relative volume, amount of enzyme (mumoles), enzyme concentration (microM) and substrate concentration (microM) in the phase where the enzyme is active. To illustrate the usefulness of this plot, the complex activity of soya bean lipoxygenase in reverse micelles acting on its interfacial substrate, octadecadienoic acid, was studied. It showed the key parameters ruling the activity profiles of lipoxygenase with respect to micelle size (omega 0), micelle concentration (theta) and the substrate/surfactant molar ratio (rho), which have never been described before.


Formyl peptides and ATP stimulate Ca2+ and Na+ inward currents through non-selective cation channels via G-proteins in dibutyryl cyclic AMP-differentiated HL-60 cells. Involvement of Ca2+ and Na+ in the activation of beta-glucuronidase release and superoxide production. In human neutrophils, the chemotactic peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) induces increases in the intracellular free Ca2+ concentration ([Ca2+]i) with subsequent activation of beta-glucuronidase release and superoxide (O2-) production. Results from several laboratories suggest that the increase in [Ca2+]i is due to activation of non-selective cation (NSC) channels. We studied the biophysical characteristics, pharmacological modulation and functional role of NSC channels in dibutyryl cyclic AMP (Bt2cAMP)-differentiated HL-60 cells. fMLP increased [Ca2+]i by release of Ca2+ from intracellular stores and influx of Ca2+ from the extracellular space. fMLP also induced Mn2+ influx. Ca2+ and Mn2+ influxes were inhibited by 1-(beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl)-1H-imidazole hydrochloride (SK&F 96365). Under whole-cell voltage-clamp conditions, fMLP and ATP (a purinoceptor agonist) activated inward currents characterized by a linear current-voltage relationship and a reversal potential near 0 mV. NSC channels were substantially more permeable to Na+ than to Ca2+. SK&F 96365 inhibited fMLP- and ATP-stimulated currents with a half-maximal effect at about 3 microM. Pertussis toxin prevented stimulation by fMLP of NSC currents and reduced ATP-stimulated currents by about 80%. Intracellular application of the stable GDP analogue, guanosine 5'-O-[2-thio]diphosphate, completely blocked stimulation by agonists of NSC currents. In excised inside-out patches, single channel openings with an amplitude of 0.24 pA were observed in the presence of fMLP and the GTP analogue, guanosine 5'-O-[3-thio]triphosphate. The bath solution contained neither Ca2+ nor ATP. The current/voltage relationship was linear with a conductance of 4-5 pS and reversed at about 0 mV. fMLP-induced beta-glucuronidase release and O2- production were substantially reduced by replacement of extracellular CaCl2 or NaCl by ethylenebis(oxyethylenenitrilo)tetra-acetic acid and choline chloride respectively. In the absence of Ca2+ and Na+, fMLP was ineffective. SK&F 96365 inhibited fMLP-induced beta-glucuronidase release and O2- production in the presence of both Ca2+ and Na+, and in the presence of Ca2+ or Na+ alone. NaCl (25-50 mM) enhanced the basal and absolute extent of fMLP-stimulated GTP hydrolysis of heterotrimeric regulatory G-proteins in HL-60 membranes. The order of effectiveness of salts in enhancing GTP hydrolysis was LiCl > KCl > NaCl > choline chloride.(ABSTRACT TRUNCATED AT 400 WORDS)


A 109-amino-acid C-terminal fragment of Alzheimer's-disease amyloid precursor protein contains a sequence, -RHDS-, that promotes cell adhesion. Amyloid beta (A beta), the major constituent of the fibrils composing senile plaques and vascular amyloid deposits in Alzheimer's disease (AD) and related disorders, is a 39-42-residue self-aggregating degradation peptide of a larger multidomain membrane glycoprotein designated amyloid precursor protein (APP). An array of biological functions has been assigned to different APP domains, including growth regulation, neurotoxicity, inhibitory activity of serine proteinases and promotion of cell-cell and cell-matrix interactions. A beta is generated through an as-yet-unknown catabolic pathway that by-passes or inhibits the cleavage of APP within the A beta sequence. We have identified a 16 kDa intermediate APP C-terminal fragment containing A beta in leptomeningeal vessels of aged normal individuals and AD patients by means of its immunoreactivity with a panel of four different anti-(APP C-terminal) antibodies, indicating a different pathway of APP processing. Previous studies have indicated that the APP C-terminal domain is the most likely to be involved in cell-matrix interactions. A 109-amino-acid construct C109 with a sequence analogous to the C-terminal of APP (positions 587-695 of APP695), similar in length and immunoreactivity to the 16 kDa fragment, was found to promote cell adhesion. By use of synthetic peptides, this activity was initially located to the extracellular 28 residues of A beta. Inhibition studies demonstrated that the sequence RHDS (amino acids 5-8 of A beta, corresponding to residues 601-604 of APP695 was responsible for the adhesion-promoting activity. The interaction is dependent on bivalent cations and can be blocked either by the tetrapeptides RHDS and RGDS or by an anti-(beta 1 integrin) antibody. Thus, through integrin-like surface receptors, APP or its derivative proteolytic fragments containing the sequence RHDS may modulate cell-cell or cell-matrix interactions.


Observations on the inhibition of HIV-1 reverse transcriptase by catechins. The sensitivity and specificity of the inhibition of HIV-1 reverse transcriptase by various catechins have been examined. As previously reported, (-)epicatechin 3-gallate inhibits the viral polymerase. However, it is noted here that this inhibition is not observed in the presence of either serum albumin or Triton X-100. Other catechins behave similarly to (-)epicatechin 3-gallate in that they inhibit polymerase activity only in the absence of these reagents. Additionally, other DNA polymerases are inhibited to a similar degree by (-)epicatechin 3-gallate. Taken cumulatively, these results suggest that these catechins, and in particular (-)epicatechin 3-gallate, bind with no apparent selectivity and that the observed inhibition of HIV-1 reverse transcriptase is non-specific in nature.


The effect of mevalonic acid deprivation on enzymes of DNA replication in cells emerging from quiescence. We have investigated the biochemical basis of the mevalonate dependence of DNA replication. Stimulating quiescent rat hepatoma cells to proliferate in the presence of compactin, an inhibitor of mevalonate synthesis, prevented DNA replication in as many as 80% of these cells. The percentage of cells that failed to replicate DNA increased with the increased duration of quiescence. Aphidicolin-sensitive DNA polymerase and ornithine decarboxylase activities were selectively decreased in compactin-treated cells, whereas RNA and protein synthesis, the level of dihydrofolate reductase and aphidicolin-resistant DNA polymerase activity were unaffected. Adding putrescine, the product of ornithine decarboxylase and the precursor of other polyamines, did not restore DNA replication. Our results demonstrate that the decreased activities of at least two DNA-replication enzymes are among the proximal causes of the failure of mevalonate-deprived cells to synthesize DNA. More importantly, our data indicate that a mevalonate-dependent factor(s) is progressively depleted during quiescence, and that inability to resynthesize this factor(s) may be the ultimate cause of the failure of resting cells to replicate DNA when stimulated to proliferate in the absence of mevalonate.


Immunological discrimination of beta-tubulin isoforms in developing mouse brain. Post-translational modification of non-class-III beta-tubulins. Individual beta-tubulin isoforms in developing mouse brain were characterized using immunoblotting, after preceding high-resolution isoelectric focusing, with monoclonal antibodies against different structural regions of beta-tubulin. Some of the antibodies reacted with a limited number of tubulin isoforms in all stages of brain development and in HeLa cells. The epitope for the TU-14 antibody was located in the isotype-defining domain and was present on the beta-tubulin isotypes of classes I, II and IV, but absent on the neuron-specific class-III isotype. The data suggest that non-class-III beta-tubulins in mouse brain are substrates for developmentally regulated post-translational modifications and that beta-tubulins of non-neuronal cells are also post-translationally modified.


Heparin increases chromatin accessibility by binding the trypsin-sensitive basic residues in histones. Recent evidence indicates that chromatin accessibility to transcription factors is of regulatory significance. The polyanion heparin is known to increase chromatin accessibility to DNAase I and to stimulate both RNA and DNA synthesis. In the present study, chromatin structure and its modification by polyanions were examined by using trypsin and micrococcal nuclease as probes. Both heparin and poly(glutamic acid) were found to be equivalent to trypsin digestion of histones in their ability to increase nuclease accessibility in chromatin. However, no increase in nuclease accessibility was observed when trypsin-digested chromatin was further treated with heparin, indicating that polyanions and trypsin are not additive in their effects on chromatin accessibility. Moreover, sucrose-gradient analysis demonstrated that heparin binds tightly to intact nucleosomes but not to trypsin-digested nucleosomes. These data suggest that polyanions interact predominantly with the trypsin-sensitive lysine and arginine residues in histone H1 and the N-terminal segments of the core histones. The possible relevance of these results to the chromatin structure of actively transcribed regions is discussed.


Phosphatidylinositol-3-kinase is a non-tyrosine phosphorylated member of the insulin receptor signalling complex. In rat HTC cells expressing a large number of human insulin receptors, insulin stimulated phosphatidylinositol-3-kinase (PI-3-kinase) activity. This activity was more effectively immunoprecipitated with anti-phosphotyrosine antibody (alpha-PY) than with anti-insulin receptor antibody (alpha-IR), suggesting that PI-3-kinase was not directly associated with the insulin receptor. alpha-PY immunoprecipitable PI-3 kinase activity, which was regulated by insulin, corresponded to a small pool of the total cellular PI-3-kinase activity. PI-3-kinase was not directly tyrosine phosphorylated by insulin treatment. A comparison of both catalytic activity and content of PI-3-kinase in alpha-PY immunoprecipitates indicated that after insulin treatment PI-3-kinase activity was enhanced by its association with tyrosine phosphorylated proteins. These studies suggest therefore that PI-3-kinase is a non-tyrosine phosphorylated member of the insulin receptor signalling complex.


Post-transcriptional regulation of insulin-like growth factor binding protein-2 mRNA in diabetic rat liver. IGFBP-1 and IGFBP-2 mRNAs are increased in the livers of streptozotocin-diabetic rats. A corresponding increase is observed in transcription of the IGFBP-1 but not the IGFBP-2 gene, indicating that the increase in steady-state levels of IGFBP-2 mRNA is a post-transcriptional effect. IGFBP-1 and IGFBP-2 mRNAs also differ in the rapidity of their response to insulin treatment: hepatic IGFBP-1 mRNA is normalized within 1 h, IGFBP-2 mRNA decreases more slowly. These differences suggest that IGFBP-2 may provide more chronic adaptation to metabolic change than IGFBP-1.


Bilayers containing calcium ionophore A23187 form channels. For the first time, based on bilayer membrane conductance experiments, it has been shown that A23187, a carboxylic calcium ionophore, incorporated in lipid bilayers gives single channel currents similar to the well known gramicidin channel. The current characteristics indicate the possibility that the transmembrane ion transport by this important calcium ionophore is initially by a carrier mechanism but with time is by a channel or pore mechanism due to the aggregation of the molecule in a lipid matrix.


A mutation generating a stop codon in the alpha-L-fucosidase gene of a fucosidosis patient. Fucosidosis is an autosomal recessive, lysosomal storage disease featured by deficient activity of alpha-L-fucosidase. Lymphoid cell lines from a fucosidosis patient (JT) and a healthy individual (control) contained alpha-L-fucosidase mRNA of the same size, 2.3 Kb, as determined by Northern blot analysis. cDNA was prepared from alpha-L-fucosidase mRNA of JT and control cells and each cDNA was amplified by the polymerase chain reaction. Direct DNA sequencing of the amplified products revealed a single mutation in JT, a G1141-->T transition. This changed the codon (GAA) for Glu-375 to a stop codon (UAA). Amplification and sequencing of the area containing the G1141-->T transition in genomic DNA of JT and control cells demonstrated that the mutation was homozygous in JT. Analysis of cDNA and genomic DNA derived from lymphoid cells of mother JT revealed her to be heterozygous (G and T) at position 1141. The G1141-->T mutation is probably responsible for disease in JT.


Identification of a novel tyrosine kinase receptor-like molecule in neuroblastomas. Growth factor receptors are important determinants of both normal and abnormal cell growth. We have now used degenerate primers designed from conserved tyrosine kinase domains to identify and clone a novel receptor-like molecule (designated Nbtk-1) from a NB41 mouse neuroblastoma cell line. Nbtk-1 is related to the met proto-oncogene family of tyrosine kinase receptors. Transcripts of approximately 2.1 and 2.6 kb have been found in mouse cell lines and one transcript of approximately 3 kb in human cell lines and in a wide range of primary human tumors, such as neuroblastomas, primitive neuroectodermal tumors (PNETs), Wilms' tumors, and melanomas and in the corresponding normal human tissues. These observations suggest that Nbtk-1 may have important roles in normal and tumor cell growth.


Duck hepatitis B virus polymerase produced by in vitro transcription and translation possesses DNA polymerase and reverse transcriptase activities. Activities of the hepadnavirus polymerases are known to include those of DNA polymerase, reverse transcriptase and RNase H. To date, it has been difficult or impossible to clone and express the product as an active enzyme. In this study, full length capped RNA encoding Duck Hepatitis B Virus (DHBV) polymerase was produced by in vitro transcription from a T7 promoter. The RNA was translated in a rabbit reticulocyte lysate system and produced an 35S-Methionine labelled 79 Kd band on SDS-polyacrylamide gel electrophoresis. The translation product showed DNA polymerase and reverse transcriptase activities on exogenous templates (respectively) of DNA or RNA with random DNA hexamer primers. The same RNA transcripts were also microinjected into Xenopus oocytes, but appeared to be toxic and gave no detectable translation product. Production of hepadnavirus polymerase by in vitro transcription/translation may provide a useful tool for structure/function and pharmacological studies on this important group of polymerases.


Epidermal growth factor enhances insulin-like growth factor binding protein-1 synthesis in human hepatoma cells. Epidermal growth factor (EGF) was found to induce a rapid 2-fold increase in the amount of insulin-like growth factor binding protein-1 (IGFBP-1) mRNA in human hepatoma Hep2G cells, and this was accompanied by a 2-fold increase in IGFBP-1 secretion. A protein synthesis inhibitor cycloheximide (CHX) caused a 2-3-fold increase in the amount of IGFBP-1 mRNA, which could be accounted for the observed stabilization in decay of IGFBP-1 mRNA after CHX treatment. In nuclear run-on transcription experiments neither EGF nor CHX affected the transcription rate of the IGFBP-1 gene. It is concluded that EGF increases IGFBP-1 secretion rapidly by enhancing IGFBP-1 mRNA accumulation, and the addition of a protein synthesis inhibitor results in a specific increment of IGFBP-1 mRNA, suggesting that a labile protein repressor protein is involved in the turnover IGFBP-1 mRNA.


ET-1 released histamine from guinea pig pulmonary but not peritoneal mast cells. Endothelin(ET)-1 triggered histamine release of mast cells from pulmonary tissue but not from the peritoneal cavity of guinea pigs. The observed difference in response to ET-1 was attributable to a quantitative difference in ET-1 binding sites between both cells. The concentrations of ET-1 required for half maximal release of histamine and half maximal binding of [125I]ET-1 were approximately 0.05 and 0.08 nM, respectively. The release of histamine by ET-1 was a Ca(2+)-dependent but not a cytotoxic process. These observations, taken together, suggest that ET-1 induces histamine release from mast cells in a receptor-dependent fashion.


Isolation of a cDNA clone for beta 1-4 galactosyltransferase from embryonic chicken brain and comparison to its mammalian homologs. Based on the observed biochemical and immunological similarities between bovine beta 1-4 galactosyltransferase (GalT) and a developmentally regulated galactosyltransferase (GalT-4; UDP-Gal:Lc3 beta 1-4 GalT) from embryonic chicken brain, a genetic similarity between the two enzymes has been postulated. To test our hypothesis, we have employed a reverse transcription-polymerase chain reaction (RT-PCR)based approach and isolated a approximately 600 bp cDNA clone from embryonic chicken brain mRNA. Our results indicate that, within the approximately 600 bp fragment, the avian cDNA nucleotide sequence is 74% homologous to the human beta 1-4 galactosyltransferase cDNA. Similarity and identity between the predicted amino acid sequences of the two galactosyltransferases are 75% and 61%, respectively. Similar to its mammalian counterparts, the embryonic chicken brain galactosyltransferase gene appears to encode multiple mRNA transcripts (2.3 and 1.4 kb) and shows multiple bands on a Southern blot (18.6, 12.9, 10.5 and 3.7 kb) indicating that the avian gene is either polyexonic and/or it belongs to a multiple gene family.


Induction of human microvascular endothelial tubular morphogenesis by human keratinocytes: involvement of transforming growth factor-alpha. Transforming growth factor-alpha(TGF-alpha), homologous to epidermal growth factor(EGF), is closely involved in hyperproliferation of human keratinocytes. Psoriasis is a common hyperproliferative skin disease characterized by hyperproliferation of keratinocytes and abnormal development of dermal capillary networks. In this study, we have examined whether keratinocytes could enhance angiogenesis. TGF-alpha or EGF efficiently stimulated formation of tubular-like structures of human omental microvascular endothelial(HOME) cells in type I collagen gels. Human keratinocytes produced TGF-alpha. To examine whether co-cultured keratinocytes could induce tubulogenesis of HOME cells in collagen gel, we have developed a co-culture system with human keratinocytes. Surprisingly, there appeared new development of many tubular-like structures of HOME cells in collagen gels when co-cultured with keratinocytes. This keratinocytes-dependent tubulogenesis was almost completely blocked when anti-TGF-alpha-antibody was present. The TGF-alpha molecules derived from keratinocytes appeared to enhance tubulogenesis of human microvascular endothelial cells. We propose the hypothesis that secretory TGF-alpha from human keratinocytes may promote an autocrine loop to proliferate the skin keratinocytes and also a paracrine loop to induce the skin angiogenesis.


Molecular cloning and characterization of the platelet-activating factor receptor gene expressed in the human heart. PAF decreases cardiac contractility and blood pressure. To characterize the cardiac PAF receptor, we screened a human ventricular cDNA library in a low stringency condition, using a PCR product derived from guinea pig lung PAF receptor as a probe. Four clones were obtained and named HV1-4. In Xenopus oocytes injected with cRNA derived from HV3 or 4 but not from HV1 or 2, PAF elicited a Ca(2+)-activated Cl- current. HV3 and HV4 were duplicate clones, encoding a 342 amino-acid polypeptide which was identical to that of the human leukocyte PAF receptor. However, a portion of the 5' untranslated region of HV3 (or 4) was different from that of the leukocyte receptor cDNA. Northern blotting of human ventricles and atria using the HV3 insert showed a single band of approximately 4 kb. These results suggest a tissue-specific translational mechanism responsible for regulation of the expression of the PAF receptor mRNA in these tissues.


Purification and characterization of a novel growth factor (FF-GF) synthesized by a rat hepatoma cell line, FF101. A rat hepatoma cell line, FF101, established in serum-free, protein-free medium, synthesizes a growth factor(FF-GF). FF-GF was purified by gel filtration chromatography, cation-exchange chromatography and isoelectric focusing. Purified FF-GF was revealed as a single band on SDS-gel electrophoresis and its molecular weight was estimated to be 70 KDa. FF-GF stimulated DNA synthesis of various cells from different origins. The growth-promoting activities of FF-GF were abolished by treating with protease, dithiothreitol, acid and heating, whereas its activity was not inhibited by antibodies against acidic and basic fibroblast growth factors. These results indicate that FF-GF is a novel growth factor.


Growth stimulation of rat fetal hepatocytes in response to hepatocyte growth factor: modulation of c-myc and c-fos expression. Hepatocyte growth factor, which is a potent growth factor for primary cultured adult hepatocytes, strongly stimulated DNA synthesis of rat fetal (20-day of gestation) hepatocytes. Its mitogenic capacity, measured as (3H)-thymidine incorporation into acid precipitable material was dose dependent, being detectable at 1 ng/ml and maximal at 5 ng/ml. Over 15% of the cells entered into S-phase and mitosis as judged by flow cytometric analysis of the cell cycle. HGF had additive effects with transforming growth factor-alpha, whereas transforming growth factor-beta strongly inhibited DNA synthesis of fetal hepatocytes stimulated by HGF. HGF induced c-fos and c-myc expression in a time-dependent manner, with a maximum at 30 min for c-fos and 8 h for c-myc. These results suggest that HGF may act as a proliferative factor during fetal liver growth.


Chromosomal band assignments of the genes encoding human FKBP12 and FKBP13. Human FKBP12 and FKBP13 are encoded by distinct genes designated FKBP1 and FKBP2, respectively. Human FKBP1 was previously characterized. The characterization of human FKBP2 is described. FKBP2 is three kb in length and contains six exons. Fluorescence in situ hybridization of FKBP1 and FKBP2 genomic probes to metaphase chromosomes localized FKBP1 to human chromosome 20 band p13 and FKBP2 to human chromosome 11 band q13.1-q13.3.


Potent synergism between vascular endothelial growth factor and basic fibroblast growth factor in the induction of angiogenesis in vitro. Vascular endothelial growth factor (VEGF), also known as vascular permeability factor or vasculotropin, is a recently characterized endothelial-specific mitogen which is angiogenic in vivo. Here we demonstrate that VEGF is angiogenic in vitro: when added to microvascular endothelial cells grown on the surface of three-dimensional collagen gels, VEGF induces the cells to invade the underlying matrix and to form capillary-like tubules, with an optimal effect at approximately 2.2nM (100ng/ml). When compared to basic fibroblast growth factor (bFGF) at equimolar (0.5nM) concentrations, VEGF was about half as potent. The most striking effect was seen in combination with bFGF: when added simultaneously, VEGF and bFGF induced an in vitro angiogenic response which was far greater than additive, and which occurred with greater rapidity than the response to either cytokine alone. These results demonstrate that like bFGF, VEGF induces an angiogenic response via a direct effect on endothelial cells, and that by acting in concert, these two cytokines have a potent synergistic effect on the induction of angiogenesis in vitro. We suggest that the synergism between VEGF and bFGF plays an important role in the control of angiogenesis in vivo.


Down regulation of CD4 expression in cultured microglia by immunosuppressants and lipopolysaccharide. Microglia, brain macrophages, are thought to be the primary target of HIV-1 infection in the brain, because they exclusively express the CD4 antigen which is effectively used for viral entry. The expression of CD4 mRNA in cultured microglia could be detected by the reverse-PCR method. Using this and immunohistochemical staining, we found that the immunosuppressants cyclosporin A and FK506 decreased CD4 expression in cultured murine microglia without causing any significant decrease in cell viability. FK506 was more potent than cyclosporin A. Lipopolysaccharide also decreased CD4 mRNA expression in microglia. The effects of immunosuppressants and lipopolysaccharide seemed to be specific for microglia since these chemicals did not alter the CD4 expression in lymphocytes or peritoneal macrophages. These agents, if modified to pass through the blood-brain barrier, may prevent viral spread of HIV-1 infection in the central nervous system and the AIDS-dementia complex.


Comparison of gene expression of extracellular matrix molecules in brain microvascular endothelial cells and astrocytes. By use of random-primed cDNA probes the expression of extracellular matrix molecules in cerebral microvascular endothelial cells (cEC) and in astrocytes from mouse brain was examined. Two phenotypically different batches of cloned cEC were used. Expression of major adhesive ECM molecules, constituting the endothelial basement membrane (i.e., fibronectin, laminin A, B and collagen IV) and of other attachment factors, such as SPARC (osteonectin), tenascin and thrombospondin 1, was examined. We have demonstrated that cEC of different morphology display variations in the expression of fibronectin (FN), thrombospondin 1 (TSP1) and collagen IV (C IV). Astrocytes were shown to contain FN, TSP1, TN and SPARC mRNA. Unexpectedly, SPARC mRNA could not be detected in any of the capillary endothelial cells examined. Therefore, we suggest that astrocytes are likely to be involved in endothelial differentiation and function in the central nervous system via ECM molecule secretion.


Molecular cloning of the carboxy terminus of a canine tracheobronchial mucin. A cDNA library constructed from canine tracheal mRNA was screened with polyclonal antiserum specific to canine tracheal apomucin (CTM-A). Eight antibody reactive clones were isolated and purified to clonality. One of the clones, designated pCTM-A, had a 1.7 kb insert and included a single open reading frame with a poly (A)+ tail. The amino acid composition of the encoded protein was consistent with that expected for CTM-A. The fusion protein produced by cloning the 1.7 kb insert in the pMALc expression vector reacted with the purified anti-apomucin CTM-A antibody. Also, polyclonal antibodies raised to the purified protein product encoded by pCTM-A reacted with deglycosylated CTM-A confirming that this clone does indeed code for apomucin CTM-A. This is the first report of a cDNA encoding the C-terminus of a canine tracheal mucin.


Inhibition of repair of bleomycin-induced DNA strand breaks by 2'-deoxycoformycin and its effect on antitumor activity in L5178Y lymphoblasts. We have observed previously that treatment of plateau-phase L5178Y murine lymphoblasts in vitro with 2'-deoxycoformycin plus deoxyadenosine (dCF/dAdo) can inhibit the repair of X-irradiation-induced DNA single-strand breaks (SSB) in these cells and that this effect is associated with synergistic cell kill. In this study we examined the effect of a combination treatment of plateau-phase L5178Y cells with bleomycin (BLM) plus dCF/dAdo. Incubation of BLM-treated cells with dCF/dAdo resulted in significant inhibition of the repair of BLM-induced DNA SSB. However, an additive, but not a synergistic, increase in cell kill was observed when cells were treated with a combination of BLM plus dCF/dAdo.


Alteration of mice L-tryptophan metabolism by the organophosphorous acid triester diazinon. Diazinon [O,O-diethyl O-(2-isopropyl-6-methyl-4- pyrimidinyl)phosphorothioate] altered the formation of several L-tryptophan metabolites associated with the L-kynurenine pathway in mice. Liver kynurenine formamidase was inhibited almost completely by diazinon (10 mg/kg). The enzyme inhibition resulted in reduced L-kynurenine biosynthesis in livers with a concomitant accumulation of N-formyl-L-kynurenine. In contrast to the liver, plasma L-kynurenine increased up to 5-fold in diazinon-treated mice. Consequently, the urinary excretion of xanthurenic acid and kynurenic acid was raised 5- to 15-fold. The revelation of this novel mechanism of diazinon action is an important piece of information needed for a better understanding of the noncholinergic toxicity of organophosphorous acid triesters and methylcarbamates.


Inhibition of RNA polymerase by captan at both DNA and substrate binding sites. RNA synthesis carried out in vitro by Escherichia coli RNA polymerase was inhibited irreversibly by captan when T7 DNA was used as template. An earlier report and this one show that captan blocks the DNA binding site on the enzyme. Herein, it is also revealed that captan acts at the nucleoside triphosphate (NTP) binding site, and kinetic relationships of the action of captan at the two sites are detailed. The inhibition by captan via the DNA binding site of the enzyme was confirmed by kinetic studies and it was further shown that [14C]captan bound to the beta' subunit of RNA polymerase. This subunit contains the DNA binding site. Competitive-like inhibition by captan versus UTP led to the conclusion that captan also blocked the NTP binding site. In support of this conclusion, [14C]captan was observed to bind to the beta subunit which contains the NTP binding site. Whereas, preincubation of RNA polymerase with both DNA and NTPs prevented captan inhibition, preincubation with either DNA or NTPs alone was insufficient to protect the enzyme from the action of captan. Furthermore, the interaction of [14C]captan with the beta and beta' subunits was not prevented by a similar preincubation. Captan also bound, to a lesser extent, to the alpha and sigma subunits. Therefore, captan binding appears to involve interaction with RNA polymerase at sites in addition to those for DNA and NTP; however, this action does not inhibit the polymerase activity.


[The effect of chemical modification on the immunochemical activity of Rm-III neurotoxin from the anemone Radiantus macrodactylus]. A chemical modification was used for studying the organization of antigenic determinants of neurotoxin Rm-III from the sea anemone Radianthus macrodactylus. Immunochemical experiments were performed using competitive enzyme-linked immunosorbent assay (ELISA) with polyclonal antibodies to Rm-III. The modification affected N-terminal amino group of Gly1, Lys4, Arg13, Trp30 residues, a residue in the Lys46-Lys47-Lys48 sequence, two different residues in the Asp6-Asp7-Glu8 sequence in two samples of the toxin, and two disulphide bonds. Only the modification of the disulphide bonds led to a considerable change in the toxin's affinity to antibodies. The modification of Trp30 resulted in two-fold decrease of the toxin concentration necessary for 50% inhibition of the test-system, whereas upon modification of any other amino acid residue this concentration increased but not more than by 2.2 times. It is suggested that Rm-III sequence lacks individual residues which are of great importance for the toxin's antigenic activity, its conformation being of vital importance for the formation of the toxin's antigenic determinants.


The intrinsic migratory capacity of memory T cells contributes to their accumulation in rheumatoid synovium. Mechanisms controlling the infiltration of T cells into rheumatoid synovium have not been fully characterized. These studies were undertaken to investigate the relationship between T cell phenotype and migratory capacity, so as to elucidate mechanisms that might contribute to the accumulation of T cells at inflammatory sites. The characteristics of in vivo migrating cells were studied by dual-immunofluorescence FACS (fluorescence-activated cell sorter) analysis of rheumatoid synovial and peripheral blood T cells. Migratory cells were also characterized using a recently developed in vitro assay, wherein peripheral blood T lymphocytes (PBTL) with the capacity to migrate through endothelial cell monolayers were retrieved and assessed. Migratory CD4+ T cells from rheumatoid arthritis (RA) and normal individuals were characterized as being CD45RA-, CD29bright, CD11abright, L-selectin-, CD54+, and CD58+. Migrating RA PBTL (compared with normal PBTL), however, were significantly enriched in activated HLA-DR+ T cells. RA synovial tissue lymphocytes exhibited a similar phenotype, but with decreased surface density of CD4 and an increase in HLA-DR and VLA-1. RA synovial lymphocytes exhibited a 2-3-fold increase in migratory capacity over normal and RA PBTL: These studies demonstrate the inherent migratory proficiency of CD4+ T cells that express a memory phenotype (CD29bright, CD11abright, and CD58+). In addition, enhanced transendothelial migration was observed for CD4+ T cells that were CD54+ and L-selectin-. These studies demonstrate that the migratory patterns of circulating lymphocytes may be correlated with their surface phenotype and that the intrinsic migratory capacity of memory T cells is one component contributing to their accumulation in the rheumatoid synovium.


Inhibition of synovial fluid T cell proliferation by anti-CD5 monoclonal antibodies. A potential mechanism for their immunotherapeutic action in vivo. Monoclonal antibodies (MAb) directed against the T cell surface molecule CD5 are able to provide accessory stimulatory signals to resting T cells. The potential role of CD5 as an immunoregulatory molecule in inflammatory synovitis was examined. Synovial fluid and peripheral blood T cells of patients with active rheumatoid arthritis (RA) were purified and stimulated with interleukin-2 (IL-2), and the effect of MAb directed against CD5 on IL-2 responsiveness was examined. IL-2-induced proliferation of synovial fluid T cells was strongly inhibited by anti-CD5 MAb, but not by anti-CD28 or anti-CD3 MAb. In RA peripheral blood T cells, MAb directed against CD5, CD3, and CD28 induced IL-2-dependent T cell growth, similar to findings in healthy controls. The difference in activity of anti-CD5 MAb on synovial fluid T cells compared with peripheral blood T cells was not due to different surface expression of CD5. Anti-CD5 has an inhibitory effect on in vivo-activated synovial fluid T cells. The disease-ameliorative effects of anti-CD5 immunotoxin treatment of RA may be partly due to "switching-off" of T cell activation in the joints.


Antilactoferrin antibodies in patients with rheumatoid arthritis are associated with vasculitis. To determine the occurrence of antineutrophil cytoplasmic antibodies (ANCA) and the specificity of these antibodies (Ab) in serum from patients with rheumatoid arthritis (RA) and patients with rheumatoid arthritis complicated by vasculitis (rheumatoid vasculitis [RV]). ANCA was detected with an indirect immunofluorescence test on ethanol-fixed granulocytes. Ab against the cytoplasmic antigens proteinase-3, elastase, lactoferrin (LF), and myeloperoxidase were measured by enzyme-linked immunosorbent assay. ANCA were found in the serum of 43% of 49 patients with RV and in 36% of 50 patients with RA. Anti-LF Ab occurred more frequently in RV patients (45%) than in RA patients (4%), whereas reactivity against the other cytoplasmic antigens did not differe significantly between these groups. Anti-LF Ab in serum of patients with RA may be useful in the diagnosis of vasculitis in RA.


Effect of miltefosine on transplanted methylnitrosourea-induced mammary carcinoma growing in Fischer 344 rats. Miltefosine (hexadecylphosphocholine, HPC, CAS 58066-85-6) was investigated in transplanted primary methylnitrosourea-induced PYH mammary carcinoma of F344 rats. The therapy was performed in the 5th and 10th passage. At first HPC (113 mg/kg body weight) significantly reduced the median tumor volume, but a loss of activity was observed in the 10th passage. To explain the loss of sensitivity and to obtain information on the mechanism of action histology, cytoskeleton and hormone receptor content were investigated. The most important change was observed in the histopathology of the tumor. The initial tubular papillary adenocarcinoma was transformed into a malignant adenoacanthoma with epithelial structure. Vimentin as an endothelial marker of the cytoskeleton was equally expressed in all passages. Cytokeratin was weakly expressed in the earlier passages and intensively present in the late passages. The histopathological change from tubular adenocarcinoma to malignant adenoacanthoma might be caused by an overgrowth of the primary epithelial tumor cells or by a real transformation in the morphological characteristics of the tumor, which may occur during repeated transplantation.


Humoral responses to the immunodominant gag and env epitopes of human T-lymphotropic virus type I among Melanesians. The immune responsiveness to the immunodominant B-cell epitopes of human T-lymphotropic virus type I (HTLV-I), derived from the external envelope glycoprotein (recombinant MTA-1(162-209), synthetic Env-1(194-214), and Env-5(242-257)) and the gag-encoded matrix protein (Gag-1a(102-117)), was analyzed in 19 HTLV-I-seropositive and 51 HTLV I-seroindeterminate Melanesians from Papua New Guinea and the Solomon Islands. The reactivities of seropositive Melanesian specimens to MTA-1 (100%), Env-5 (89%), and Gag-1a (79%) were similar to that seen with U.S. specimens, while reactivity to Env-1 was lower in Melanesian specimens (68%). Minimal reactivity was found to the env epitopes among the 51 HTLV-I-seroindeterminate Melanesians, but 29 (57%) reacted to Gag-1a. The failure to detect HTLV-I gag, pol, env, and tax gene sequences by polymerase chain reaction among the seroindeterminate Melanesians suggests that such reactivities to the Gag-1a epitope represent cross-reacting antibodies with closely related microbial or cellular proteins.


Antibody-dependent cellular cytotoxicity (ADCC) is directed against immunodominant epitopes of the envelope proteins of human immunodeficiency virus 1 (HIV-1). In this study, epitopes of HIV envelope proteins that are involved in ADCC were identified. Peripheral blood mononuclear cells (PBMC) were obtained from adults with asymptomatic HIV infection or early symptoms of AIDS. These PBMC, which were reported to be "armed" in vivo with HIV-specific antibodies, were used as effector cells in 51Cr release assays. Target cells consisted of CD4 lymphocytes from healthy seronegative donors, coated with the IIIB strain of HIV-1 or with one of seven synthetic peptides. Cytotoxicity was detected against CD4 lymphocytes coated with HIV-1 IIIB or with the peptides env aa 507-518, corresponding to the carboxy-terminus of gp120, and env aa 597-611, corresponding to the region of the cysteine loop of gp41. The magnitude of target cell lysis was directly related to the quantity of peptide used. In contrast, target cells coated with the peptide gag aa 129-135, corresponding to the p17/p24 cleavage region of the gag precursor, were not killed. The same immunodominant regions which were involved in ADCC were recognized in enzyme-linked immunoabsorbent assays (ELISA) by the majority of 107 sera from HIV-infected adults. We conclude that the immunodominant epitopes located at the carboxy-terminus of gp120 and the cysteine loop of gp41 serve as recognition structure for antibodies, capable of mediating ADCC against HIV-infected cells.


Basal forebrain neurons and memory: a biochemical, histological, and behavioral study of differential vulnerability to ibotenate and quisqualate. The differential vulnerability of basal forebrain cells to ibotenate (IBO) or quisqualate (QUIS) was investigated in rats. IBO was also coinjected with cystine (CYS) or zinc (Zn). Cortical choline acetyltransferase (ChAT) and glutamate decarboxylase (GAD) activity, neurotensin receptors, and high-affinity choline uptake sites were quantified in conjunction with radioimmunoassays for neurotensin, substance P, and somatostatin; immunocytochemistry for neurotensin-, somatostatin-, Leu-enkephalin-, and ChAT-positive cells; and in situ hybridization histochemistry of somatostatin, substance P, and enkephalin mRNAs. Compared with the performance of controls, continuous alternation performance in a T maze of IBO+Zn or IBO+CYS rats was better than that of IBO rats, whereas the performance of QUIS rats was unimpaired. Of those neurotransmitter systems examined, only ChAT-immunoreactive cells were vulnerable to IBO or QUIS. However, cholinergic cell loss did not correlate with impaired performance.


Biochemical and behavioral effects of a sensorimotor cortex injury in rats pretreated with the noradrenergic neurotoxin DSP-4. The role of the noradrenergic (NE) system in recovery of motor function after sensorimotor cortex (SMCX) injury was investigated. After training on a beam-walking task to assess changes in motor function, animals were given DSP-4 or saline and tested for 2 weeks; both groups then received unilateral SMCX suction ablations. Animals that received DSP-4 were significantly retarded in motor recovery compared with the saline group. At 24 days after injury (after motor recovery), the animals' deficits were significantly reinstated with NE-blocking drugs. DSP-4 significantly depressed NE levels in the hippocampus and cerebellum. A Timm histochemical analysis revealed glutamatergic sprouting in the hippocampus of animals that were pretreated with DSP-4, which suggests the possibility that similar glutamatergic plasticity in other pathways may occur and that excitotoxicity might also play a role after the DSP-4 induced NE deafferentation.


HIV-1 biological phenotype and the development of zidovudine resistance in relation to disease progression in asymptomatic individuals during treatment. To determine which parameters are associated with clinical progression during zidovudine treatment of asymptomatic HIV-1-infected individuals. Twenty-four initially asymptomatic HIV-1-infected individuals were treated with zidovudine and followed until the development of AIDS or for approximately 3 years. HIV-1 phenotype was determined by cocultivation of patient cells with donor lymphocytes, and by a new assay of direct cocultivation with MT-2 cells. Specific mutations in the HIV-1 reverse transcriptase (RT) gene conferring resistance to zidovudine were detected using a selective polymerase chain reaction. Progression to AIDS was more rapid in individuals harbouring syncytium-inducing (SI) viral isolates or showing a conversion from non-syncytium-inducing (NSI) to SI viral isolates. One out of 20 patients who spent a total of 559 months harbouring an NSI phenotype progressed to AIDS, whereas eight out of 12 patients who spent a total of 223 months harbouring an SI phenotype progressed to AIDS (P < 0.001). There was no significant difference between SI and non-SI isolates in the frequency of five mutations causing zidovudine resistance. However, all SI isolates obtained after 2 years of treatment contained mutations in codons 41 and 215 of the RT gene, whereas only five out of 11 (45%) NSI isolates obtained at that time had this combination of mutations. Conversion to the SI phenotype cannot be prevented by zidovudine treatment. The presence or appearance of an SI virus heralded disease progression in zidovudine-treated individuals. Further research is required to investigate the relationship between virus phenotype and development of zidovudine resistance.


An Algerian child homozygous for the M470V polymorphism and for a deletion of two nucleotides in exon 10 of the CFTR gene, shows severe cystic fibrosis symptoms. When screening for the presence of major cystic fibrosis mutations in Algerian cystic fibrosis families by heteroduplex formation, aberrant heteroduplexes were observed for exon 10 in one family. Here we describe the clinical and molecular findings in a severely affected child of this family, homozygous for the 1609delCA and for the M470V polymorphism.


Techniques for studies on growth characteristics of human prostatic cancer cells. The methods described in this article seem to be useful for studies on the growth characteristics of malignant epithelial prostate cells which are still under the influence of healthy cells. Before establishing primary cultures, pieces of tissue were sectioned in such a way that they could be unfolded to obtain a large surface. These pieces were treated enzymatically and then incubated for at least 4-6 weeks. In this time, cells grew or migrated out of the tissue and spread over the surface of the culture flasks. The viable single cells harvested from these primary cultures were characterized flow cytometrically, fractionated by countercurrent centrifugal elutriation, or further incubated above agarose so that they formed three-dimensional spheroids. Cytometric determinations of cellular cytokeratin, vimentin, and DNA were performed before and after incubation. They suggested that the percentages of cytokeratin-positive (epithelial) cells, vimentin-positive cells (fibroblasts), and aneuploid cells remained at levels (in vitro) similar to those within the pieces of tissue used for the culturing experiments, respectively. Since our culture technique allows the propagation of human epithelial prostate cells in vitro as they would grow in vivo under the control of the surrounding tissue, the method should help to investigate which particular treatment of the cells influences the growth of the malignant cells, while they are still surrounded by other cells of the same prostatic organ.


A T-helper cell epitope overlaps a major B-cell epitope in human papillomavirus type 18 E2 protein. Cultivated CD4+ T-helper cells from two patients with cervical adenocarcinoma showed responses to a peptide EKTGILTVTYHSETQRTK derived from an E2 protein of human papillomavirus type 18 (HPV 18), but not to a corresponding HPV 16 peptide (HKSAIVTLTYDSEWQRDQ). Serum antibodies in the HPV 18 peptide were also demonstrated in these patients. The GILT motif resembles a common pattern present in many T-cell epitopes, and is located at the beginning of an 11-amino acid-long A-helix structure close to the carboxyterminal end of HPV 18 E2. We conclude that two epitopes (a T-helper cell epitope and a B-cell epitope) overlap in the HPV 18 E2.


Dextran retention in the rat brain following release from a polymer implant. Intracranial controlled release polymers may improve drug administration to the brain, where therapy is frequently limited due to the low permeability of brain capillaries to therapeutic agents. On the basis of drug transport and elimination rates, we proposed that high molecular weight, water-soluble molecules would be retained in the brain space following release from an intracranial implant. To test this hypothesis, solid particles of different molecular weight fractions of fluorescein isothiocyanate labeled dextran (FITC-dextran; 4 x 10(3) Da (4 kDa) < weight-averaged molecular weight (Mw) < 150 kDa) or fluorescein were uniformly dispersed in matrices of a polyanhydride copolymer synthesized from a fatty acid dimer and sebacic acid in a 50:50 ratio, P(FAD:SA). When incubated in buffered saline, FITC-dextran fractions of 70 kDa Mw were released from the polymer within 48 h; 4 kDa Mw FITC-dextran and fluorescein were released more slowly. Following implantation of P(FAD:SA) matrices containing either 70 kDa Mw FITC-dextran, 4 kDa Mw FITC-dextran, or fluorescein into the brains of normal rats, fluorescent tracers were continuously released into the brain tissue for 30 days. Tracer concentrations within the brain were significantly higher for large molecular weight tracers (70 kDa Mw FITC-dextran >> 4 kDa Mw FITC-dextran > fluorescein). The rate of elimination, kapp, of each tracer from the brain was determined by comparing experimental data with a model describing tracer diffusion/elimination in the brain extracellular space; kapp decreased with increasing molecular weight (fluorescein > 4 kDa Mw FITC-dextran > 70 kDa Mw FITC-dextran).


Expression of cytokeratin and erbB-2 oncoprotein in Paget's disease of the nipple. An immunohistochemical study. The occurrence of epithelial membrane antigen (EMA), low molecular weight cytokeratin (CK) and erbB-2 oncoprotein was investigated in twenty-eight cases of Paget's disease of the nipple (PD) to determine their diagnostic usefulness. The ABC technique with monoclonal antibodies was used on formalin-fixed, paraffin-embedded tissue. The Paget's cells showed positive immunoreactivity for all three antigens investigated in a high percentage of PD. Immunoreactivity for CK and erbB-2 oncoprotein was restricted to the Paget's cells, whereas EMA in some cases also stained the adjacent keratinocytes. Since CK and/or erbB-2 oncoprotein occurred in 93% of the cases, we conclude that demonstration of both antigens is useful in the diagnosis of PD. ErbB-2 oncoprotein was also found to be expressed in a high percentage of the underlying intraductal and invasive carcinomas.


Altered distribution of mitochondria and actin fibers in 3T3 cells cultured on microcarriers. The mitochondria and actin fibers of 3T3 fibroblasts cultured on microcarriers in spinner flasks were visualized using fluorescent stains. In contrast to cells grown on planar surfaces under static or steady laminar flow conditions, cells exposed to higher levels of turbulent agitation do not form actin stress fibers. Greater agitation also leads to a more diffuse appearance of the mitochondria and a wider distribution of them throughout the cytoplasm. This response may indicate damaged mitochondria, as similar results have been reported for chemical toxins.