Renal tumors with pseudohermaphroditism and glomerular disease. From 1972 through 1991, 404 patients with renal tumor (Wilms' tumor and clear cell sarcoma) were seen at the Pediatric Oncology Unit of Hacettepe Children's Hospital. The genital abnormalities and renal diseases in these patients were evaluated retrospectively. Eight patients with renal tumor had genital abnormality and/or renal disease of various types and degrees. One of these patients had a clear cell sarcoma while all the others had Wilms' tumor. Two patients had all components of the Drash syndrome. Two patients with Wilms' tumor had genital abnormality and mild proteinuria. Another two patients had only Wilms' tumor and renal disease. Two patients had only renal tumor and genital abnormality of whom one was the patient with clear cell sarcoma.


Carboplatin and etoposide (CarE) combined with radiotherapy and vincristine, doxorubicin and cyclophosphamide (VAC) in limited small cell lung cancer. A phase II study. Sixty-eight patients with limited small cell lung cancer were treated between April 1988 and October 1990 with combination carboplatin 450 mg/m2 i.v. on day 1 and etoposide 100 mg/m2 i.v. on days 1-3 (CarE) for two courses, followed by thoracic radiotherapy (TRT) 50 Gy, and then vincristine 1 mg/m2, doxorubicin 50 mg/m2 and cyclophosphamide 750 mg/m2 on day one (VAC) for four courses. Prophylactic cranial irradiation (30 Gy) was given to patients with CR after completion of chemotherapy. Sixty patients (89%) achieved an objective response (40% complete responses). The median time to progression was 8.5 months and median survival time 12.1 months. Predicted one- and two-year survival was 50% and 12% respectively. Myelosuppression was the main toxicity, with WHO grade 3 and 4 leukopenia occurring in 32% of VAC courses. There were 5 (7%) treatment-related deaths, all of them during VAC. We conclude that the present combination is active in terms of response rate, but it did not demonstrate any superiority in survival. The frequency of haematological toxicity was substantial during VAC courses.


Ki-1 positive (anaplastic, large cell) lymphoma (case reports and review). Ki-1 positive (anaplastic, large cell) lymphoma is a subgroup of non-Hodgkin lymphomas identified recently by Ki-1 (or BER-H2) (CD 30) monoclonal antibody. The clinicopathological features of two such pediatric cases of lymph node origin described here, and also the available literature emphasize the heterogenous nature of Ki-1 positive lymphomas, in almost every respect. Nevertheless, the Ki-1 antibody serves as an important diagnostic tool to differentiate lymphomas from other anaplastic, large malignancies.


Thyroid hormones and active calcium transport of inside-out red cell membrane vesicles. Thyroid hormones may influence the active transport of Ca2+ across the cell membrane. To test the physiologic relevance of this mechanism, we used inside-out human red cell membrane vesicles as a model of the cell membrane Ca2+ pump. We monitored by spectrophotometric methods the kinetics of the uptake of Ca2+ in the presence of 10(-5)-10(-10) M thyroid hormones or their analogues. Vesicles freed of calmodulin and protein inhibitor(s) of the Ca2+ pump were also obtained. The results are as follows: (1) Thyroxine inhibits the active Ca2+ uptake; (2) this effect antagonizes that of soluble calmodulin; and (3) triiodothyronine and other analogues of the thyroid hormones are less active than thyroxine. We conclude that the thyroid hormones may influence cell Ca2+ homeostasis by direct action on the Ca2+ pump.


Erythrocyte transmembrane Na and K fluxes in pseudohypoaldosteronism. Pseudohypoaldosteronism (PHA) is a disease characterized by hyponatremia, hypotension, and dehydratation, despite the presence of hyperreninemic hyperaldosteronism. The membrane-bound Na,K ATPase activity and the transmembrane Na and K transport systems have been studied in vitro in red blood cells of two subjects, son and mother, affected by pseudohypoaldosteronism with different degrees of clinical involvement. Both parameters were significantly altered suggesting that the refractory response to mineralocorticoids is detectable, not only in kidneys and salivary and sweat glands, but also in red blood cells. Since pseudohypoaldosteronism, in its asymptomatic form, may be much more common than expected, we suggest the use of the tests described herein as a practical approach to the early diagnosis of pseudohypoaldosteronism in the investigation of sodium wasting syndromes.


Structural framework for the protein kinase family. In this review, we have summarized the general structural features of the catalytic subunit of cAMP-dependent protein kinase, emphasizing those features that will very likely be conserved in all members of the protein kinase family. The overall secondary structure of the catalytic core will probably be conserved throughout the catalytic core, as will the active site regions associated with MgATP binding and catalysis. The mechanisms for activation and the role of protein phosphorylation are unique for each kinase. The structure of the catalytic subunit now provides a general framework for modeling other protein kinases. Although this is no substitute for a crystal structure for each protein kinase, this one structure, nevertheless, does provide major insights to the molecular organization of each of these enzymes.


Lymphoproliferative responses to human papillomaviruses in patients with cutaneous warts. In vitro lymphoproliferative responses of peripheral blood mononuclear cells (PBMC) from patients with cutaneous warts, caused by infection with human papillomavirus type 1 (HPV-1) or type 2 (HPV-2), were assayed during the course of treatment. Purified HPV-1 and HPV-2 were used as antigens, as well as herpes simplex virus (HSV) and concanavalin A (Con A). All patients had normal percentages of subsets within the PBMC population and normal lymphoproliferative responses to Con A, and those with a clinical history of HSV infections had positive lymphoproliferative responses to HSV. Responses to both HPV antigens were poor. Only 10 of 100 assays of PBMC from 26 patients showed a stimulation index greater than 2. Addition of interleukin 2 made little difference in most cases. No correlation of clinical status of warts, i.e. improving, unchanged or resolved, with lymphoproliferation was found. When the PBMC were depleted of plastic-adherent cells and enriched for T cells, some samples which had not shown a lymphoproliferative response to HPV-1 or HPV-2 became positive; this response was abolished when the adherent cells were re-added. Thus it is possible that the adherent cell population from a proportion of patients contains cells which suppress lymphoproliferation, or that an immunoregulatory network is present so that lymphoproliferation does not take place in vitro without prior activation and cloning of T cells.


Adenylate cyclase induces intracellular calcium increase in single human epidermal keratinocytes measured by fluorescence microscopy using Fura 2-AM. Intracellular calcium ([Ca2+]i) is an important second messenger of extracellular signals to induce various cellular responses. Extracellular and intracellular Ca2+ are considered to be important for cellular differentiation and proliferation of epidermal keratinocytes. Several mechanisms which increase [Ca2+]i have been demonstrated in various tissues, but in epidermal keratinocytes these mechanisms are poorly understood. In epidermal keratinocytes the adenylate cyclase-cyclic AMP response is thought to regulate cell proliferation and differentiation. However, the series of reactions which follow the cyclic AMP response remain unknown. Beta-adrenergic agonists increase [Ca2+]i in cultured epidermal keratinocytes, and we have therefore studied whether stimulation of keratinocyte adenylate cyclase could induce [Ca2+]i increase, by using fluorescence microscopy with Fura 2-AM. Adenosine and histamine, which are known to be keratinocyte adenylate cyclase receptor agonists, induced transient [Ca2+]i increase, as did epinephrine. In addition, forskolin, a direct adenylate cyclase activator, and dibutyryl-cyclic AMP also induced an increase in [Ca2+]i. In a calcium-free medium epinephrine, adenosine, histamine and dibutyryl-cyclic AMP induced an increase in [Ca2+]i. These results suggest that cyclic AMP in human epidermal keratinocytes regulates [Ca2+]i, which is released from intracellular stores.


Patch averaging of electron images of GP3*I crystals with variable thickness. The combination of Fourier and correlation averaging techniques with multivariate statistical analysis and classification, a method known as patch averaging, is used to analyze untilted and tilted images of negatively stained GP32*I crystals, which exhibit variable thicknesses in a single crystal. Within a single image, coherent areas of the same apparent thickness can be distinguished from areas of differing thicknesses. Analysis using the phase relationships among symmetry-related reflections from reconstituted images obtained from untilted micrographs confirms the ability of the method to classify these variable thicknesses properly. Furthermore, the phases from some of the reconstituted images obtained from both untilted and tilted micrographs were found to match well with the phases in a previously determined three-dimensional data set of this crystal with pg symmetry along the crystallographic b axis. These results indicate the utility of the patch averaging procedures in the structural determination of protein crystals with different thicknesses.


Herpes simplex-1 virus thymidine kinase gene is unable to completely eliminate live, nonimmunogenic tumor cell vaccines. Recent experiments with genetically engineered tumors have generated renewed interest in active cellular immunotherapy as a cancer treatment modality. In order to consider the use of live tumor cells for immunotherapy in human cancer patients, it will be important to ensure that these cells do not themselves produce morbidity in the event the immune system fails to eliminate them. Toward this end, we have examined a strategy for eliminating genetically manipulated nonimmunogenic tumors in vivo. When B16F10 melanoma cells were transfected with the Herpes simplex virus 1 thymidine kinase (HSV-TK) gene, cells were rendered susceptible to killing by the nucleoside analogs acyclovir (ACV) and ganciclovir (GCV). B16-HSV-TK+ tumors established in C57BL6 mice were successfully "suicided" in vivo when GCV was administered by continuous infusion. However, late recurrences were observed even after 1 month of continuous GCV treatment. In vivo growth kinetics suggested that the recurrences resulted from a tiny number (< 20) of cells that had survived the GCV treatment. Interestingly, recurrent tumors were as sensitive to GCV as the parental B16-HSV-TK+ line. While these results demonstrate potential feasibility of the suicide gene strategy for active immunotherapy with live tumor cells, they also illustrate that approaches dependent on the intracellular generation of cell cycle-dependent toxins may fail to eliminate small numbers of cells that temporarily exit cell cycle or that are pharmacologically sequestered.


[Mucinous metaplasia of fallopian tube mucous membrane as a cause of pseudomyxoma peritonei]. A woman aged 49 years was relaparotomized at the Gynaecological Department of the "Rudolf Virchow" Medical Centre of the Free University of Berlin, following diagnosis of metastatic mucinous adenocarcinoma. Proliferative mucinous metaplasia of the Fallopian tube and consecutive peritoneal pseudomyxoma were established as clinical findings and were hypothetically attributed to mucinous metaplasia of the salpingofimbrial mucosa. Neither mucocele of the appendix nor ovarian tumour were found. This possible site origin has been rarely mentioned in any case report in the past.


Screening for antibodies against Aleutian disease virus (ADV) in mink. Elucidation of dubious results by additive counterimmunoelectrophoresis. In order to distinguish true positive results in counterimmunoelectrophoresis from false positive ones an additive counterimmunoelectrophoresis was developed. The method was tested on selected mink serum samples as part of a routine testing for antibodies towards Aleutian disease virus on 3 million blood samples. The procedure of the method is, that a known positive serum sample is mixed with the patient serum to be tested. The result from a false positive sample will be one precipitin line towards virus and one nonspecific line. If the serum sample is a true positive one, the antibodies originating from the patient serum will be added to the antibodies in the standard positive serum giving only one precipitin line. The system is further extended by testing the serum samples towards an antigen preparation containing all the cellular components but free from virus.


Does cruciform DNA provide a recognition signal for DNA-topoisomerase II? Topoisomerase II displays higher affinity for supercoiled DNA compared to the same relaxed DNA. Moreover, cruciform structures are formed in topologically constrained DNA. Here we report that, using S1 nuclease experiments on supercoiled DNA, hairpin structures are located close to numerous topoisomerase II cleavage sites on the BPV I genome. Therefore, DNA secondary structure may play a role in the recognition mechanism of DNA by topoisomerase II.


Multiplication and immunogenicity of a temperature-sensitive and thymidine kinase-deficient strain of Aujeszky's disease virus in pigs. Hysterectomy-produced colostrum-deprived 5- and 27-day-old pigs were inoculated intramuscularly (IM) or intranasally (IN) with the temperature-sensitive and thymidine kinase-deficient ZHtsTK- strain of Aujeszky's disease virus (ADV), and the nasal swabs and organs of the pigs were periodically collected for virus isolation. No abnormal clinical signs were observed in these pigs, except for a mild febrile response. Viral shedding in the nasal swabs with low titers was detected in the pigs inoculated IN between postinoculation day (PID) 1 and 5, but not in those of the pigs inoculated IM. No contact infection, however, occurred in the cohabiting pigs. Viruses with low titers were isolated only from the muscles and lymph nodes at the site of inoculation in the pigs inoculated IM on PID 2 and 4, but not from any organs of the pigs inoculated IN. To investigate the ability of the ZHtsTK- strain to establish a latent infection in pigs, the pigs inoculated IM or IN with the ZHtsTK- strain were treated with prednisolone. No virus was detected in the trigeminal ganglia or the nasal swabs collected after prednisolone treatment by the cocultivation method. The immunological evaluation demonstrated that immunization of pigs with this strain was effective in preventing clinical signs caused by ADV infection. The duration of virus shedding was markedly shortened in immunized pigs, particularly in those immunized twice and the total quantity of virus recovered from immunized pigs was reduced in comparison with unimmunized pigs.


Encephalomyocarditis (EMC) virus-induced myocarditis by different virus variants and mouse strains. The mode of occurrence of encephalomyocarditis (EMC) virus-induced myocarditis in mice was pathologically and virologically investigated using 2 virus variants (highly diabetogenic EMC-D and non-diabetogenic EMC-B) and 2 mouse strains (diabetes-susceptible BALB/c and diabetes-resistant C57BL/6). Mice were inoculated with 10(5) PFU/head of the virus intraperitoneally and observed up to 7 days post inoculation (7DPI). As compared with EMC-B-infected BALB/c and EMC-D-infected C57BL/6 mice, EMC-D-infected BALB/c mice developed marked myocarditis and exhibited a heart virus titer of more than 100 times above that of the others after 4DPI. Electron microscopically, small aggregations of virus-like particles, with 20-25 nm in diameter, were found in the cytoplasm of degenerated cardiomyocytes showing mitochondrial and myofibrillar degeneration in EMC-D-infected BALB/c mice.


Use of a soluble tetrazolium/formazan assay for chicken cells. We evaluated a soluble tetrazolium/formazan assay using 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)-carbonyl ]-2H- tetrazolium hydroxide (XTT) for chicken cell growth. Fifty microliter of solution containing 1 mg/ml of XTT and 0.025 mM phenazine methosulfate was added to the cells in a well of 96-well microplate. After 4 hr incubation at 37 degrees C, the absorbance was measured at 490 nm. Under this condition, absorbances were well correlated with cell number of Marek's disease tumor cells and chicken embryo fibroblasts. Proliferation of chicken lymphocytes stimulated with mitogens was also effectively measured. The formazan of XTT is water-soluble and can be quantitated in culture medium without the necessity for extraction with organic solvents. Thus XTT assay is simple and useful for the quantity assay with chicken cells.


Replication of porcine cytomegalovirus in the 19-PFT cell line. Replication of porcine cytomegalovirus was examined in fibroblast- and epithelial-like cell lines of the 19-PFT cell line derived from pig fallopian tube. The virus grew well in the fibroblast-like cell line than the epithelial-like cell line. Cytomegalic cytopathic effects of the virus were clearly observed under the microscope after dispersion of the infected cell culture by trypsin-versene and it was demonstrated that cytomegalic cytopathic effects could be used for infectivity titration. Intranuclear inclusions were formed in the infected cells and herpetic virus particles were observed in the nucleus and cytoplasm when infected cells were observed under the transmission electron microscope. Infected cells formed characteristic red plaque.


Down-modulation of cell surface expression of p80 form of the tumor necrosis factor receptor by human immunodeficiency virus-1 tat gene. Tumor necrosis factor-alpha (TNF-alpha) induces the expression of human immunodeficiency virus type-1 (HIV-1) in vitro in chronically infected cells of T and monocytic origin. The tat protein from the HIV-1 virus has been shown to be essential for HIV replication and in the immunosuppression associated with the virus infection. Previous studies in our laboratory have shown that HIV-1 tat gene induces TNF-beta (lymphotoxin) in human B-lymphoblastoid cells (Sastry et al., 1990, J. Biol. Chem. 265, 20091-20093). In an attempt to characterize further the relationship between the host and HIV-1, we investigated the effect of the functional HIV-1 tat gene on the expression of TNF receptors in a human B lymphoblastoid cell line (Raji). We report here that Raji cells transfected with HIV-1 tat gene express fewer cell surface TNF receptors than control cells. At least a 5-fold decrease in the receptor number without any significant change in receptor affinity was observed. The decrease in TNF receptors in tat-transfected Raji cells (Raji-tat cells) was found not to be due to receptor occupancy by the autocrine production of TNF-beta. The decrease in the cell surface expression of TNF receptors in Raji-tat cells was also found to be not due to a decrease in the gene expression of the receptor. The kinetics, amount of TNF binding and its internalization were temperature dependent, and it was different in Raji-tat cells than in the control cells.(ABSTRACT TRUNCATED AT 250 WORDS)


Establishment of a human T-cell line constitutively producing several eosinophil chemotactic lymphokines and their functional heterogeneity on eosinophils. We established a T-cell line, STO-2, by human T-cell lymphoma-leukemia virus-induced transformation of normal human T cells. Partial purification with isoelectric electrophoresis revealed that STO-2 liberated several eosinophil chemotactic factors (ECF) for eosinophils from healthy individuals with different isoelectric point of PI5, PI6, PI7, PI8, and PI9. Molecular weight of all the ECF was about 30,000 to 45,000. None of the ECF except ECF-PI5 was suppressed when they were incubated with monoclonal antibodies against IL-3, IL-5, and GM-CSF together, suggesting that ECF activity of ECF-PI5 is mainly comprised of IL-3, IL-5, and GM-CSF. ECF-PI5, PI6, and PI7 also exhibited enhancing activity on ex vivo eosinophil survival whereas ECF-PI8 and PI9 failed. Expression of Fc epsilon receptor II on eosinophils was potentiated by ECF-PI6 and ECF-PI7. In contrast, expression of Fc gamma receptor III was potentiated by ECF-PI7, ECF-PI8, and ECF-PI9. ECF-PI6 could also change an eosinophilic cell line, EOL-1, to eosinophilic granule-positive cells, whereas the rest of ECF failed. The above results suggested that eosinophils attracted by an ECF exhibit their biological functions, which differ from those of eosinophils attracted by other ECF. In further experiments, the chemotactic response of eosinophils from patients with eosinophilia was compared to that of eosinophils from healthy individuals.(ABSTRACT TRUNCATED AT 250 WORDS)


Blocking factors (soluble membrane receptors) for tumor necrosis factor and lymphotoxin detected in ascites and released in short-term cultures obtained from ascites and solid tumors in women with gynecologic malignancy. Studies were conducted to identify and establish the cell and tissue source of blocking factors (BF), materials that inhibit the bioactivity of human TNF and LT in vitro. Ascites and samples of solid tumors were collected from women with various gynecologic malignancies. Supernatants were collected from cultures of tumor and ascites cells after 24, 48, and 72 h. Cell-free ascites (CFA) and culture supernatants were tested for their ability to block human recombinant TNF and LT-induced lysis of L929 cells in vitro. Levels of soluble forms of the 55- and 75-kDa TNF/LT receptors were measured by ELISA assay in the same samples. CFA and culture supernatants contained TNF/LT blocking factors and high levels of one or both soluble 55- and 75-kDa TNF/LT membrane receptors. Levels of BF bioactivity and receptors appeared rapidly, peaked at 24 h, and declined thereafter. Soluble TNF/LT receptors may be the active BF in these samples, and tumor tissues and ascitic cells may be a source of these receptors in the ascites fluid of these patients.


Evidence of a role for phosphatidylinositol synthesis in human amnion cell proliferation. Phosphatidylinositol (PtdIns) is the key precursor of phosphoinositide-derived intracellular mediators. The effects of changing the rate of PtdIns synthesis on mitogenic activity of human amnion-derived WISH cells were investigated. Incubation of the cells with [3H]inositol caused a time- and dose-dependent PtdIns labeling. Exogenous Ca2+ inhibited [3H]inositol incorporation in a dose-dependent fashion; half-maximal inhibition occurred with 0.3-1.0 mM Ca2+. In contrast, removal of cytosolic Ca2+ by ionophore A23187 and 1 mM EGTA induced enhancement of the PtdIns labeling as a function of A23187 concentration, perhaps through release of inhibitory effects of endogenous Ca2+. The A23187-stimulated PtdIns labeling with [3H]inositol was not abolished by additional unlabeled inositol, suggesting that [3H]inositol labeling of PtdIns occurred mainly through de novo synthesis catalyzed by PtdIns synthase (EC 2.7.8.11). In cells with PtdIns synthase activity decreased by exogenous Ca2+, [3H]thymidine incorporation was also inhibited, while A23187 caused dose-dependent enhancement of thymidine incorporation. The changes in PtdIns synthase activity occurred in parallel with changes in mitogenic activity caused by increasing the dose of exogenous Ca2+ or A23187. A similar lowering of mitogenic activity was observed upon suppression of PtdIns synthase by pemirolast potassium (9-methyl-3-1H-tetrazol-5yl-4H-pyrido[1,2-a]pyridin-4-one potassium) via a Ca(2+)-independent mechanism. These data demonstrate that changes in PtdIns synthase activity by some agents acting via different mechanisms are associated with parallel changes in thymidine incorporation, and suggest that PtdIns production is tightly coupled to cell proliferation in human amnion cells.


A novel hypothalamic peptide, pituitary adenylate cyclase activating peptide, modulates Sertoli cell function in vitro. Pituitary adenylate cyclase-activating peptide (PACAP), a novel hypothalamic peptide that has been shown to exist in several tissues including the testis, was examined for its effects on cultured rat Sertoli cells. PACAP stimulates cAMP accumulation in Sertoli cells cultured from 15-day-old rats in the presence or absence of methylisobutylxanthine, a phosphodiesterase inhibitor, and in the presence of pertussis toxin, a blocker of the adenylate cyclase inhibitory pathway. Maximal stimulation, which is 20-40% of that attainable with FSH, occurs at PACAP concentrations of 10 nM: the ED50 is approximately 100 pM. The ability of PACAP to stimulate Sertoli cell cAMP declines with increasing age of donor animals (15-60 days of age) in a fashion similar to the FSH effect. PACAP stimulation of Sertoli cell cAMP accumulation is additive with submaximal, but not maximal, concentrations of FSH or forskolin. PACAP also stimulates the secretion of lactate, estradiol, and inhibin in a concentration-dependent manner. The stimulation of Sertoli cell cAMP accumulation by PACAP is not altered by a vasoactive intestinal peptide antagonist, and vasoactive intestinal peptide alone does not stimulate cAMP accumulation, indicating that PACAP is not acting via vasoactive intestinal peptide receptors. Further experiments are needed to determine whether PACAP is synthesized within the testis and if so, in which cell types; however, the present data clearly demonstrate that PACAP can modulate Sertoli cell function in vitro.


[An etiopathogenic and therapeutic update in chronic viral hepatitis in children]. The author presents the new prospects in the treatment of the chronic viral hepatitis in children, which is an up-to-date-problem in the last decade. There are emphasized the present aspects concerning the etiopathogenesis of the disease, which represent a constellation of achievements as a scientific foundation for the therapy: new notions about the viral genome, the mutant viruses, the host's cell-mediated immunity. There are reminded the classifications of the chronic hepatitis and especially the new one of the chronic type B hepatitis, the carries state risks, the vertical transmission from mother to child as an epidemiological source, valid in our country, too. There are specified the findings of the inefficiency and the contraindication of the cortisone and immunosuppressive therapy in patients with proved viral markers (the improvement of the HBV replication and thus, worsening the disease). The present and the future therapy is represented by the antiviral and immunomodulator drugs (alfa-IFN); there are presented the doses, the mode of administration, the predictive and responsivity factors, as well as the combined therapy (prednisone and antivirals). There is concluded that the passive and active immunoprophylaxis in the newborns of HBs Ag-positive mothers in the replicative phase is a great necessity.


[Common dermatitis in infants (dermatitis seborrheica, atopic dermatitis and "diaper dermatitis")]. The most common dermatoses during early infancy are: seborrheic dermatitis, atopic dermatitis and diaper dermatitis. It is important to differentiate these conditions for prognostic and therapeutic reasons.


[The indications and limits for echographic studies in pediatrics]. Echography is an non-invasive investigation with many advantages, but also with its limits. It allows diagnosis in numerous diseases and in other illness may suggest the choice of the next investigations, thus shortening considerably the diagnosis steps. The information provided by the echographic examination must be correlated with the clinical findings and other data furnished by the laboratory tests or other imagistic investigations, to obtain an integrated holistic evaluation, able to give a complete and correct final diagnosis.


[The hematological manifestations in human immunodeficiency virus (HIV) infection in children]. The hematologic disorders that occur in HIV infected children are analysed by the authors, on the basis of some recent studies from current literature. There are emphasized the importance of the frequency of anemia, leukopenia and granulocytopenia. There are also stressed the pathogenic mechanisms of these disorders, their predictive significance and the rationale basis for therapeutics (actual and future treatment methods).


[Neonatal convulsions: the current concepts and classification]. The authors presented a new classification of the neonatal convulsions, based on prolonged EEG registrations related to concomitant clinical or videorecording of fits. They focussed on the atypical neonatal convulsions and on the differential diagnosis between epileptic and nonepileptic seizures. Finally, they discussed the therapeutical consequences of these new conceptions about the conclusions in the neonate.


[Recurrent abdominal pains (RAP) in children (their definition, incidence and classification)]. The authors presents the particular aspects of recurrent abdominal pain in children. After the definition, they underline the main elements that characterise this syndrome. An important place is assigned to the classification of recurrent abdominal pain, three different conceptual methods being analysed in this article, concerning the etiopathogenesis of this syndrome. The methodological errors that occurred from the practical study of recurrent abdominal pain are emphasised in order to the first bivariant model (psychogenic and organic, J. Apley, 1958). Advances in investigations of children with recurrent abdominal pain in the last ten years demonstrate that organic recurrent abdominal pain occur more often than in was considered before, in other studies. More than that, in the last years, a new concept has been considered--dysfunctional recurrent abdominal pain (without organic or psychogenic support)--in order to the trivariant model purposed by R. Barr (1981). Finally, the authors present the last conceptual model (1984) which presumes the complex interactions between four primary forces that interfere in recurrent abdominal pain modulation.


[The clinico-echocardiographic aspects in acute pericarditis in a small child]. The authors present a case of pericarditis in a 12-months-old child. They underlined the peculiarities of the onset and of the evolution.


[The metabolic characteristics of low-birth-weight infants]. The authors studied the protein and fat metabolism in 2-6-weeks-old premature (100 cases) and "small for date" babies (20 cases), all being artificially fed with isocaloric diets. Weight curves were related to dietary intake of proteins in small for date babies. The premature born infants showed no similar relationship between dietary intake of proteins and weight charts. BUN had superior values in premature babies; it means probably that the kidneys of the small for date infants responded better to a protein charge. The values of both serum total fats and of the cholesterol were related to the dietary intake of fats. In conclusion, the milk formula have to be adapted to metabolic peculiarities of these categories of infants.


[A case of true precocious puberty in a child]. The authors present a case of true precocious puberty in a 2 1/2-years-old female child, probably due to a neurogenic cause (sequela of a purulent meningitis). The girl showed telarche, pubarche, menarche and an advanced statural development. The diagnosis was confirmed by laboratory. On treatment with Depo Provera (medroxy-progesterone acetate long acting) 200 mg/day for 10 days i.m., the evolution was favourable (cessation of precocious menstra).


Paramagnetic probes attached to a light chain on the myosin head are highly disordered in active muscle fibers. We have measured the orientation of a region of the myosin head, close to the junction with the rod, during active force generation. Paramagnetic probes were attached specifically to a reactive cysteine (Cys 125) of purified myosin light chain 2 (LC2) and exchanged into myosin heads in glycerinated rabbit psoas muscle. Electron paramagnetic resonance spectroscopy was used to monitor the orientation of the probes. Previous work has shown that the LC2 bound spin probes are significantly ordered in rigor and muscle in the presence of adenosine diphosphate (ADP). In contrast, there is a nearly random angular distribution in relaxed muscle. We show here that during the generation of isometric tension, all of the LC2 bound spin probes (98 +/- 1.6%) show an angular distribution similar to that of relaxed muscle. These findings contrast with results obtained from probes attached to Cys 707 on the cross-bridge, located close to the actin binding site, where, during active force generation, a proportion of the spin probes were ordered as in rigor, whereas the remaining probes were disordered as in relaxation. To test the hypothesis that this ordered component is due to modification of Cys 707, we measured the spectra obtained from probes attached to LC2 in fibers modified at Cys 707. The modification of Cys 707 did not produce an ordered component in these spectra. The absence of an ordered component at the LC2 site limits the populations of some states in active fibers. An actin/myosin/ADP state is thought to be the major force-producing state. Our present results show that the populations of states with ordered probes on LC2 are < 2% in active fibers; thus, the major force-producing state is different from the one obtained by addition of ADP to rigor fibers.


High molecular weight proteins in the nematode C. elegans bind [3H]ryanodine and form a large conductance channel. Single-channel properties of a polypeptide fraction from the nematode Caenorhabditis elegans highly enriched in binding sites were studied in planar bilayers. [3H]Ryanodine binding sites were purified by sucrose gradient centrifugation of C. elegans microsomes solubilized in CHAPS detergent. The highest [3H]ryanodine binding activity sedimented at approximately 18% sucrose (wt/vol), and was composed of a major polypeptide with a M(r) of 360,000 and a minor polypeptide with a M(r) of 170,000. The ryanodine-binding polypeptide(s) formed a Ca(2+)-permeable channel with a permeability ratio P(divalent)/P(monovalent) = 4 and two conductance states of 215 pS and 78 pS in 0.25 M KCl. Ryanodine locked the channel in the 78 pS state and inhibited transitions between the 215 pS and 78 pS states. These data demonstrated the presence of a ryanodine receptor in C. elegans with functional properties comparable to those described in mammalian muscle.


Modulation of the Ca2+ channel voltage sensor and excitation-contraction coupling by silver. Ag+ (0.5-10 microM) is known to produce a transient contraction of intact frog skeletal muscle fibers followed by complete inhibition of excitation-contraction (E-C) coupling. We have carried out physiological and biochemical experiments to investigate the basis of this effect. Dihydropyridine (DHP) Ca2+ channel blockers, which inhibit the voltage sensor of the Ca2+ channel, completely inhibit Ag+ contractions. Removal of extracellular Ca2+, or blockade of Ca2+ entry with cadmium, does not inhibit Ag+ contractions. Activation of the Ca2+ channel's voltage sensor with the Ca2+ channel agonists Bay K 8644 or with perchlorate, potentiates the Ag(+)-induced contraction. Ag+ binds to the partially purified rabbit skeletal muscle Ca2+ channel and inhibits DHP binding (IC50 = 1.1 microM) and sulfhydryl (SH) reactivity (IC50 = 0.11 microM) over the concentration range where it inhibits E-C coupling. Oxidation of free SH groups by H2O2 or their reaction with DTNB prevents Ag+ contractions, while DTT reduction of oxidized SH groups restores Ag+ contractions. These results suggest that Ag+ binds to critical SH groups on the DHP receptor Ca2+ channel, resulting in modification of the channel's voltage sensor and the failure of E-C coupling.


[The diagnostic difficulties in a case of a hemorrhagic syndrome in an infant]. The authors present a case of cystic fibrosis in a 3 months old infant. Clinically, the first manifestation was a severe anemia secondary to iron deperdition through a hemorrhage due to an acquired trouble of thrombin formation. The late was explained by the vitamin K malabsorption and by the hepatocellular insufficiency.


Protein kinase C regulates secretion of lymphotoxin (LT/TNF beta) and TNF alpha by human EBV-transformed B cells. Epstein-Barr virus (EBV)-transformed cell lines constitutively secrete lymphotoxin (LT/TNF beta) and not tumor necrosis factor-alpha (TNF alpha). To analyze the cellular processes that regulate LT and TNF alpha secretion by lymphoblastoid cell lines, we studied the role of two signal transduction pathways leading to either protein kinase C (PK-C) or PK-A activation. We demonstrate that PK-C activation, either after cross-linking of surface Ig or by direct activation with phorbolester, leads to increased production of both LT and TNF alpha, whereas no prominent role for PK-A was found. Interleukin (Il)-4 was found to synergize with PK-C activation in raising levels of secreted LT and TNF alpha. Increased levels of LT and TNF alpha did not correlate with augmented levels of immunoglobulin secreted by the cell lines nor with improved proliferation. These observations demonstrate that EBV B cells respond to B cell activation signals leading to PK-C activation with increased production of both LT and TNF alpha. It is, however, unlikely that these molecules serve as autostimulatory factors for EBV B cells, but in contrast might play a role in downregulation of biological functions in these cells.


Susceptibility of human monoclonal antibody-producing B cell lines to infection by human immunodeficiency virus. Seven immortalized B cell clones, five of which secreted specific human monoclonal antibodies (MAbs) against hepatitis B, tetanus toxoid, and Rhesus D antigens, were evaluated for their susceptibility to infection by human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2). Infection was confirmed in three human MAb-producing lines by detection of infectious virus and p24 antigen in culture supernates, by immunofluorescence, and by detection of viral DNA in cells by polymerase chain reaction. The infectable lines were as susceptible to HIV-1 infection as several T cell lines and remained persistently infected for several months, but in contrast to T cell controls, viral cytopathic effects were not observed. Levels of unintegrated viral DNA in the HB1 B cell line were significantly lower than in the HUT78 T cell line. Cell lines that were susceptible to HIV expressed HLA DR, CD20, and CD21, whereas the uninfectable cell lines did not express any of the markers tested. CD4 was undetectable or present on a small percentage of cells in two of the infectable cell lines. However, infection with HIV-1 was blocked more efficiently in B cells than in T cells by soluble CD4, anti-CD4 MAb, and dextran sulphate. The effect of HIV infection on human MAb secretion was variable, being reduced on a per-cell basis in one line, increased in another, and unchanged in a third.(ABSTRACT TRUNCATED AT 250 WORDS)


Manipulation of human B cells to confer immortality. Electroporation was used to deliver genomic DNA from a lymphoid tumor to activated/stimulated human peripheral blood lymphocytes to create immortalized lymphoid cell lines. Activation of the recipient lymphocytes was essential for efficient immortalization. A panel of human B cell transfectant clones, each phenotypically representing specific stages of differentiation, resulted from the transfection. Monoclonal antibody production was measured, and the level produced depended on the phenotype of the cells, with the more mature B cell transfectants secreting up to 10 micrograms/mL of immunoglobulin. The transfectants were stable with respect to their morphological appearance, growth rate, and antibody production. Chromosome analysis indicated that the transfectants displayed a normal karyotype, devoid of abnormalities. We have shown that electroporation is an effective method of immortalizing human lymphocytes at different stages of differentiation. The transfectants provide a panel of cells that can readily be studied with respect to their phenotypic/karyotypic stability, regulation, and production of immunoglobulin, lymphokines, and growth factors. These data demonstrate the feasibility of generating immortalized human B cells to provide an important resource for the study of B cell differentiation and immortalization.


Oligonucleotide fingerprinting as a means to identify and survey long-term cultured B cell hybridomas and T cell lines. Common problems encountered during cell culture are cross-contamination, instability, and inadvertent exchange of cells. Here we report on the application of oligonucleotide fingerprinting as a simple and efficient method to screen hybridomas and T cell lines. Among the fingerprint probes tested, the simple repetitive oligonucleotide (CAC)5/(GTG)5 proved to be most useful for obtaining many fragments specific for each cell line. Because of variable loss of chromosomes, cloned hybridoma cells from one fusion exhibit different fingerprint patterns. Thus, antibody-secreting B cell hybridomas can be distinguished easily even when they originate from the same fusion. Furthermore, we were able to monitor the genomic integrity of myelomas and hybridomas over a period of more than 2 years, thereby highlighting long-term stability. Another application of the method is the control of T cell lines requiring irradiated or mitomycin-treated feeder cells for continuous growth or cloning. There cell lines are always threatened to be overgrown by feeder cells that have escaped from the lethal pretreatment. T cell clones of one individual, known to display differently rearranged T cell receptors, did not show differences in their fingerprint patterns. However, in EBV-transformed cloned B cells, slight differences between clones from the same donor were identified.


Human papillomaviruses and genital cancer. Human papillomaviruses (HPVs) are epitheliotropic agents which preferentially infect either mucosal surfaces or the skin. A subset of the mucosotropic HPVs, in particular HPV types 16 and 18, are strongly linked with various forms of genital carcinomas. The great majority of these tumours carry integrated HPV DNA sequences and constitutively express two early viral genes, E6 and E7. DNA transfection studies show that these same genes can co-operate to immortalise human epithelial cells in vitro and that immortalised cells subsequently acquire a malignant phenotype through additional cellular genetic changes. Cell lines established from HPV-positive tumours also express E6 and E7, upon which continued tumour cell proliferation appears to depend. The development of molecular and serological markers specific for 'high risk' HPV types should allow the natural history of infection with these viruses, and their role in tumour development, to be better understood.


Human papillomaviruses and skin cancer.

Human papillomaviruses and skin cancer. Human papillomavirus (HPV)-induced skin warts are classically benign lesions. However an association between specific HPV types and skin cancer becomes obvious in epidermodysplasia verruciformis (EV). The analysis of this disease suggests that lesions infected with HPV types 5 and 8 carry a high risk of developing squamous cell carcinomas. The oncogenes of EV-viruses appear to be E6 and E2, rather than E7. The 'high risk' EV-viruses, HPV 5, 8, and 47, differ from related HPV types in the transforming activity of the E6 gene and in the density of positive transcription control elements in the non-coding region (NCR) of the genome. The extrachromosomal viral DNA in cancers may show deletions affecting regulatory sequences. EV-specific lesions occasionally occur in immunosuppressed patients and HPV 5 or 8 persist in some of the skin cancers to which these patients are prone. DNAs of HPV 2, 16, 34, or 41 were identified in few premalignant and malignant skin tumors of the general population.


Epstein-Barr virus, lymphomas and Hodgkin's disease. Epstein-Barr virus (EBV) is implicated in the pathogenesis of a number of human lymphoid malignancies, including the immunoblastic B lymphomas that arise in immunocompromised individuals, Burkitt's lymphoma, Hodgkin's disease, and certain T cell lymphomas. The immunoblastic lymphomas are most likely a direct consequence of EBV-driven B cell lymphoproliferation that would in normal circumstances be eliminated by virus-specific cell-mediated immune responses. The other EBV-associated malignancies arise in individuals with more or less intact cellular immune responses and appear to have a complex multi-step pathogenesis. Throughout the EBV-positive lymphoid malignancies there is an intimate association between tumour cell phenotype and virus latent gene expression, with each of the three forms of latency seen in in vitro models being exemplified in vivo. Tumours with these different forms of virus latency will differ in their susceptibility to EBV-specific immune T cell control.


Epstein-Barr virus and Burkitt's lymphoma. Recent investigations indicate that Burkitt's lymphoma consists of several subtypes, defined by their clinical and molecular features. Each geographical region so far studied appears to consist of a different mixture of subtypes. Interestingly, there appear to be geographic 'gradients' with respect to the fraction of tumors associated with EBV and the type of 8;14 chromosomal translocation. The rate of EBV association is highest in Equatorial Africa, lowest in North America and intermediate in South America. The fraction of tumors with breakpoints far upstream of the c-myc gene follows a similar pattern. These findings strongly suggest that the subtypes of Burkitt's lymphoma are environmentally determined, and we propose that the pattern of infection (e.g. malaria) to which the young child is exposed influences the tumor subtype distribution by altering the relative and absolute numbers of various B cell precursors at sites of B cell ontogeny (the bone marrow, and possibly mesentery). These B cell precursors are the cells which are susceptible to the specific chromosomal translocations associated with Burkitt's lymphoma. We further propose that immunoglobulin enhancers (recognized and unrecognized) both influence the likelihood of the translocation occurring, and in at least a fraction of cases, contribute to the deregulation of a c-myc. EBV, via EBNA-1, the only invariably expressed latent-gene in Burkitt's lymphoma, probably influences c-myc expression in Burkitt's lymphoma by increasing immunoglobulin enhancer function. Thus, in effect, EBV collaborates with the translocations associated with Burkitt's lymphoma in causing c-myc deregulation. This collaboration is independent of the breakpoint location. While other molecular abnormalities must be able to contribute to myc deregulation in the same way, EBV association in Burkitt's lymphoma is probably determined by the age at which EBV infection occurs (being more likely when infection occurs in very young children) and perhaps also by other infectious diseases that numerically influence the fraction, and predominant stage of differentiation (and hence translocation breakpoint sites) of immature B cells infected by EBV. The presence of EBV in many such cells greatly increases the incidence rate of Burkitt's lymphoma, since one of the genetic lesions needed to deregulate c-myc is already present.


Epstein-Barr virus and nasopharyngeal carcinoma. Nasopharyngeal carcinoma, an epithelial tumor which is characterized by marked geographic and population differences in incidence, is consistently associated with the Epstein-Barr virus (EBV). Within the tumor, the EBV DNA is homogeneous and clonal with regard to repeat sequences suggesting that the tumor is also clonal. Expression of specific viral mRNAs or gene products are consistently detected within all of the tumor cells. These data suggest that EBV is an essential component in the development of nasopharyngeal carcinoma. Genetic or environmental co-factors may influence the ability of the virus to express these genes in infected epithelial tissue or may contribute to clonal predominance.


Mammalian hepatitis B viruses and primary liver cancer. Hepatitis B virus is a major etiologic agent in the development of human hepatocellular carcinoma, but the precise role of the virus in the tumorigenic process is still unclear. Recent studies of naturally occurring animal models, such as woodchucks and squirrels infected with hepatitis B-like viruses (hepadnaviruses) have revealed different oncogenic strategies and outlined the predominant role of myc genes in rodent hepatomas. Higher oncogenicity of woodchuck hepatitis virus has been correlated with a direct contribution of the virus as an insertional mutagen of myc genes: c-myc, N-myc and predominantly the woodchuck N-myc retroposon. In contrast, rare viral integration events but frequent amplifications of c-myc characterize ground squirrel hepatitis virus-induced tumors, indicating that hepadnaviruses may contribute in malignant transformation through different, direct or indirect ways.


Retroviruses and human cancer.

Retroviruses and human cancer. Retroviruses have long been associated with cancer in many species. Human T cell leukaemia virus type I causes adult T cell leukaemia. Human immunodeficiency virus infection is associated with lymphoma and Kaposi sarcoma, but oncogenesis is largely secondary to its effect on the immune system. The incidence of Kaposi sarcoma varies greatly in different social groups with AIDS, being most frequent among male homosexuals; an unknown, sexually transmissible agent may be responsible. Human endogenous retroviral genomes are linked with myeloproliferative disease. Finally, the risk is discussed that cancer could be a side-effect of the use of retroviral vectors in human gene therapy.


[Anoxia-induced changes in cAMP contents in the cat cerebral cortex]. Effect of the cessation of oxygen supply on cAMP content and neuronal spike activity (NSA) in the cortex brain was studied. The interruption of oxygen supply during in first decades of seconds evoked changes in the pattern of NSA, followed with the decrease of cAMP content (to 56 +/- 10%). Then the phase of neuronal hyperactivity and increase of cAMP level (to 198 +/- 26%) took place. The content of cAMP approximated the basal one in 2.5 min anoxia. Anoxia during 5 min resulted in direct opposite shifts of cAMP content in two groups of cats (an increase up to 223 +/- 11%, and decrease up to 75 +/- 8%, respectively, which correlated with individual features of NSA recovery in postanoxic period and values of cAMP basal level in the cortex of different animals. Upon 30 min reoxygenation after 2.5 min anoxia a decline of the content of cAMP (to 63 +/- 12%) accompanied enhance of NSA. This period of reoxygenation after 5 min anoxia demonstrated two types of reactions, observed in different groups of cats: the first type--NSA tended to normalization with the level of cAMP 44 +/- 8% below basal level, and the second type--insufficient recovery of NSA attended by value of cAMP 90 +/- 13% above basal level.


[Effects of corazol on the regulation of GABAa receptor-channel complex]. Effects of corazol (pentylenetetrazole, PTZ) on the GABAA receptor-channel complex mediated 36Cl- influx into synaptoneurosomes isolated from rat cerebral cortex, were studied. PTZ inhibited the 36Cl- influx stimulated by a GABA agonist, muscimol, concentration-dependently with an IC50 of 2.11 +/- 0.25 mM. In addition, the rate of muscimol-dependent desensitization of the GABAA receptor-channel complex was significantly reduced in the presence of PTZ. Apparently, the superimposition of these two mutually opposing effects is the cause of a mixed (competitive/noncompetitive) type of inhibition displayed by PTZ. The rate of desensitization of GABAA receptor-channel complex is suggested to be regulated by intracellular concentration of Cl- through a feed-back mechanism.


[Two-phase effect of endothelin-1 on resistance of coronary vessels in anesthesized rats with intact chest]. Effects of intracoronary infused (2 pM/kg/min for 5 min) endothelin-I on coronary blood flow was studied using modification of the method of Vetterlein and Schmidt. Blood flow in extracorporeal circuit was measured by 20 MHz pulsed Doppler flowmeter. One end of the circuit was connected to the left common carotid artery and the other was connected to the especially curved glass cannule which was placed to the origin of the coronary artery and through the right common carotid artery. Five-minute infusion of endothelin was followed by transitory dilatation and then by constriction of coronary vessels. Blockade of dihydropyridine-sensitive Ca-channels potentiated endothelin-induced vasodilation and decreased the constrictor response three-fold.


[Differences in the characteristics of opiate and catecholaminergic receptors of the striatum and cerebral cortex of rats of Fisher-344 and WAG/GSto strains may cause differences in reinforced action of morphine]. It has been shown that Fischer-344 rats more than WAG/GSto inbred rats preferred to consume the solution of morphine. In intravenous self-administration testing, Fischer-344 rats had a higher rate of reinforced responses that resulted in morphine infusion. Bmax values for mu- and alpha 2-adrenoreceptors were significantly higher in the cortex of WAG/GSto rats. The sensitivity of the serotonin and dopamine receptors in the cortex and striatum of WAG/GSto rats was lower than that in Fischer-344 rats. These findings suggest that the difference between morphine consumptions in two inbred rat strains may be due to individual genetic patterns determining opioid and catecholamine receptors binding in the brain.


Overexpression of cellular retinoic acid-binding protein type II (CRABP-II) and down-regulation of CRABP-I in psoriatic skin. Cellular retinoic acid-binding proteins (CRABPs) might exert a physiological function by controlling the intracellular levels of free retinoic acid. The aim of this study was to analyze the relative expression of CRABP-I and CRABP-II in lesional (LPS) and nonlesional (NLPS) psoriatic skin. CRABP-I and -II proteins were analyzed by a PAGE-autoradioblotting technique, and their respective mRNA were studied by RNA blot and in situ hybridization. We found that CRABP-II levels were 6-fold higher in LPS and 2-fold in NLPS as compared to normal skin, whereas CRABP-I levels were decreased in NLPS and LPS. CRABP-II mRNA were grossly overexpressed in all LPS and some NLPS specimens. These results indicate a switch to the overexpression of CRABP-II mRNA in psoriasis which induces high levels of the protein mainly in LPS; these observations may be relevant to the pathophysiology and therapy of psoriasis as CRABP-I and -II have different ligand-binding affinities.


Primitive neuroectodermal tumor arising in the skin. Differentiation from neuroendocrine carcinoma of the skin. We report a 25-year-old male patient with primitive neuroectodermal tumor presenting as a subcutaneous tumor on the right buttock, which was initially diagnosed as neuroendocrine carcinoma of the skin. Subsequently, local recurrence and metastatic spread to the lung occurred. The tumor was resistant to highly aggressive chemotherapy. The patient died 6 months later with severe pulmonary and lymph node involvement.


Lisinopril-induced "scalded mouth syndrome". To report a case of "scalded mouth syndrome" (SMS) caused by lisinopril. A woman being treated with lisinopril for hypertension developed a burning sensation of her lips and buccal mucosa. The condition persisted with continued use of lisinopril and subsided when the medication was discontinued. The symptoms described by our patient were similar to those reported in previous cases of SMS associated with the use of enalapril and captopril, two other angiotensin-converting enzyme (ACE) inhibitors. This reaction to ACE inhibitors appears to be dose related, and subsides with a decreased dosage or discontinuation of the medication.


Renal potassium wasting in distal renal tubular acidosis: role of aldosterone. The pathogenesis of renal potassium wasting and hypokalemia in classic renal tubular acidosis (type 1 RTA) remains uncertain. The prevailing theory is that K(+)-Na+ exchange is stimulated due to an inability of the distal tubule to establish a normal steep lumen-peritubular H+ gradient. We encountered a 42-year-old woman with type 1 RTA associated with Sjögren's syndrome, in whom renal potassium wasting and hypokalemia persisted despite sustained correction of systemic acidosis with alkali therapy and increased intake of potassium. In addition, plasma renin activity was markedly increased and the serum aldosterone level was upper-normal despite the hypokalemia. Increased intake of sodium resulted in suppression on the serum aldosterone and correction of renal potassium wasting and hypokalemia. This case shows that secondary hyperaldosteronism, possibly due to an impairment of sodium conservation in the distal tubule, may contribute to the loss of potassium from the distal tubule even after the correction of acidosis.


Identification of a family of avirulence genes from Xanthomonas oryzae pv. oryzae. Races of Xanthomonas oryzae pv. oryzae, the causal agent of bacterial blight of rice, interact with cultivars of rice in a gene-for-gene specific manner. Multiple DNA fragments of various sizes from all strains of X. o. pv. oryzae hybridized with avrBs3, an avirulence gene from Xanthomonas campestris pv. vesicatoria, in Southern blots; this suggests the presence of several homologs and possibly a gene family. A genomic library of a race 2 strain of X. o. pv. oryzae, which is avirulent on rice cultivars carrying resistance genes xa-5, Xa-7, and Xa-10, was constructed. Six library clones, which hybridized to avrBs3, altered the interaction phenotype with rice cultivars carrying either xa-5, Xa-7, or Xa-10 when present in a virulent race 6 strain. Two avirulence genes, avrXa7 and avrXa10, which correspond to resistance genes Xa-7 and Xa-10, respectively, were identified and partially characterized from the hybridizing clones. On the basis of transposon insertion mutagenesis, sequence homology, restriction mapping, and the presence of a repeated sequence, both genes are homologs of avirulence genes from dicot xanthomonad pathogens. Two BamHI fragments that are homologous to avrBs3 and correspond to avrXa7 and avrXa10 contain a different number of copies of a 102-bp direct repeat. The DNA sequence of avrXa10 is nearly identical to avrBs3. We suggest that avrXa7 and avrXa10 are members of an avirulence gene family from xanthomonads that control the elicitation of resistance in mono- and dicotyledonous plants.


Parathyroid hormone-related protein as a cause of hypercalcemia in a B-cell type malignant lymphoma. Hypercalcemia occurred in a patient with non-Hodgkin's (B-cell type) lymphoma when generalized lymphadenopathy developed. Despite low normal plasma parathyroid hormone (PTH), nephrogenous cAMP (NcAMP) was not suppressed, and serum and urine PTH-related protein (PTH-rP) levels were elevated. The plasma level of 1,25(OH)2D was within normal range. The combined chemotherapies successfully reduced the tumor size, serum Ca, PTH-rP, and lactic dehydrogenase. Serum osteocalcin was suppressed while the patient was hypercalcemic, and increased after chemotherapy. In the extract of the tumor tissue obtained post mortem, bioactivity stimulating the production of cAMP in osteoblasts was demonstrated along with the immunoreactive PTH-rP. This is the first report of a B-cell lymphoma producing PTH-rP and its association with humoral hypercalcemia of malignancy.


Allergic granulomatous angiitis and peripheral nerve lesion. We examined 8 cases of allergic granulomatous angiitis (AGA). All cases showed peripheral nerve lesion, comprising damage of all myelinated fibers, which was more severe in larger ones. Immunofluorescent deposits of IgE were detected in the peripheral myelin. There was lymphocyte infiltration both around the endoneural capillaries and in the endoneurium, and an increase of endothelial cells. Nerve ischemia due to obstruction of the vasa nervorum, circulation insufficiency of the small vessels, or immunological abnormality through IgE may play a pathogenetic role in the peripheral neuropathy of AGA.


Transposable elements.

Transposable elements. Transposable elements comprise a major fraction of eukaryotic genomes. They are studied both because of their intrinsic biological interest and because they can be exploited as valuable research tools. Many interesting papers dealing with various aspects of the biology of these elements have been published during the past year and a number of new elements have been reported. Four areas in which particularly valuable contributions have been made are the mechanisms of transposition, the regulation of transposition, the use of transposable elements as research tools, and the biological function of transposable elements.


Horizontal transfer.

Horizontal transfer. Eukaryotic transposable elements provide some of the best documented examples of the occasional horizontal transfer of DNA sequences between both closely and distantly related species. Although the mechanisms involved in such a transfer remain a puzzle, new ideas are beginning to emerge. The rapidly expanding number of reports of transposable elements that may have been transferred horizontally raises questions both about whether these elements are more prone to this mode of transfer than non-mobile genes, and about the possible evolutionary significance if such a difference is real.


Transcriptional regulation and transpositional selection of active SINE sequences. Alu repeats are short interspersed elements whose transposition has lead to genetic variability and heritable disorders in humans. A select subset of the nearly one million Alu sequences in human DNA actually produce new transpositions. The evolution of newly inserted Alu repeats is currently a key subject for study. Mechanisms of RNA polymerase III activity and the sequence environment into which an Alu inserts might select for transcriptional and posttranscriptional determinants of Alu transposition.


Population genetics of microbial organisms. Population data suggest that many parasitic protozoa (e.g. Trypanosoma, Leishmania, Entamoeba and Giardia) reproduce clonally, but this hypothesis has been highly controversial for Plasmodium falciparum. Although reproduction is predominantly clonal in the enteric bacteria Escherichia coli and Salmonella, the level of recombination affecting short (< 1 kb) regions of the chromosome is sufficient such that many genes are obviously mosaics of different ancestries. Transposable insertion sequences in E. coli are examples of selfish DNA whose short-term population dynamics are determined mainly by transposition and horizontal transmission among strains balanced against the regulation of transposition as a function of copy number, and negative effects on fitness. Occasional advantageous effects of transposable elements have also been documented.


Presynaptic control as a mechanism of sensory-motor integration. In studies of central nervous system networks, it is synaptic transmission to the postsynaptic soma-dendritic membrane that has received the most attention, in particular in relation to the analysis of sensory-motor integration. Sensory transmission is gated during ongoing movements in both invertebrates and vertebrates, such that it may be depressed in one phase of a cyclic movement and facilitated in another, in order to optimize the execution of the ongoing motor task. This presynaptic modulation is not limited to sensory afferents, but also occurs in synapses of both excitatory and inhibitory premotor interneurons. The modulation can be mediated by the release of different transmitters at axo-axonal synapses, which activate different types of receptors. In addition, presynaptic sensory axons can be coupled via gap junctions, which under certain conditions may mediate a presynaptic facilitation.


2.5 years survival with sequential ganciclovir/foscarnet treatment in a patient with acquired immune deficiency syndrome and cytomegalovirus retinitis. The case with the longest survival time (30 months) after the diagnosis of cytomegalovirus retinitis in a group of 53 patients with the acquired immune deficiency syndrome and cytomegalovirus retinitis (median survival time 8.4 months) is described. The patient developed cytomegalovirus retinitis in his left eye and received intravenous virustatic treatment for 29.5 months. Treatment was started with ganciclovir. After withdrawal from maintenance treatment, a relapse of cytomegalovirus retinitis occurred, which was again successfully treated with ganciclovir. A secondary cataract developed, and cataract extraction was performed. After 12.5 months on treatment with ganciclovir, a change to foscarnet was necessary because of neutropenia. Maintenance treatment with foscarnet was well tolerated for 17 months. The cytomegalovirus retinitis showed no signs of reactivation during this period. Vision in the right eye was preserved until death, and no sign of cytomegalovirus retinitis developed in the right eye. This case report demonstrates that an unusual long survival time is possible in a patient sequentially treated with ganciclovir and foscarnet.


Amplimers with 3'-terminal phosphorothioate linkages resist degradation by vent polymerase and reduce Taq polymerase mispriming. The 3'-->5' exonuclease activity of Vent, a thermostable polymerase from Thermococcus litoralis, enhances DNA replication fidelity but also diverts PCR primers (amplimers) from targeted amplification by degrading their 3' termini. We demonstrate that amplimers with a 3-base 3'-terminal mismatch can be efficiently truncated by Vent to prime DNA polymerizations that compete with the specific amplification reaction. However, amplimers with phosphorothioate bonds joining their 3'-terminal residues are resistant to degradation and demonstrate greatly enhanced priming specificity. Slight destabilization of base-pairing by phosphorothioate bond-linked residues also diminishes extension of mispaired 3' amplimer termini in Taq polymerase-mediated amplifications.


Galactocerebroside mediates Ca2+ signaling in cultured glioma cells. Expression of galactocerebroside (GalC) was detected in human glioma cell line (U-87 MG). Exposure of cells to antibody against GalC and fluoresceinated second antibody showed intense fluorescence on the plasma membrane. Possible involvement of GalC in receptor-mediated transmembrane signaling was explored in this cell line. Antibodies raised against GalC were used as ligands. Binding of anti-GalC to these cells caused a transient increase in intracellular free calcium ([Ca2+]i). The response was observed both in the presence and absence of extracellular calcium demonstrating that the rise in [Ca2+]i induced by anti-GalC was due to an influx of Ca2+ through plasma membrane as well as the release of Ca2+ from intracellular pools. Ca2+ influx was blocked by verapamil, indicating that influx is mediated by voltage-sensitive channels. Our results suggest that GalC can play a role in transmembrane signaling by modulation of voltage-sensitive Ca2+ channels.


[3H]Rauwolscine binding sites in the brains of male tree shrews are related to social status. The aim of the present study was to investigate the influence of social status on central nervous alpha 2-adrenoceptors. Using the specific alpha 2-adrenoceptor antagonist [3H]rauwolscine, binding sites in the brains of dominant and subordinate male tree shrews were quantified by in vitro autoradiography. In 5 of the 14 brain structures investigated, subordinates had significantly lower numbers of binding sites than dominants. These structures were the solitary tract nucleus, the dorsal motor nucleus of the vagus, the periaqueductal gray, the perifornical region of the hypothalamus and the medial nucleus of the amygdala. These brain areas are all intimately involved in the regulation of autonomic functions and of emotional behavior. Also the affinities for [3H]rauwolscine differed between the groups. In 3 nuclei, the solitary tract nucleus, the periaqueductal gray and the medial nucleus of the amygdala, dominants had significantly higher Kd-values than subordinates. This demonstrates the presence of low affinity binding sites in dominants which do not exist in subordinates. It is suggested that the low number of [3H]rauwolscine binding sites in subordinates results from down-regulation of alpha 2-adrenoceptors by high levels of noradrenaline and/or adrenaline. The disappearance of low affinity [3H]rauwolscine binding sites may play an important role in the etiology of psychosocial stress.


Stress-induced increase of extracellular levels of cyclic AMP in rat cortex. Previous studies have suggested that stress may increase levels of cyclic AMP (cAMP) in the brain but these findings have been controversial due to the use of stressful procedures to inactivate brain enzymes. The present experiment therefore used a non-stressful technique, microdialysis, to assay extracellular levels of cAMP in the rat cortex after stress. Experiments were conducted 2 days after implantation of probes in the frontal cortex. Significant increases were found after the mild stressors of restraint or intraperitoneal injection of saline suggesting that increased tissue levels of cAMP had occurred. These responses were potentiated by local infusion of the phosphodiesterase (PDE) inhibitor, rolipram. It is concluded that one or more adenylate cyclase-coupled receptors in the cortex is activated by mild stress and that this activation can be detected in vivo by microdialysis.


Different Na+ currents in P0- and P7-derived hippocampal astrocytes in vitro: evidence for a switch in Na+ channel expression in vivo. Hippocampal astrocytes, derived from postnatal day zero (P0) rats, appear to be pluripotential with respect to sodium current expression in vitro, and display Na+ currents with h infinity midpoints close to -65 up to 5 days in vitro (DIV), and Na+ currents with midpoints close to -85 mV at 6 DIV and thereafter. These astrocytes also exhibit a biphasic pattern of Na+ current density, which is expressed at moderate levels at early times in vitro and decreases throughout the first 5 DIV, prior to expressing a second peak for the duration of time in culture. These observations have been interpreted as suggesting that astrocytes in these cultures display a 'switch' in Na+ channel biosynthesis, so that they express different types of Na+ current (with different h infinity curves) at early and later times in culture. To test the hypothesis that a similar switch in Na+ channel expression occurs in vivo, we have used patch-clamp methods to study Na+ current expression in astrocytes derived from rat hippocampus at various stages of postnatal development, P0, P4, P5 and P7. We observed a biphasic distribution of Na+ current density, which was highest in P0- and P7-derived astrocytes (18 pA/pF and 10.3 pA/pF, respectively); astrocytes derived at P4 and P5 did not express sodium currents. While P0-derived astrocytes show depolarized h infinity curves (midpoints close to -65 mV) at early times in culture, P7-derived astrocytes, studied at comparable times in vitro, display hyperpolarized h infinity curves (midpoints close to -85 mV).(ABSTRACT TRUNCATED AT 250 WORDS)


Evidence for hippocampal calcium channel regulation by PKC based on comparison of diacylglycerols and phorbol esters. Studies using phorbol esters imply that hippocampal Ca2+ channels are regulated by protein kinase C (PKC); however concerns have been raised because in some circumstances phorbol esters have non-specific effects on ion channels. We have tested the hypothesis that PKC modulates Ca2+ channel activity in hippocampal neurons by conducting a detailed comparison of the effects of the diacylglycerols, diC8 and OAG, with those of the phorbol ester, PDBu, on whole-cell and single-channel Ca2+ currents. Close similarity of action of these different activators would support the hypothesis. We found that, like PDBu, the diacylglycerols (DAGs) suppressed whole-cell Ba2+ current (IBa) in a dose-dependent and reversible manner and caused a hyperpolarizing shift in the voltage dependence of steady-state IBa inactivation. Suppression of IBa by diC8 and OAG was not mimicked by an enzymatically inactive diacylglycerol isomer, EGD. The effects of both PDBu and DAGs could be blocked by a specific peptide inhibitor of PKC, and both types of activator depressed IBa when it was recorded in the nystatin perforated-patch mode. In single-channel recordings, DAGs enhanced L-type Ca2+ channel activity in a manner indistinguishable from that of PDBu. Finally, DAGs as well as PDBu markedly increased spontaneous synaptic activity in tissue-cultured hippocampal neurons. The numerous similarities between the effects of DAGs and PDBu strongly support the general conclusion that PKC mediates the effects of these activators and the specific conclusion that PKC modulates Ca2+ channel activity in hippocampal neurons.


Muscimol injections into the nucleus basalis magnocellularis of rats: selective impairment of working memory in the double Y-maze. Anatomical and neurochemical results suggest that the cortico- and amygdalopetal cholinergic neurons of the nucleus basalis magnocellularis (NBM) may receive GABAergic inputs. The present experiments were undertaken to evaluate the possible influence of intra-NBM injections of the GABAA agonist, muscimol, on memory. In two experiments, rats were chronically implanted with guide cannulae placed bilaterally into the NBM. Rats were trained to a criterion of at least 83% correct on each component in a double Y-maze task that allowed a dissociation of working and reference memory. The task began with placement into one of the two end arms of the first Y-maze and the reference memory task was to go to the stem for food. Access to the second Y was then given and the working memory task was to go to the goal arm opposite the arm in the first maze from which that trial began. In experiment 1, pre-trained rats (n = 7) received muscimol (0.5 microliter) in doses of 0, 0.01, 0.1 and 1.0 microgram in a counterbalanced order with re-training to criterion between injections. In experiment 2, pre-trained rats (n = 8) received saline, muscimol (0.1 microgram), the GABAA antagonist, bicuculline (0.01 microgram), and muscimol + bicuculline. Results of experiment 1 revealed that intra-NBM muscimol produced a dose-dependent and differential impairment of working and reference memory. A dose of 0.1 microgram impaired working memory without significantly affecting reference memory; doses of 0.01 microgram and 1.0 microgram affected neither and both types of memory, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)


Chronic selective activation of excitatory opioid receptor functions in sensory neurons results in opioid 'dependence' without tolerance. We previously showed that mouse sensory dorsal root ganglion (DRG) neurons chronically exposed to 1 microM D-ala2-D-leu5-enkephalin (DADLE) or morphine for > 2-3 days in culture become tolerant to the usual opioid inhibitory receptor-mediated effects, i.e. shortening of the duration of the calcium-dependent component of the action potential (APD), and supersensitive to opioid excitatory APD-prolonging effects elicited by low opioid concentrations. Whereas nanomolar concentrations of dynorphin(1-13) or morphine are generally required to prolong the APD of naive DRG neurons (by activating excitatory opioid receptors), femtomolar levels become effective after chronic opioid treatment. Whereas 1-30 nM naloxone or diprenorphine prevent both excitatory and inhibitory opioid effects but do not alter the APD of native DRG neurons, both opioid antagonists unexpectedly prolong the APD of most of the chronic opioid-treated cells. In the present study, chronic exposure of DRG neurons to 1 microM DADLE together with cholera toxin-B subunit (which selectively blocks GM1 ganglioside-regulated opioid excitatory, but not inhibitory, receptor functions) prevented the development of opioid excitatory supersensitivity and markedly attenuated tolerance to opioid inhibitory effects. Conversely, sustained exposure of DRG neurons to 1 nM DADLE, which selectively activates excitatory opioid receptor functions, resulted in characteristic opioid excitatory supersensitivity but no tolerance. These results suggest that 'dependence'-like properties can be induced in chronic opioid-treated sensory neurons in the absence of tolerance. On the other hand, development of some components of tolerance in these cells may require sustained activation of both excitatory, as well as inhibitory, opioid receptor functions.


The membrane properties and Ca-currents of the trigeminal root ganglion cells in primary culture of the marine catfish, Plotosus, studied with whole-cell recordings. Neurons of the trigeminal root ganglion (TRG) were isolated from the marine catfish Plotosus. Collagenase treatment and culture in L15 medium, modified for higher tonicity, were required to remove their myelin sheath. TRG neurons were spherical 15-20 microns in diameter after 1-4 days culture, although they later developed extensive neurites. The membrane properties were studied by whole-cell recording technique. The resting potential was about -63 mV. The specific membrane resistance and capacitance, 5.9 K omega.cm2 and 1.2 microF/cm2, were similar to those of mouse dorsal root ganglion (DRG). The action potential, however, was usually humped, and followed by a long afterhyperpolarization. The maximum firing rate reached only about 70 Hz. Voltage-clamp study revealed TTX-sensitive Na current and TEA-sensitive K current, and in addition, two types of Ca currents: low- and high-voltage activated (LVA and HVA). The HVA current seemed to be involved in hump formation. The LVA current was similar in kinetics to T-type current of chick DRG, and was presumably inactivated at the resting potential, which might be removed during the afterhyperpolarization.


Na+ conductance and the threshold for repetitive neuronal firing. The Hodgkin-Huxley equation for electrogenesis in the voltage clamped squid giant axon was used to predict the effect of altering maximal Na+ conductance (gNa+max) on the repetitive firing process. The main finding was that increasing gNa+max, without changing any other membrane parameter, reduced the threshold current required to evoke repetitive firing. That is, it rendered the membrane hyperexcitable. Threshold for evoking single action potentials was also affected, but much less so. Other consequences of increasing gNa+max were a decrease in the minimum sustainable rhythmic firing frequency (mRFF), a monotonic increase in firing frequency at any given suprathreshold stimulus intensity, an increase in the current value at which intense depolarizing stimuli block rhythmogenesis, an increase in the maximal sustainable firing frequency using intense currents (MRFF), and the consequent expansion of the dynamic range for stimulus encoding. Thus, the control of gNa+max through the regulation of Na+ channel synthesis and membrane incorporation at sites of rhythmogenesis (e.g. axon hillock-initial segment region, or peripheral sensory endings) is a potential regulatory mechanism for neuronal excitability and stimulus encoding.


GABA-induced responses in Purkinje cell dendrites of the rabbit cerebellar slice. Pressure applications of GABA localized to Purkinje cell somas in a rabbit cerebellar slice produced uniphasic hyperpolarizing responses, whereas applications of GABA that were directed at the Purkinje cell dendrites produced complex, triphasic responses with hyperpolarizing and depolarizing components. Both somatic and dendritic application of GABA elicited fast hyperpolarization (GABAhf), but dendritic application also elicited a slower depolarization (GABAd) and a later, long-lasting hyperpolarization (GABAhl). All three types of responses were accompanied by increased conductance. Use of either GABA antagonist, bicuculline or picrotoxin, eliminated the GABAhf and GABAd responses but left the GABAhl response intact. Pressure delivery of the GABA agonist, baclofen, to the dendrites but not the soma elicited a GABAhl response. Application of baclofen paired with membrane depolarization sufficient to elicit local, calcium-dependent dendritic spiking produced a persistent reduction in the GABAhl response, whereas alternating presentations of baclofen and membrane depolarization or presentations of baclofen alone could not. The fact that GABA and baclofen inhibited Purkinje cell activity in the rabbit cerebellar slice and that picrotoxin and bicuculline eliminated some, but not all of the components of the GABA response suggests the presence of both GABAA and GABAB receptors. The ability of baclofen to inhibit Purkinje cells if it was applied to the dendrites but not if applied to the soma suggests that GABAB receptors are located predominantly on Purkinje cell dendrites. The pairing-specific change in the baclofen response suggests the existence of GABAB-mediated modifiability of Purkinje cell dendrites.(ABSTRACT TRUNCATED AT 250 WORDS)


Characterization of monoclonal antibodies to the outer membrane protein (OmpD) of Salmonella typhimurium. A panel of monoclonal antibodies, seven against the trimeric and seven against the monomeric forms to outer membrane protein D (OmpD) of Salmonella typhimurium were produced. The specificities of these monoclonal antibodies for the porin proteins of S. typhimurium and their cross-reactions with Salmonella porins OmpC and OmpF were determined by Western immunoblotting and enzyme-linked immunosorbent assay. We observed that OmpD shared more epitopes and had greater structural similarity with OmpC than with OmpF.


Mutagenesis and chromosome mobilization in Hyphomicrobium facilis B-522. Spontaneously derived antibiotic-resistant mutants of Hyphomicrobium facilis B-522, a restricted facultative methylotroph, occurred at a high frequency on agar plates with low antibiotic concentrations. Mutants specifically defective in methanol oxidation have been obtained using an allyl alcohol direct selection technique. By chemical mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine in the presence of chloramphenicol several stable auxotrophic mutants could be isolated: three leucine auxotrophs, two threonine auxotrophs, and two leucine-methionine double auxotrophic mutants. Optimal conditions for transposon mutagenesis have been developed by comparing several transposon delivery vectors. With the suicide plasmid pRK2013 as a vector, the tetracycline resistance conferring transposon Tn5-132 was introduced into the genome of H. facilis B-522. The following insertion mutants have been obtained: leu-3::Tn5-132, ilv-1::Tn5-132, and pur-1::Tn5-132. Broad host range IncP-1 plasmids could be successfully transferred by interspecific matings. Chromosome mobilization was demonstrated with the conjugative IncP-1 plasmids RP1, R68.45, pMO60, and H. facilis 2189 (leu-2, met-1, mox-1, nfs-1, str-12) as recipient strain. Transconjugants occurred at frequencies ranging from 10(-6) to 10(-8) for each marker.


Further evidence for a possible role of trypsin-like activity in the adherence of Porphyromonas gingivalis. The present study was undertaken to evaluate a possible correlation between the level of trypsin-like activity and the adherence properties of Porphyromonas gingivalis. It was demonstrated that strains with high cell-associated trypsin-like activity attach in higher numbers to human epithelial cells than strains with low levels of trypsin-like activity. To a lesser extent, the same tendency was also noted for the agglutination of human erythrocytes. The ability of P. gingivalis to attach to erythrocytes and epithelial cells was found to be affected by the presence of arginine and thiol protease inhibitors (leupeptin, p-chloromercuriphenylsulfonic acid). The inhibition profile was partially dependent on the age of the bacterial cells used in the adherence assay. It is suggested that adherence of mid-log P. gingivalis cells involves primarily trypsin-like proteases, whereas 2-day-old cells possess additional specific attachment mechanism(s).


Detection of equine herpesvirus and differentiation of equine herpesvirus type 1 from type 4 by the polymerase chain reaction. Although both equine herpesvirus type 1 (EHV-1) and equine herpesvirus type 4 (EHV-4) can be associated with respiratory disease, epizootics caused by EHV-1 are much more serious because the virus can cause abortions and paralysis. It is, therefore, important to identify the type of EHV involved in an outbreak by a test that is quick, sensitive, and reliable. We have adapted the polymerase chain reaction (PCR) to detect and distinguish between EHV-1 and EHV-4 in the same reaction. Primers for PCR were designed from the sequences of the glycoprotein B genes of EHV-1 and EHV-4. The PCR products derived from EHV-1 and EHV-4 were 135 and 326 base pairs, respectively, and could be readily separated by electrophoresis. The identity of the PCR products was confirmed by determining their nucleotide sequence, which agreed with the published sequence of the gB genes. The test could be performed directly on virus pelleted from small volumes (300 microL) of medium in which nasal swabs were transported and did not rely on the presence of infectious virus. The PCR was unaffected by conditions that reduced the infectivity of a virus preparation by 99%. The PCR detected EHV-4 in 5 of 10 nasal mucous samples taken from an outbreak of respiratory disease in race horses. Virus isolation in indicator cells was successful in detecting virus in four of the five samples positive by PCR.


The N-linked oligosaccharides of human lactoferrin are not required for binding to bacterial lactoferrin receptors. The oligosaccharides of human lactoferrin were enzymatically removed with glycopeptidase F, resulting in a preparation containing partial and fully deglycosylated human lactoferrin. The derivatives were separated by Concanavalin A affinity chromatography and compared with native human lactoferrin with respect to their ability to bind to bacterial receptors. Competitive binding experiments demonstrated that the lactoferrin derivatives were equally capable as native lactoferrin in binding to receptors of Neisseria meningitidis, Neisseria gonorrhoeae, and Moraxella catarrhalis. This result indicates that the oligosaccharides on human lactoferrin are not essential for binding to the bacterial receptors.


Serological titers to bovine herpesvirus 1, bovine viral diarrhea virus, parainfluenza 3 virus, bovine respiratory syncytial virus and Pasteurella haemolytica in feedlot calves with respiratory disease: associations with bacteriological and pulmonary cytological variables. Acute and convalescent serum samples were taken from 59 calves with signs of respiratory disease (cases) and 60 clinically normal animals (controls) during their first month in the feedlot. Sera were analyzed for antibodies to bovine parainfluenza 3 (PI3) virus by hemagglutination inhibition, to bovine viral diarrhea (BVD) virus, bovine respiratory syncytial (BRS) virus and bovine herpesvirus 1 (BHV1) by virus neutralization, and to Pasteurella haemolytica by indirect agglutination (PhIA) and cytotoxin neutralization (PhCN) tests. There was minimal evidence of serological activity to BHV1. Serological activity to the other agents occurred commonly and the prevalence of acute titers and their mean values was similar in case and control groups. Mean convalescent PI3 and P. haemolytica (PhIA) titers were higher in controls than cases (p < 0.01) but, otherwise, convalescent titers did not differ between groups. The incidence of seroconversion was similar in both groups for all agents except for PI3 virus which was more frequent in controls than cases (p < 0.0001). There was a positive association between PhIA and CN seroconversion and isolation of P. haemolytica from bronchoalveolar lavage (BAL) fluid (p < 0.1). The measure of agreement (kappa) between seroconversion with the P. haemolytica PhIA and PhCN tests was 0.51. Bacteriological and cytological evaluations of the respiratory tract were made using BAL. No associations were evident between serological titers and pulmonary cytology. A multivariate logistic analysis was used to evaluate associations between disease status and serological, bacteriological and cytological data. Cases were positively associated with the presence of neutrophils and Pasteurella multocida in BAL fluid and negatively associated with PI3 virus and PhIA seroconversion.


Effects of pentoxifylline on equine neutrophil function and flow properties. Pentoxifylline has been reported to improve peripheral vascular circulation by altering the flow properties of blood. To determine if the hemorrheological effects of pentoxifylline were mediated by alterations in neutrophil function and/or flow properties, we evaluated the drug's effects on equine neutrophils in vitro. Pentoxifylline, at a concentration of 1 x 10(-1) M, but not at concentrations of 1 x 10(-6) M to 1 x 10(-2) M, markedly suppressed neutrophil superoxide production, zymosan phagocytosis and adherence to nylon wool. Pentoxifylline failed to improve neutrophil filterability through 3 mu polycarbonate filters at any concentration tested. We conclude that equine neutrophil function and flow properties are unlikely to be affected by pentoxifylline concentrations achievable in vivo.


Antigenic change in feline calicivirus during persistent infection. To determine if antigenic variation occurred during persistent infection of cats with feline caliciviruses (FCV), nine persistent (progeny) isolates from nine different carrier cats were compared antigenically to the original infecting parent strain, FCV 255, by two-way cross-neutralization tests with rabbit antisera. Five of the nine progeny viruses isolated 35 to 169 days after initial infection were antigenically different from the parent strain. These five isolates represented four distinct antigenic phenotypes. The emergence of four distinctly different antigenic variants from a single parent strain indicates that FCV, like many other RNA viruses, exhibits considerable antigenic heterogeneity during replication in its natural host, and supports the hypothesis that antigenic variation contributes to chronic FCV infection.


Proviral detection and serology in bovine leukemia virus-exposed normal cattle and cattle with lymphoma. Twenty-seven cattle with lymphoma and 46 cows from a known bovine leukemia virus (BLV)-infected herd were tested for anti-BLV antibody by the agar gel immunodiffusion (AGID) test and an enzyme-linked immunosorbent assay (ELISA). The polymerase chain reaction (PCR) and Southern hybridization were used to detect BLV provirus in the tumor DNA of the 27 cattle with lymphoma. The PCR was used to detect BLV provirus in the peripheral blood mononuclear cell DNA of the 46 normal known-exposed cattle. Two presumed false negative AGID test results compared to ELISA were found. Of ten cattle three years of age or less with "sporadic" forms of lymphoma, four had BLV provirus in tumor DNA, detectable by PCR. In two of these four, BLV provirus was clonally integrated based on digestion of tumor DNA with restriction enzymes followed by Southern hybridization. The BLV provirus was not detected by PCR in 5 of 17 cattle with "enzootic" lymphoma and two of these five were seronegative. Among normal BLV-exposed cows, 6.5% (3 of 46) were serologically positive and PCR negative; serologically negative and PCR positive cows occurred with the same frequency. Serological and PCR test results, when considered in all cattle (n = 73), had a concordance rate of 83.6%. Discordant test results occurred with approximately equal frequency between serologically positive and PCR negative (7 of 73, 9.6%) and serologically negative and PCR positive (5 of 73, 6.8%) groups. These data suggest that the role of BLV in some "sporadic" bovine lymphomas, previously unassociated with BLV, should be reexamined. The BLV provirus was not demonstrable in the tumor DNA from five adult cattle with lymphoma, suggesting that BLV may not be the etiological agent in all adult bovine lymphomas. The findings of persistently seronegative PCR positive and seropositive PCR negative cattle indicate that further work is needed to more fully understand the host-virus interaction. Present serological screening methods may not have sufficient sensitivity for determining BLV status in some circumstances.


Detection of multiple retroviral infections in cattle and cross-reactivity of bovine immunodeficiency-like virus and human immunodeficiency virus type 1 proteins using bovine and human sera in a western blot assay. Bovine antibovine immunodeficiency-like virus (BIV) antibodies were detected by Western blot analysis (WBA) using a chemiluminescence protocol. Bovine sera with anti-BIV activity, obtained from cows in two dairy herds, had antibodies directed against a variety of BIV-specific antigens indicating chronic infections. These sera were also tested for serological reactivity against bovine leukemia virus (BLV) and bovine syncytial virus (BSV). Cows most commonly had anti-BSV antibodies (12 of 39). Evidence for infection with BSV and BIV or BSV and BLV occurred with almost equal frequency (5 of 39 and 4 of 39, respectively) while only one instance of BIV and BLV coseropositivity was detected. The high prevalence of BSV seropositivity is consistent with a relatively infectious virus, which, as is known, may be transferred congenitally. Similar rates of coseropositivity of BIV or BLV with BSV in this population suggest that BIV is no more infectious than BLV and probably requires prolonged close contact for transmission. Seven of nine cows with anti-BIV antibodies detected primarily human immunodeficiency virus type 1 (HIV-1) p51 and p63 antigens by WBA using an alkaline phosphatase detection system, suggesting that HIV-1 proteins have potential usefulness in screening cattle for BIV seropositivity. Six human sera that showed strong reactivity against multiple HIV-1 proteins and the serum from one of three patients considered to be an "indeterminate" HIV-1 reactor, cross-reacted primarily with BIV p26. This is the first report of human sera with antibody to BIV-specific proteins.(ABSTRACT TRUNCATED AT 250 WORDS)


Pestivirus is a common contaminant in maedi-visna and caprine arthritis-encephalitis virus stocks. Eight different laboratory stocks of maedi-visna or caprine arthritis-encephalitis virus were examined for the presence of pestiviruses by a fixed-cell immunoperoxidase assay with polyclonal and monoclonal antibodies. All of the viral stocks examined were found to contain noncytopathic pestivirus contaminants. The panel of monoclonal antibodies could not type the isolates as being more related to bovine virus diarrhea virus or border disease virus. However, the results did indicate that all isolates were not the same, except for two from the same laboratory where the source of pestivirus contamination may have been common.


Synaptic plasticity in the rat striatum following chronic haloperidol treatment. Administration of the dopamine (DA) antagonist haloperidol leads to the development of behavioral hypersensitivity as well as enhanced neuronal growth when striatal extracts from these animals are incubated with mesencephalic cultures. For determining if alterations in neuronal growth also occur in vivo, the ultrastructure of the neuropil in the dorsolateral quadrant of the striatum from rats treated (24 days) with haloperidol (1.25 mg/kg) was examined by electron microscopy. Haloperidol-treated rats developed statistically significant behavioral hypersensitivity relative to vehicle-treated controls (p < 0.01). Evaluation of the neuropil revealed that haloperidol treatment enhanced, relative to vehicle-treated controls, the overall number of synaptic boutons by 9% (p < 0.01). The number of perforated synaptic profiles as well as the number of double synapses was increased by 20 and 50%, respectively, although this increase was not statistically significant. The number of myelinated axons remained unchanged, while the number of dendritic spines was increased by 21% (p < 0.05). These data suggest that chronic haloperidol treatment enhanced the growth and possible sprouting of presynaptic neurons and also induced postsynaptic plastic changes. These ultrastructural changes may contribute in part to hypersensitivity behaviors.


Scintigraphic evaluation of left ventricular function and correlation with autonomic cardiac neuropathy in diabetic patients. Left ventricular function of 20 diabetic patients was investigated at rest and during hand-grip test using radionuclide ventriculography. The aim of the study was to discuss the correlation of cardiac function with autonomic cardiac neuropathy (ACN) in diabetic subjects. ACN was tested using heart rate response to valsalva maneuver, standing up, deep breathing; blood pressure response to standing up, sustained hand-grip, and additionally corrected QT (QTc) measurements. Plasma glucose regulation was screened with fructosamine levels. Ejection fraction (EF), peak ejection (PER) and filling rates (PFR), times to peak ejection (TPE) and filling (TPF), time to endsystole (TES), TES/T, TPE/T, TPF/T, 1/3 PER, 1/3 PFR, 1/3 EF, 1/3 FF (filling fraction) we calculated. Thirteen patients had ACN. Six patients (30%) had a low EF at rest. As a response to hand-grip, 14 patients (70%) showed a decrease in EF (9 ACN). PFR was low in 10 patients (50%) at rest and in 12 (60%) during hand-grip. The mean rest PER value of ACN+ patients (4.4 +/- 1.3) was significantly higher than that of controls (2.9 +/- 0.5) and patients without ACN (3.4 +/- 0.4; p < 0.05) as well as the mean 1/3 PER value (1.7 +/- 0.5 vs. 1.3 +/- 0.5; p < 0.05). Fourteen patients (70%) had a fall in PER 10 ACN) as a response to hand-grip. The mean TES/T value of patients with ACN (0.44 +/- 0.05) was significantly higher than of those without ACN (0.38 +/- 0.05; p < 0.05). In conclusion, diastolic dysfunction was detected frequently at rest. Systolic parameters were markedly impaired as a response to hand-grip in patients with ACN. Sympathetic overactivity was noted in ACN+ group at rest. Our results indicated that the patients with diabetes and ACN have subclinical left ventricular diastolic dysfunction and symphatic overactivity.