Scanning electron microscopic observations of the periodontal ligament fibers and cells in rat molar teeth. The periodontal ligament of rat molar teeth was observed with scanning electron microscopy (SEM) using two different methods: NaOH maceration and KOH-collagenase treatments. Rat jaws with molar teeth were fixed, demineralized with 10% EDTA, and cut into several pieces. After maceration with 5% NaOH for 5 days at room temperature, the cellular elements were completely removed and the periodontal ligament fibers appeared as bundles of collagen fibrils. The fibers branched and anastomosed but did not spread fibrils randomly. In some regions near the alveolar bone, collagen fibrils circularly binding the fibers were found. When treated with 30% KOH for 7 to 10 minutes at 60 degrees C and with 0.1% collagenase, the periodontal ligament fibers were removed and the cells appeared as spindle and stellate shapes, and combined with the irregular cell processes of each other. Thus, the interaction between the periodontal ligament fibers and cells were three-dimensionally visualized by using the two different methods.


[Detecting and monitoring leprosy neuropathy: which test to chose?]. The purpose of the study is to propose a simple and reproducible test for assessing nerve damage in leprosy. It is applied to the sensory branch of the radial nerve of leprosy patients, prior to any treatment. Skin sensitivity is measured by means of a needle, a drop of ether and some calibrated filaments. These three tests are collated and compared with the results of electromyographic examination of the nerve. The filament calibrated to 0.2 grams gives optimum sensitivity (0.79) and excellent specificity (0.95) in relation to the electromyographic test. Its routine use in the field is simple and reproducible and should result in a greater number of patients receiving the treatment they need.


[Prolonged crushing of the head (the biomechanics, clinical picture, diagnosis and treatment)]. Analysis of the victims of the earthquake in Armenia made it possible to distinguish a form of trauma, unique in biomechanics and pathogenesis, which was not described earlier, namely, prolonged compression of the head (PCH). Its clinical picture is characterized by superimposition and mutual aggravation of general organismic, general brain, cerebral and extracerebral focal symptomatology. Variants of the relations of the syndrome of prolonged compression of the soft tissues of the head and the craniocerebral damages proper are described. A detailed CT characterization of PCH is given. The principles of stage treatment of victims and the indications for compelled nonoperative and surgical tactics in PCH are suggested for the first time.


The pathology of the posterior root ganglia in AIDS and its relationship to the pallor of the gracile tract. The spinal cord and the thoracic and lumbar posterior root ganglia (PRGs) of 14 HIV-positive men and 7 age- and sex-matched controls were studied by routine histology, morphometric analysis of the number of nodules of Nageotte (nN) and the diameters of sensory ganglion cells, immunohistochemistry and in situ hybridization. In 7 patients (2 of whom had evidence of cytomegalovirus ganglionitis) there were increased numbers of nN and diffuse, mild infiltration with CD45R+ T lymphocytes; no B lymphocytes were observed. Macrophages were increased in number in all cases. Whenever more than one ganglion was examined from the same patient, the appearances were similar in all. There was no alteration in the distribution of ganglion cell diameters. Changes in the spinal cord included vacuolar myelopathy (5 cases), HIV myelitis (1 case), microglial nodules (3 cases) and pallor of the gracile tracts (GTP) in 7 cases, in 6 of whom it co-existed with increased numbers of nN. Seven cases had no abnormalities, except the increase in number of macrophages in PRGs. In spite of a correlation between sensory nerve cell loss and GTP our findings suggest that other mechanisms, such as 'dying back' may contribute to the pathogenesis of GTP. Moreover, sensory disturbances were found most commonly in association with nerve cell loss; however, loss of sensory ganglion cells was not necessarily associated with evidence of sensory impairment.


Histologic confirmation of endometriosis in different peritoneal lesions. The aim of this study was to compare the macroscopic evaluation of peritoneal abnormalities suspected of endometriosis with the histologic examination of the tissue. Peritoneal biopsies from 152 patients investigated for gynecological problems (94) or undergoing tubal sterilization (58) were taken from macroscopically abnormal peritoneum, and examined for the presence of endometriosis. Endometriosis was histologically confirmed by the presence of both endometrioid glands and stroma. Endometriosis was confirmed in 78 of the patients (51%). If the diagnostic criteria were extended also to include endometrioid stroma lacking glands, but containing iron pigment and/or hemorrhage, 82 patients (54%) were positive for endometriosis. Endometriosis was histologically confirmed in 76% of pigmented lesions, in 57% of clear or red papules, in 12% of peritoneal pockets, and in 16% when only fibrosis or scarring was observed. Half of the samples with confirmed endometriosis exhibited cyclic activity. The reproducibility of the histologic evaluation was satisfactory. A high rate of negative biopsies emphasizes the need of taking biopsies from peritoneal lesions suspected of endometriosis for confirmation of the diagnosis.


Microalbuminuria as a predictor of preeclampsia. Urinary albumin concentration (UA) and albumin/creatinine ratio (UA/UC) in early morning specimens were assayed in 225 consecutive pregnant women at 20, 26, 28 and 30 weeks of gestation. 193 did not develop preeclampsia (control group), 14 developed preeclampsia later (preeclamptic group), 9 were excluded and 9 dropped out. Reference intervals of UA and UA/UC of healthy pregnant women (wk 20-30) was obtained. A statistically significant increase in urinary albumin excretion was observed with increasing gestational age as a normal phenomenon. There was no significant difference in the values of UA and UA/UC in the preeclamptic group when compared with the control group at the same stage of gestation. This indicates that microalbuminuria cannot be used as a predictor of preeclampsia.


Triplet, quadruplet and quintuplet pregnancies. Management and outcome. The management and outcome of 46 pregnancies, 37 triplets, 7 quadruplets and 2 quintuplets, were analysed. Management of pregnancies, initiated upon diagnosis of multiple pregnancy, included bed rest, beta-mimetic agents, dexamethasone late in the second trimester and selective cerclage. The mean gestational age at labor was 235 days in triplet pregnancies, 241 for quadruplets and 220 days for quintuplets. Fifty-four percent of the deliveries were by cesarean section and the remainder per vaginam. The mean weight of the neonates was 1809 g for the triplets, 1837 g for quadruplets and 1284 g for the quintuplets. The mean overall Apgar score was 8.13, total perinatal mortality 14.8% and 9.4% in cases more than 28 weeks. There was no statistically significant difference in the outcome for triplets born vaginally or by cesarean section. In recent years there has been a pronounced reduction in neonatal mortality, dropping from 17.3% during 1970-78 to 5.9% from 1979 to 1983 (p less than 0.05), probably due to the improved neonatal treatment.


Preinductive cervical ripening with prostaglandin E2 in women with one previous cesarean section. Thirty term pregnant women with one previous cesarean section and with unripe cervices were given 0.5 mg prostaglandin E2 in gel strictly intracervically for cervical ripening and labor induction. Fifteen out of these 30 women (50%) were vaginally delivered within 24 hours. The cervical ripening/labor induction was considered a failure in two women. In one woman, an episode of hypercontractility was registered. After tocolytic therapy the uterine activity was normalised and the woman had a normal vaginal delivery. The frequency of cesarean sections was 8/30 (27%). At the operations no insufficiencies in the uterine scars were noted. Conclusively, strict intracervical application of 0.5 mg prostaglandin E2 in gel can be used for cervical ripening and labor induction also in women with one previous cesarean section.


Survey of the treatment of Chlamydia trachomatis infection of the female genital tract. A questionnaire was sent to every tenth (n = 302) general practitioner in Norway. The physicians were to indicate their choice of antibiotic regimen for the treatment of genital chlamydial infections in women. Sixty-nine percent of the practitioners responded. The choice of treatment varied widely between the respondents. Forty-two different regimens were used for chlamydial cervicitis, 34 for cervicitis in pregnancy and 63 for probable pelvic infection. Of the prescribed treatments for these three diagnoses 14%, 5% and 46%, respectively, were compatible with the advice of the World Health Organization or the Centers for Disease Control. For the three previously mentioned diagnoses 49%, 79% and 43%, respectively, of the practioners would prescribe an antibiotic in either smaller doses, fewer daily doses or shorter duration than recommended. There is an urgent need to improve and standardize Norwegian general practitioners' treatment of chlamydial infection in women.


Tumor debulking followed by immediate combination chemotherapy in advanced ovarian carcinoma. Phase II. Fifty-one consecutive, previously untreated patients with FIGO stage III or IV epithelial ovarian carcinoma were enrolled in a prospective study over a period of 7 years (1981-1988). Significant improvement has been noted in patients with advanced ovarian cancer following the administration of modified PAC 1 (adriamycin, cytoxan and cyclophosphamide) immediately following primary debulking surgery (within 24 hours) and repeated for 11 monthly cycles. A second look operation was found as an important prognostic indicator. Thirty-one patients (all Stage III) completed the chemotherapy course and were eligible for second look operation. Of these, 21 patients (68%) showed negative second look. Of the patients with negative second look, 17 of 21 (81%) are alive with a mean survival of 61 months (range 19-103 months) after diagnosis. Among those with positive second look only 3 of 10 are alive with a mean of 41 months after diagnosis. The remaining 20 patients (13 stage III and 7 stage IV) did not undergo second look laparotomy. Only 2 of these 20 patients are alive with a mean of 35 months after diagnosis. Other factors of significant importance were: age and completion of chemotherapy course. Patients appeared to benefit from the combined regimen of optimal debulking surgery, completion of 12 courses of chemotherapy with the first course administered immediately after surgery and second look operation. Tumor type or histologic grade did not seem to influence results. The early use of chemotherapy was well tolerated and toxicity was minimal and acceptable.


Oral contraceptives and cancer of the cervix uteri. A meta-analysis. Because the findings of epidemiologic studies of the relationship between oral contraceptive use and cervical cancer have not been consistent, we reanalyzed the relationship. Meta-analysis of studies published to date. Papers were located by searching the MEDLINE data base, supplemented by a hand search of all the references in the articles recovered. Studies were graded as to quality. Two meta-analyses were performed: one including all the studies gathered and one including methodologically acceptable studies only. The method of Woolf was used to combine relative risks. Heterogeneity of the effect was assessed. Fifty-one published studies were collected: 21 case-control, 18 cross-sectional, and 12 cohort designs. Twenty-one of these were considered as methodologically acceptable, but only 18 could be pooled. The main results observed were: relative risks was 1.52 (1.3-1.8) for dysplasia, 1.52 (1.3-1.8) for carcinoma in situ, and 1.21 (1.1-1.4) for invasive cancer. A significant linear dose-response effect was observed in dysplasia, carcinoma in situ, and invasive cervical cancer. Heterogeneity of the effect was present in some of the former estimates. Oral contraceptive use may be a risk factor for all stages of the natural history of cervical cancer, which may imply an initiator effect. Limitations to this research are discussed.


Transvaginal sacrospinous colpopexy for vaginal vault and complete genital prolapse in aged women. Twenty-five women (mean age 72.8 years) with massive eversion of the vagina were treated with transvaginal sacrospinous ligament colpopexy between 1986 and 1990. Nine of them had a posthysterectomy vaginal prolapse; 16 had complete genital prolapse and coincident vaginal hysterectomy was performed. The operation was performed under spinal anesthesia in all cases except one with general anesthesia. Simultaneous anterior colporrhaphy was done in 88%, repair of enterocele in 72% and posterior colpoperineorrhaphy in 88% of all cases. There were no intra- or post-operative complications. Vaginal vault prolapse did not recur during a mean follow-up period of 2.8 years in 22 cases. Three patients developed asymptomatic cystocele or enterocele, and 5 (23%) women had a curtailed vagina. Sacrospinous ligament colpopexy under regional anesthesia is an effective and suitable operation for aged women with vaginal vault and complete genital prolapse. The operation is also a safe and fairly simple procedure if the anatomic relationship of the nearby structures is known.


Fetal heart rate patterns preceding intrauterine death during labor. A fetus of 37 weeks died in labor during recording of the fetal heart rate patterns. Five hours earlier a reactive non-stress test had been obtained. No other cause of death than a tight nuchal cord was found.


Pasteurella multocida chorioamnionitis from vaginal transmission. A 21 year old primigravida with a twin pregnancy developed Pasteurella multocida chorioamnionitis. Infection occurred at 27 weeks gestational age after prolonged rupture of membranes. The twin in the separate sac presenting proximal to the cervix suffered infection and died shortly after birth whereas the other twin was not infected. The bacterium is believed to have caused ascending infection from asymptomatic colonization of the vaginal tract.


Spurious elevation of follicle-stimulating hormone. Serum concentrations of follicle-stimulating hormone (FSH) were measured in a 33-year-old eumenorrhoeic woman with primary infertility. Postmenopausal levels were obtained. Using an alternative assay, the patient was found to have normal levels of FSH. The probable cause of the misleading result is discussed.


The coexistence of stromomyoma and uterine tumor resembling ovarian sex-cord tumors. Report of a case and immunohistochemical study. The case of a 40-year-old female with a uterine tumor resembling an ovarian sex-cord tumor, located within another neoplastic nodule having the histopathological features of a stromomyoma is reported. Light microscopic examination of the uterine sex-cord-like tumor revealed a highly cellular tumor tissue with no specific differentiation, consisting of solid nests and anastomosing cords and a few pseudo-tubular structures. The vacuolated cytoplasm of the lipid-rich cells proved vimentin-positive, but desmin- and keratin-negative. The coexistence of the two variants of endometrial stromal tumor was interpreted as arising from endometrial stroma or multipotential uterine mesenchyme that showed a diverse differentiation toward ovarian sex-cord stroma and uterine smooth muscle with interspersed stromal cell clusters.


Postpartum rupture of a subcapsular hematoma of the liver. We report a case of postpartum rupture of a subcapsular hematoma of the liver. The etiology, pathology, diagnosis and treatment of this condition are discussed. We emphasize the modern concepts in treatment of hepatic hemorrhage. This knowledge and an awareness of the possible diagnosis will help to decrease the high morbidity and mortality rates associated with this disorder.


Relapsing thymic carcinoma during pregnancy. A 23 year old woman, who was known to be in remission from thymic carcinoma, presented in her first trimester. She had previously been treated with radiation and chemotherapy. Her pregnancy was complicated by fever and contractions at 30 weeks and ended with the birth of a live preterm male infant. Subsequent investigation revealed a massive relapse of her mediastinal tumor. This case reinforces the devastating consequences that pregnancy has on thymic hyperplasia. Of nine previously reported cases, five of the women died either during pregnancy or within six months and only one was alive longer than five years. Therapeutic abortion should be considered in the pregnant woman with known thymic neoplasia.


Metastatic choriocarcinoma with coexistent term pregnancy following four recurrent consecutive moles. A case is reported of a 38-year-old woman with metastatic choriocarcinoma which followed four recurrent consecutive hydatidiform moles occurring within the preceding 5-year period. She conceived while having pulmonary metastasis, had a coexistent normal pregnancy and delivered a normal infant at term. Surgical resection of the lung lesion together with chemotherapy accomplished a sustained remission, with no evidence of disease after 20 months' follow-up.


[A case of metastatic urinary bladder tumor from gastric carcinoma]. We report a case of metastatic bladder tumor from gastric carcinoma. A 55-year old male patient was referred to our urological clinic with a complaint of frequent urination and voiding pain. He had undergone total gastrectomy for poorly differentiated adenocarcinoma, signet-ring cell type, 9 months earlier. Computed tomographic scan revealed a thick bladder and rectum wall all around. Punch biopsies from vesical and rectal wall revealed metastatic adenocarcinoma, signet-ring cell type. There were no other metastatic sites. Systemic chemotherapy was done with a combination of mitomycin-C, 5-fluorouracil and cytosine arabinoside. This chemotherapy was effective and complete remission was obtained at bladder and rectum. Six months after chemotherapy, peritoneal recurrence developed and he died 9 months after chemotherapy. However no recurrence of bladder tumor was detected. This was a quite rare case of metastatic bladder tumor characterized by good response to systemic chemotherapy.


Intensive etoposide and carboplatin chemotherapy for advanced non-small-cell lung cancer. A phase II trial of the Cancer and Leukemia Group B. From April 2 to July 9, 1989, Cancer and Leukemia Group B (CALGB) conducted a Phase II study of etoposide and carboplatin in advanced (AJC Stage IIIb-IV) non-small-cell lung cancer (NSCLC) patients whose performance status (PS) was 0-2. The combination was given at the maximum tolerated dose as defined in a prior CALGB study. Of 76 eligible patients with follow-up data, complete responses were achieved in three patients (4%) and partial responses, in five patients (7%). One patient (1%) with evaluable disease showed improvement. There was only one partial response in the PS 2 patients. Performance status was a predictive factor for response or improvement (p = 0.0368). A high incidence (74%) of severe or life-threatening hematologic toxicity and fatal sepsis in four patients was a reflection of the intensity of the chemotherapeutic regime. The median survival from study entry was 7.4 months. Thirty-seven percent of the patients survived beyond 1 year; the median survival for the PS 0-1 patients was 11.7 months for the PS 2 patients, 4.1 months. Median time to treatment failure was 3.9 months, but treatment had not failed in 9% of the patients after 1 year, all of whom were PS 0-1 at time of study entry. Although the response rate with this dose-intensive chemotherapy regimen was disappointing, the median survival of PS 0-1 patients was equivalent to that of Stage III NSCLC patients in prior CALGB studies. In patients with NSCLC who are treated with chemotherapy, PS may be as important a prognostic factor as stage, when median survival is used as an endpoint.


The impact on survival by adjuvant chemotherapy and radiation therapy in stage II non-small-cell lung cancer. Forty-nine consecutive patients with pathologic Stage II non-small-cell lung cancer treated over a 15-year period were retrospectively reviewed. The treatment strategy evolved during the period of review. Early patients were treated with surgery alone (S); subsequent patients were treated with adjuvant radiation therapy (SR); and more recent patients were treated with postoperative chemotherapy and radiation therapy (SRC). Fifteen patients received S alone, 10 patients received SR, and 24 patients received SRC. The median survival time (MST) of all 49 patients was 20 months, and the estimated 5-year survival was 25%. The MST of patients in each of the three treatment arms was S-6 months; SR-19 months; and SRC-25 months. The majority of patients died from systemic relapses or second primary lung cancers. The addition of adjuvant therapy (SR, SRC) significantly improved the MST of patients compared to surgery alone (S). The overall survival of patients did not change between treatment arms.


Fast neutron radiation for recurrent pleomorphic adenomas of the parotid gland. Between 1984 and 1989, six patients with recurrent pleomorphic adenomas of the parotid gland were treated with fast neutron radiotherapy. All of the patients in this series had one or more unfavorable prognostic features, which would predict suboptimal local tumor control rates with the utilization of standard surgical and adjuvant photon/electron radiation therapies. These prognostic features included multiply recurrent disease in three patients, known gross postoperative residual disease in one patient, extensive unresectable disease in one patient, and multiply recurrent disease plus gross postoperative residual disease in one patient. Patients had an average of three surgeries for recurrent adenomas before their neutron irradiation. The median period from the original diagnosis to the time of neutron treatment was 11 years, with a range of 1.3-32 years. With a median follow-up of 52 months, an overall local control rate of 100% was achieved. Only one grade III (European Organization for Research and Treatment of Cancer-Radiation Therapy Oncology Group scale) late complication occurred secondary to the neutron irradiation. Similar to their malignant counterparts, benign salivary gland neoplasms appear to display excellent response and control rates to treatment with fast neutron irradiation. Neutron irradiation of pleomorphic adenomas should be considered an appropriate therapeutic approach in situations, such as recurrent disease and postoperative gross residual disease, where one may be concerned about potential local failures with the use of standard surgical and radiotherapeutic modalities. Neutron radiation also carries a low risk of facial nerve damage, a consideration that may argue for limiting the extent of surgical resection of recurrent disease.


Activity of azithromycin (CP-62,993) and erythromycin against chloroquine-sensitive and chloroquine-resistant strains of Plasmodium falciparum in vitro. Several antibiotics, including the macrolide erythromycin and the azalides azithromycin (CP-62,993) and CP-63,956, that inhibit protein synthesis on 70S ribosomes demonstrated antimalarial effects in vitro against two strains of Plasmodium falciparum, one sensitive to chloroquine and the other resistant. In 48-hr incubations, erythromycin was 10-fold less potent than the azalides against the chloroquine-resistant strain. Erythromycin and the azalides were essentially equipotent against the chloroquine-sensitive strain. An additive effect occurred with the azalides in combination with chloroquine against both strains, but this was not seen with erythromycin.


Biotinylated 1012-S conjugate as a probe ligand for benzodiazepine receptors: characterization of receptor binding sites and receptor assay for benzodiazepine drugs. A nonisotopic receptor assay using the biotin-1012-S conjugate was developed and the usefulness of this conjugate as a probe ligand for the benzodiazepine receptor was evaluated. The conjugate was incubated in a receptor suspension, and then the concentration of free conjugate in the supernatant was determined nonisotopically with a solid-phase avidin-biotin binding assay. Studies on the ligand saturation with the conjugate demonstrated that the conjugate has very high affinity and specificity for the receptors and the biotin labeling does not decrease the affinity of 1012-S. This assay method was applied to the characterization of binding sites of benzodiazepine receptors in cow brain. Competition interactions between the conjugate and benzodiazepine drugs gave well-defined dose-response curves. These results confirm the possibility that this conjugate could serve as a probe for the study of receptor-ligand interactions and provide the basis of a new nonisotopic receptor assay for benzodiazepine drugs.


Detection of the neor gene expression in animal cells after genetic transformation. A reliable assay is reported for the detection of the marker gene aminoglycoside phosphotransferase activity in cells that express this enzyme transiently or as a result of stable genetic transformation. This method combines the simplicity of the dot assays with the reliability of the more elaborate and time consuming electrophoretic or chromatographic methods. Inhibition of phosphatases and protein kinases during the reaction reduces labeled ATP consumption by these enzymes. As a result, this assay allows the detection of approx 10 times lower levels of the enzyme than currently used methods. To detect the expression of reporter genes in transformed cells aminoglycoside phosphotransferase can be used as well as the widely used chloramphenicol acetyltransferase enzyme.


Enzymatic syntheses of DNA-silicas using DNA polymerase. Oligothymidylic acids couple to an activated ester silica (N-hydroxysuccinimidyl-silica) only when they contain an added aminoalkyl group. Heteropolymeric oligomers containing other nucleotide bases were shown to also couple by way of the nucleotide base (adenine, cytosine, or guanine); however, when a heteropolymeric oligonucleotide also contains a 5'-aminoalkyl moiety, coupling by way of the latter is the favored reaction. When duplex hybrids of oligonucleotides are formed, the nucleotide bases are protected from chemical coupling. Coupling by way of nucleotide bases would be detrimental to some chromatography experiments. On the basis of these observations, two different procedures were developed to produce DNA-silicas in which a single strand of the DNA is coupled by only its 5'-terminus. In the first of these, the polymerase chain reaction was used with a 5'-aminoalkyl primer to make a duplex DNA with one strand containing the 5'-aminoalkyl group and the duplex DNA is then coupled to the activated ester silica. This yielded a silica containing about 0.17 nmol of a 242-mer per gram silica which bound only probes specific for the coupled strand. In the other procedure, a template DNA strand was poly(A) tailed and hybridized to (dT)18-silica. DNA polymerase I (Klenow large fragments) was then used to copy the template-specified sequence directly onto the 3'-terminus of the (dT)18. This procedure yielded about 1.2 to 2.7 nmol DNA copied/g of silica of a specific 21-mer sequence. The DNA-silica produced selectively hybridized only with complementary sequences and not with DNA lacking that sequence. Either of these procedures thus produces DNA-silicas from heteropolymeric DNA sequences with a predetermined, specific 5'-terminal site of attachment.


A separation method for the assay of adenylylcyclase, intracellular cyclic AMP, and cyclic-AMP phosphodiesterase using tritium-labeled substrates. A method for the separation of cyclic AMP from adenosine and polyvalent adenine nucleotides is described. The method consists of the sequential elution of adenosine and cyclic AMP from a single column of acidic aluminum oxide (alumina) with dilute hydrochloric acid and ammonium acetate. Adenosine, adenine, xanthine, and hypoxanthine are rapidly eluted with the application of 0.005 N hydrochloric acid while cyclic AMP remains adsorbed to the alumina. A subsequent application of 0.1 M ammonium acetate elutes more than 90% of the cyclic AMP. Under these conditions, polyvalent nucleotides (AMP, ADP, and ATP) remain adsorbed to the alumina. The method permits the measurement of adenylylcyclase activity using [3H]ATP as the labeled substrate. The same technique can be used to measure the accumulation of cyclic AMP in intact cells after labeling the ATP pool with [3H]adenine. With slight modification, the technique can be used to measure the activity of cyclic-AMP phosphodiesterase using [3H]cyclic AMP as the substrate. The proposed technique provides rapid, highly reproducible assays using inexpensive, disposable columns.


Effect of postdischarge surveillance on rates of infectious complications after cesarean section. Decreases in length of stay for surgical procedures and increased outpatient surgery affect accuracy of surgical wound infection rates. To assess accuracy of rates for infectious complications after delivery by cesarean section, we implemented postdischarge surveillance at our hospital (4800 annual deliveries). Physician questionnaires were used. Response rate was greater than 90%. During the 5 months before postdischarge surveillance the overall infection rate was 1.6%; afterward the rate increased to 6.3% (p = 0.0003). Approximately 59% of infectious complications would have gone undetected with only inpatient surveillance. We conclude that postdischarge surveillance is necessary for an accurate determination of rates of infectious complications. The need among this population reflects relatively short postpartum hospitalization and emphasis on outpatient management of postoperative complications.


Analysis of genetic variation of epizootic hemorrhagic disease virus and bluetongue virus field isolates by coelectrophoresis of their double-stranded RNA. Thirty-two bovine field isolates of bluetongue virus (BTV), 6 field isolates of epizootic hemorrhagic disease virus (EHDV) from deer, 4 BTV prototype serotypes (10, 11, 13, and 17), and 2 EHDV prototype serotypes (1 and 2) were coelectrophoresed, using polyacrylamide gels. Field isolates were obtained from various regions of the United States. Analysis of polyacrylamide gels and scattered plots generated for comparison of migration patterns for different isolates within each serotype of BTV revealed wide variation among the individual segments. The BTV serotypes 10 and 11 had more variation, compared with BTV serotypes 13 and 17, especially for migration of genome segment 5. A definitive correlation was not seen between the double-stranded RNA migration profiles on polyacrylamide gel electrophoresis, geographic origin, herd of origin, or year of collection. One BTV field isolate contained more than 1 electropherotype, with 2 bands at the segment-7 position, and it was further characterized as BTV serotype 11. Segments 2 and 5 of EHDV isolates were more variable in their migration than were the other gene segments. Generally, migration profiles for EHDV double-stranded RNA were more variable, compared with those of BTV isolates. Although a correlation was found between migration profiles and serotype of 2 isolates of EHDV, a study of additional EHDV isolates is required before the diversity of electrophoretic patterns of EHDV can be determined.


Response of colostrum-deprived cynomolgus monkeys to intragastric challenge exposure with simian rotavirus strain SA11. The infectivity and pathogenic potential of a cell culture-adapted simian rotavirus was evaluated in colostrum-deprived newborn and infant cynomolgus macaques (Macaca fascicularis). Intragastric challenge exposure with the simian rotavirus strain SA11 on postpartum day 2 induced diarrhea in 5 of 5 colostrum-deprived newborn monkeys. Compared with sham-inoculated controls, 3 of the 5 inoculated monkeys also manifested reduced body weight gain during the initial 5 days after challenge exposure. Rotavirus was detected in feces of 3 challenge-exposed monkeys for up to 2 days after inoculation. Evaluation of antibody response after rotavirus inoculation was obscured by high but variable prechallenge-exposure serum titers of rotavirus-specific antibody. Preexisting serum titer of neutralizing antibody in newborn monkeys was not predictive of clinical response to inoculation with rotavirus SA11. Two 90-day-old infant monkeys with low serum neutralizing antibody titer did not have diarrhea, reduced weight gain, or antibody response after oral inoculation with rotavirus SA11. Results of these challenge-exposure studies in newborn cynomolgus monkeys were consistent with a heterologous host-rotavirus model and indicate that neonatal serum antibody of maternal origin may not be associated with resistance to rotavirus-induced disease.


Effect of changes in urine pH on plasma pharmacokinetic variables of ampicillin sodium in horses. The effect of urine pH on plasma disposition of ampicillin sodium was evaluated. A single dose of 10 mg/kg of body weight was administered IV to Thoroughbreds with alkaline (pH greater than 8.0) or acidic (pH less than 4.5) urine. Urine alkalinity was achieved and maintained by oral administration of up to 400 mg of sodium bicarbonate/kg/d, and acidity was achieved and maintained by oral administration of up to 400 mg of ammonium chloride/kg/d. Ampicillin sodium was measured in the plasma of horses by use of an agar diffusion microbiological assay with Bacillus subtilis as the test organism. The plasma disposition kinetics of ampicillin sodium best fitted a 2-exponential decay pattern, and statistically significant differences were not evident in elimination half-life, area under the plasma concentration time curve, volume of distribution, or body clearance rate between horses with alkaline or acidic urine. Results indicate that changes in urine pH over a range encountered in clinically normal horses are unlikely to affect plasma pharmacokinetic variables of ampicillin sodium after IV administration of the drug.


[A failure of conduction anesthesia in a patient with latent pathology of the peripheral nervous system]. Patients who have been long exposed to ultrasound and high-frequency currents at work are likely to develop diseases of the peripheral nervous system and thus the risk of nerve damage during conduction anesthesia. Profound examination of the peripheral nervous system before surgery is necessary in such patients. In patients with this pathology general anesthesia will be the technique of choice during surgery on the lower extremities.


Duration of pituitary and adrenocortical suppression after long-term administration of anti-inflammatory doses of prednisone in dogs. Duration and magnitude of hypothalamic-pituitary-adrenal axis suppression caused by daily oral administration of a glucocorticoid was investigated, using an anti-inflammatory dose of prednisone. Twelve healthy adult male dogs were given prednisone orally for 35 days (0.55 mg/kg of body weight, q 12 h), and a control group of 6 dogs was given gelatin capsule vehicle. Plasma cortisol (baseline and 2-hour post-ACTH administration) and plasma ACTH and cortisol (baseline and 30-minutes post corticotropin-releasing hormone [CRH] administration) concentrations were monitored biweekly during and after the 35-day treatment period. Baseline plasma ACTH and cortisol and post-ACTH plasma cortisol concentrations were significantly (P less than 0.05) reduced in treated vs control dogs after 14 days of oral prednisone administration. By day 28, baseline ACTH and cortisol concentrations remained significantly (P less than 0.05) reduced and reserve function was markedly (P less than 0.0001) reduced as evidenced by mean post-CRH ACTH, post-CRH cortisol, and post-ACTH cortisol concentrations in treated vs control dogs. Two weeks after termination of daily prednisone administration, significant difference between group means was not evident in baseline ACTH or cortisol values, post-CRH ACTH or cortisol values, or post-ACTH cortisol values, compared with values in controls. Results indicate complete hypothalamic-pituitary-adrenal axis recovery 2 weeks after oral administration of an anti-inflammatory regimen of prednisone given daily for 5 weeks.


Increased numbers of duodenal mucosal mast cells in turkeys inoculated with hemorrhagic enteritis virus. The relation between average duodenal mast cell count, duodenal mucosal mast cell numbers, duodenal connective tissue mast cell numbers, circulating basophil numbers, heterophil-to-lymphocyte ratio, and lesion score were studied to gain an understanding of the events that may lead to intestinal lesion formation associated with hemorrhagic enteritis virus (HEV) infection. Changes in vascular permeability in the duodenum in birds inoculated with HEV were examined, using colloidal carbon and ferritin as vascular markers. Turkeys inoculated with HEV had significantly (P less than 0.05) higher duodenal mast cell counts than did noninfected controls. Birds inoculated with HEV had significantly (P less than 0.05) more mucosal mast cells than did phosphate-buffered saline solution-inoculated birds. Connective tissue mast cell and basophil numbers were unaffected by viral inoculation. Thermal stress did not have significant effect on lesion severity, but did increase number of birds that developed the characteristic intestinal lesions. The heterophil-to-lymphocyte ratio was significantly (P less than 0.05) higher in HEV-inoculated birds, compared with phosphate-buffered saline solution-inoculated controls. Increase in vascular permeability was only detected in HEV-inoculated birds with intestinal lesions. Results indicate that mast cells, and the vasoactive mediators contained within mast cells, may be important in the early manifestation of HEV infection. They also provide a possible mechanism through which biochemical and physiologic changes characteristic of HEV infection can occur.


Do leukotrienes mediate bile acid-induced gastric mucosal injury? Leukotrienes C4 and D4 are potent vasoconstrictors and have been proposed as mediators of the severe gastric mucosal injury caused by a variety of necrotizing agents. The purpose of this study was to investigate the role of leukotrienes on the less severe gastric mucosal injury caused by low concentrations of bile acid. Prior to injury with 5 mM acidified taurocholate (pH 1.2), rat stomachs were pretreated with either normal saline, leukotrienes C4 or D4 (10(-6), 10(-8), and 10(-9) M), or SKF-104353 (a leukotriene D4 receptor antagonist 10(-7) M). Injury was assessed by measuring net transmucosal hydrogen ion flux, luminal appearance of DNA, and histologic injury. Topical pretreatment with LTC4 and LTD4 significantly increased bile acid-induced luminal hydrogen ion loss and DNA accumulation in a dose-dependent manner. Leukotriene receptor blockade with SKF-104353 significantly decreased these parameters. Thus, both LTC4 and LTD4 exacerbate the gastric mucosal injury caused by the application of low concentrations of bile acid while leukotriene receptor blockade reduces this injury (corroborated by histologic injury analysis). This study suggests that leukotrienes may be mediators of bile acid-induced gastric mucosal injury.


Hepatitis C virus infection in patients with essential mixed cryoglobulinemia. To study the association between hepatitis C virus (HCV) infection and essential mixed cryoglobulinemia. Wards and clinics of the Ospedali Riuniti di Bergamo and Ospedale di Treviglio e Caravaggio, Italy. Fifty-one patients with essential mixed cryoglobulinemia associated with glomerulonephritis and 45 controls with noncryoglobulinemic glomerulopathies. Antibodies to hepatitis C virus (anti-HCV) in sera from patients with essential mixed cryoglobulinemia and from controls, using two enzyme-linked immunosorbent assays (c100 ELISA and c22/c200 ELISA) and a recombinant immunoblot assay (4-RIBA); cryoprecipitate anti-HCV before and after use of dithiothreitol, a substance able to destroy IgM antibodies with rheumatoid factor activity, in patients with essential mixed cryoglobulinemia; serum HCV RNA by polymerase chain reaction in patients with essential mixed cryoglobulinemia. In patients with essential mixed cryoglobulinemia, the c22/c200 ELISA detected anti-HCV in 98% of serum samples (95% CI, 90% to 100%), whereas the rate of reactivity remained at 2% (CI, 0% to 12%) in the control group (P less than 0.0001). These results were confirmed by the 4-RIBA in 66% of patients with essential mixed cryoglobulinemia. The study of cryoprecipitate by c100 ELISA showed anti-HCV in 41% (Cl, 28% to 56%) of patients. After dithiothreitol, the rate of reactivity increased to 94% (CI, 84% to 99%; P less than 0.0001 by the McNemar paired chi-square test), suggesting that the elimination of rheumatoid factor leads to unmasking of anti-HCV in cryoprecipitate. Polymerase chain reaction detected HCV RNA in 13 of 16 sera from patients with essential mixed cryoglobulinemia. The extremely high prevalence of anti-HCV in serum and cryoprecipitate along with the frequently associated serum HCV RNA suggests a close relation between essential mixed cryoglobulinemia and chronic HCV infection.


[Autoimmune diseases after unusual exposure to silica or silicones. 3 cases]. A woman, who had intensively used an abrasive cleansing powder rich in silica for 12 years, developed a connective tissue disease consisting of destructive inflammatory oligoarthritis, Gougerot-Sjögren's syndrome, Raynaud's syndrome, anti-nuclear antibodies and hypocomplementemia. Another woman, who had sprayed silicone-glaze on cables for 15 years, suffered from edematous scleroderma. A man, who had sprayed the same silicone-glaze on cables over a 20 year period, had a subluxing arthropathy. These autoimmune syndromes were compared with diseases classically induced by exposure to silica and silicone. To the best of our knowledge, these means of exposure have not been reported previously and merit being known in the aim of prevention.


[An unusual tumor of the larynx: malignant fibrous histiocytoma. Apropos of a case. Review of the literature]. On the basis of one case, the authors report about an exceptional tumor of the larynx: malignant fibrous histiocytoma. This tumor, belonging to the soft-tissue sarcomas, usually develops in the limbs. The histological diagnosis is still difficult, in spite of the help provided by immunocytochemistry and fine structural analysis. A histological grading associated to other deciding prognostic factors, such as the size and depth of the tumor and the quality of exeresis, would demonstrate a prognostic correlation. The evolution is characterized by frequent local recurrence and metastases. The treatment is mainly surgical, with as extensive an exeresis as possible at once. Radiation therapy and chemotherapy, though often associated, have not decisively proven their value.


Frey's syndrome: prevention with conservative parotidectomy and superficial musculoaponeurotic system preservation. Between 1980 and 1990, 35 patients underwent conservative parotidectomy combining a rhytidectomy incision and a superficial musculoaponeurotic system preservation technique to reduce the postoperative incidence of Frey's syndrome. All patients were evaluated by questionnaire for subjective symptoms of gustatory sweating and flushing as well as satisfaction with the aesthetic appearance of their cheek. Six percent of patients (2 of 35) complained of symptoms of Frey's syndrome. Ninety-four percent of patients (33 of 35) noticed minimal or no contour deformity of the surgical area. Twenty patients underwent Minor's starch iodine testing to identify objective evidence of aberrant nerve regeneration at the postoperative site. Fifteen percent of these patients (3 of 20) demonstrated a positive starch iodine test; however, one of these three patients was unaware of symptoms of Frey's syndrome. Symptoms of gustatory sweating are a reliable indicator of aberrant nerve regeneration. Conservative parotidectomy with superficial musculoaponeurotic system preservation for benign parotid disease produces a low incidence of Frey's syndrome and satisfactory cosmetic results.


Biosynthesis and endocytosis of lysosomal enzymes in human colon carcinoma SW 1116 cells: impaired internalization of plasma membrane-associated cation-independent mannose 6-phosphate receptor. The human colon adenocarcinoma cell lines SW 948, SW 1116, and SW 1222 were tested for their ability to sort and internalize lysosomal enzymes. The biosynthesis of the lysosomal enzymes cathepsin B, arylsulfatase A, and beta-hexosaminidase in these cell lines exhibits no significant differences to that in human fibroblasts. The intracellular targeting of newly synthesized hydrolases to the lysosomes relies in colon carcinoma cells on the mannose 6-phosphate receptor system. Both the cation-independent mannose 6-phosphate receptor (CI-MPR) and the cation-dependent mannose 6-phosphate receptor are expressed in all colon carcinoma cell lines investigated. Endocytosis of lysosomal enzymes via mannose 6-phosphate receptors is reduced in colon carcinoma cells as compared with human fibroblasts. SW 1116 cells were shown to be deficient in receptor-mediated endocytosis of mannose 6-phosphate containing ligands. Ligands of other endocytic receptors as well as the fluid-phase marker horseradish peroxidase were internalized at normal rates. While antibodies against CI-MPR bind to the surface of SW 1116 cells, these antibodies cannot be internalized. These data suggest that the cycling of CI-MPR is specifically impaired in SW 1116 cells.


Na pump and plasma membrane structure in L-cell fibroblasts expressing rat liver fatty acid binding protein. Although the intracellular fatty acid binding proteins have been investigated for nearly two decades and purified proteins are now available, little is known regarding the function of these proteins in intact cells. Therefore, L-cell fibroblasts transfected with cDNA encoding for rat liver fatty acid binding protein (L-FABP) were examined as to whether L-FABP expression in intact cells modifies plasma membrane enzyme activities, fluidity, and lipids. Plasma membrane Na/K-ATPase activity was 65.9 +/- 18.7 and 38.6 +/- 22.8 (P less than 0.001) nmol/mg protein x min for control and high-expression transfected cells, respectively. Consistent with this observation, [3H] ouabain binding to whole cells was significantly decreased from 3.7 +/- 0.3 to 2.0 +/- 0.8 pmol ouabain bound/mg cell protein in control and high-expression cells, respectively, whereas the cell's affinity for ouabain was not significantly altered. Unexpectedly, Western blot analysis indicated that transfected cells had higher levels of Na+, K(+)-ATPase protein; in contrast, the activities of 5'-nucleotidase and Mg-ATPase were unaltered. The effects of L-FABP expression on plasma membrane Na/K-ATPase function appeared to be mediated through alterations in plasma membrane lipids and/or structure. The plasma membrane cholesterol/phospholipid ratio decreased and the bulk plasma membrane fluidity increased in the high-expression cells. In conclusion, plasma membrane Na/K-ATPase activity in L cells may be regulated in part through expression of cytosolic L-FABP.


Stimulus-specific enhancement of luminol chemiluminescence in neutrophils by phosphatidylserine liposomes. When stimulated with different stimuli, neutrophils generate various active oxygen species. These active oxygen molecules can be analyzed by luminol chemiluminescence (LCL). Phosphatidylserine (PS)-liposomes increased the formylmethionyl-leucyl-phenylalanine-induced LCL of guinea pig peritoneal neutrophils without affecting their oxygen consumption and superoxide (O2.-) generation. Similar effects of PS-liposomes were also observed in LCL of neutrophils stimulated by phorbol myristate acetate or arachidonic acid but not by opsonized zymosan. Kinetic analysis revealed that the PS-liposome-induced increase in LCL depended on extracellulary generated O2.-. Moreover, the stimulatory effect of PS could be seen only when it formed liposomal membranes. The effect of PS-liposomes was also inhibited by superoxide dismutase, catalase, and deferoxamine, an iron chelator, but not by azide, an inhibitor of myeloperoxidase. Similar enhancement of stimulation-dependent LCL response was also observed with Fe3+ and ADP-Fe3+, but the degree of enhancement was much greater with PS-liposomes than with iron and its complex. The increase in hydroxyl radical generation by PS-liposome-treated neutrophils was confirmed by experiments with EPR spectrometry using spin-trapping agents. These results suggested that the interaction of neutrophils with PS-containing membrane surface might generate reactive oxygen species that enhance the stimulus-dependent LCL response of neutrophils.


Laser flash photolysis studies of electron transfer mechanisms in cytochromes: an aromatic residue at position 82 is not required for cytochrome c reduction by flavin semiquinones or electron transfer from cytochrome c to cytochrome oxidase. The influence of an aromatic side chain at position 82 of yeast iso-1-cytochrome c on the kinetics of its electron transfer reactions has been investigated using laser flash photolysis methods to compare a series of site-specific mutant cytochromes in their reduction by free flavin semiquinone and in electron transfer from reduced cytochrome to bovine cytochrome c oxidase. Although small (approximately 10%) but significant differences are observed between some of the mutants (S82, Y82, I82) and wild-type (F82) or G82 cytochrome in the second-order rate constant for reduction by lumiflavin semiquinone, these do not correlate with side-chain aromaticity. In the reaction between the ferrocytochromes and cytochrome c oxidase, significantly larger deviations from exponentiality are found for those mutants having aliphatic residues at position 82 than for wild type or Y82. We interpret the nonexponential behavior in terms of multiple orientations of the cytochromes within the oxidase binding site; the extent to which this occurs is apparently influenced by the character of the residue at position 82. However, a comparison of the average rate constants for electron transfer to cytochrome oxidase for the various mutants reveals that all are closely comparable to WT, except for I82 which is significantly slower (approximately threefold). These results, combined with those obtained previously from steady-state kinetic and thermodynamic measurements, suggest that the observed differences among the mutants are due to alterations in the mode of binding of the cytochrome to the oxidase, rather than to a specific requirement for the presence of an aromatic group at position 82.


Colloid adenocarcinoma of the urethra associated with mucosal in situ carcinoma. Adenocarcinoma is a rarity among the primary urethral carcinomas. It usually exhibits a papillary or glandular pattern and is often believed to arise from the mucosal glands. We report a unique case showing the histologic features of colloid adenocarcinoma that appears to have evolved from carcinoma in situ of the urethral epithelium.


Cerebral malakoplakia associated with neonatal herpes virus infection. Cerebral malakoplakia has been recognized as a complication of probable neonatal herpes encephalitis and may be associated with persistent neurologic deficits including seizures. At autopsy, an eight-month-old boy had a discrete cystic gliotic lesion in his left inferior frontal lobe with malakoplakia. Perinatal herpetic cutaneous lesions were seen and were successfully treated with medication at two weeks of age. Subsequent discontinuation of his anti-epileptic drugs was followed by sudden unexpected death. Immunohistochemical stains and electron microscopy of his brain failed to reveal herpes simplex virus.


Properties of a previously undescribed grapevine nepovirus from Tunisia. A virus with isometric particles c. 30 nm in diameter and angular contour was isolated by inoculation of sap from a Tunisian grapevine with mild mottling and leaf deformation. The virus sedimented in sucrose density gradients as three components: T (empty shells), M (particles containing a molecule of ssRNA with an apparent size of 5,800 nucleotides, constituting 35% of the particle weight) and B (particles containing a molecule of ssRNA with apparent size of 6,800 nucleotides, constituting 41% of the particle weight). Virus particles had buoyant densities of 1.31 (T), 1.45 (M), and 1.49 g/cm3 (B) in cesium chloride equilibrium gradients. The coat protein subunits consisted of a single polypeptide with mol. wt. of c. 59,000 daltons. An antiserum was produced with a titer of 1:256, which did not react with healthy plant antigens. Cells of artificially infected herbaceous hosts showed cytoplasmic vesiculate-vacuolate inclusion bodies, virus-containing tubules, mostly associated with plasmodesmata and/or cell wall protrusions, and crystalline aggregates of virus particles and empty capsids. The physicochemical and ultrastructural properties of this virus resemble very much those of nepoviruses. However, it was serologically unrelated to 19 different members of the group, including all those reported to infect grapevines. Therefore, the virus is possibly a hitherto unreported nepovirus for which the name of grapevine Tunisian ringspot virus (GTRV) is proposed.


Increased growth of permanent mouse fibroblasts in soft agar after transfection with hepatitis B virus DNA. Previously we have shown that a nontumorigenic mouse hepatocyte line harboring simian virus 40 large tumor antigen (SV 40 TAg) could be converted to a full-malignant phenotype by transfection with HBV DNA. Using a permanent SV 40 TAg-negative mouse fibroblast cell line (LTK-), we studied whether the in vitro-oncogenicity of HBV was dependent on simultaneous expression of SV 40 TAg or not. Three fibroblast lines stably transfected by full-length HBV DNA formed four times more colonies of large size in soft agar than nontransfected LTK- cells. All three clones expressed high levels of HBx protein, but variable levels of other HBV proteins. A second type of clone that was transfected by a partial HBV genome and that expressed HBV surface but no HBx proteins, did not acquire increased growth in soft agar. These data reveal that HBV DNA can enhance malignant growth independent of SV 40 TAg and suggest that HBx protein may act as an HBV oncogene at least in vitro.


Phylogenetically different strains of a variant of coxsackievirus A 24 were repeatedly introduced but discontinued circulating in Japan. Variations in the nucleotide sequence of 3 C proteinase of coxsackievirus A 24 variant (CA 24 v) were analyzed to define the route of transmission and spread of the virus which was introduced to Japan on three separate occasions, 1985-86, 1988, and 1989. The nucleotide sequences of isolates from the same year's outbreak in Japan were identical or closely related, while the isolates from different outbreaks were less closely related to one another than to those from other countries in the same year. All Japanese isolates from Okinawa and other prefectures in 1985 and 1986 were closely related to the Taiwan strains in those same years, indicating common-source outbreaks. Two 1988 isolates from Chiba Prefecture, Japan, were closely related to those from Singapore in 1987, China in 1988 and Hong Kong in 1988. All seven Japanese isolates from Chiba Prefecture in 1989 comprised a group together with the Taiwan and Singapore strains in 1988. The results indicate that CA 24 v was introduced into Japan on each occasion from the outside. Furthermore, in contrast to the explosive epidemics in Okinawa Prefecture in 1985 and 1986, the virus which was repeatedly introduced to other areas in Japan did not circulate endemically, and disappeared within a short time.


Characterization of the structural proteins of hemorrhagic enteritis virus. The structural proteins of hemorrhagic enteritis (HEV), a turkey adenovirus, were analyzed by polyacrylamide gel electrophoresis (PAGE) and Western blotting using polyspecific, monospecific and monoclonal antibodies for detection. In purified HEV preparations, eleven polypeptides with apparent molecular weights ranging from 96,000 to 9,500 (96k to 9.5k), were specifically recognized by convalescent turkey serum. Six of these polypeptides were further characterized by PAGE, Western blotting, ELISA, sucrose gradient centrifugation and electron microscopy. The 96k polypeptide was identified as the hexon polypeptide which is a monomer of the major outer capsid or hexon protein. The 51/52k and 29k polypeptides, identified as the penton base and fiber polypeptides respectively, were the components of the vertex or penton protein. The 57k polypeptide was identified as a homologue of the human adenovirus type 2 (Ad 2) IIIa protein with which it shares a common epitope. Two core proteins with molecular weights of 12.5 and 9.5k were present in purified HEV nucleoprotein cores. The proteins of two HEV isolates, one apathogenic (HEV-A) and one virulent (HEV-V), resembled each other in most respects. However, differences between HEV-A and HEV-V were found in electrophoretic migration of the penton base protein both under native and denatured conditions, and in the electrophoretic migration of the 43/44k polypeptide. Moreover, homologous antiserum against the fiber protein reacted stronger than heterologous antiserum in an ELISA. Single fibers were detected by electron microscopy attached to the penton base proteins of HEV virions and in isolated pentons. The feature of having single fibers is shared with the mammalian adenoviruses and the avian egg drop syndrome 1976 virus (EDS 76 V), but not with the fowl adenoviruses which have double fibers attached to their penton base proteins.


Non-cytopathic infection of rhabdomyosarcoma cells by coxsackie B5 virus. Infection of rhabdomyosarcoma (RD) cells by coxsackie B5 virus (CBV5) was non-cytopathic, although low titres of infectious virus were produced after 24 h post-infection. The extent of CBV5 replication in RD cells increased after sequential passage of the virus in these cells. The RD cells from the first cycle of CBV5 infection were recovered and maintained in culture for 3 months (equivalent to 21 passages) releasing infectious virus throughout this period; these cells were considered to be persistently infected with CBV5 and were designated piRD cells. Coxsackie virus antigen was demonstrated in a small proportion of piRD cells by immunofluorescence staining. High resolution two-dimensional polyacrylamide gel electrophoresis was used to analyse the intracellular proteins prepared from piRD cells, three proteins were detected which were absent in uninfected RD cells. These new proteins were similar in charge to virus proteins induced during CBV5 lytic infection of HEp-2 cells. Quantitative densitometry of 2-dimensional protein profiles of piRD and uninfected cells showed no significant disruption of RD cell protein synthesis by the persistent virus infection. Three cloned cell lines were recovered from piRD cells, none of which showed evidence of infectious virus or virus-induced protein synthesis suggesting that the parental cell line was a carrier culture for CBV5.


Exclusive asymptomatic neonatal infections by human rotavirus strains having subgroup I specificity and "long" RNA electropherotype. A large number of stool specimens, of healthy newborn infants, collected from various hospitals and clinics in Bangalore City, India, have been examined for the presence of asymptomatic rotaviral excretion. Out of 370 samples analysed during a three year period from 1988 to 1991, 133 specimens (36%) were positive for rotavirus RNA. All these asymptomatic neonatal strains, without exception, showed "long" RNA pattern, but subgroup I specificity. Serotype analysis by ELISA or by hybridization with serotype-specific probes indicated that these strains probably represent a new serotype in newborn children. We find an exclusive association of human rotaviruses having "long" RNA pattern and subgroup I specificity with asymptomatic neonatal infections in contrast to the earlier observations of association of such unusual strains with acute gastroenteritis in young children.


Detection of IL-1 beta, TNF-alpha, and IL-6 gene transcription by the polymerase chain reaction in keratinocytes, Langerhans cells and peritoneal exudate cells during infection with herpes simplex virus-1. Interleukin-1, tumour necrosis factor-alpha and interleukin-6 are considered to be major mediators of inflammatory processes. In the present study, cytokine gene transcription was detected by the polymerase chain reaction technique during cutaneous and intraperitoneal infection with herpes simplex virus-1. Epidermal cell suspensions obtained from mice infected with herpes simplex virus-1 in the ear pinna were enriched or depleted in Langerhans cells by immunomagnetic fractionation. Herpes simplex virus-1 infection in the skin was found to induce interleukin-1 beta, tumour necrosis factor-alpha and interleukin-6 gene transcription in keratinocytes at 24 hours post-infection. Gene transcription declined by 48 hours post-infection. Induction of interleukin-1 beta and tumour necrosis factor-alpha but not of IL-6 gene transcription was detected in Langerhans cells obtained from infected mice at 24 hours post-infection. In order to study cytokine gene transcription during intraperitoneal infection with herpes simplex virus-1, peritoneal exudate cells were obtained from infected mice. Maximal levels of interleukin-1 beta, tumour necrosis factor-alpha, and interleukin-6 mRNA were found in peritoneal exudate cells 6 hours after infection. RNA transcription declined at 24 hours post-infection and was no longer detectable at 48 hours post-infection. Since the higher susceptibility of newborn mice to intraperitoneal herpes simplex virus-1 infection has been suggested to be related to defective cytokine production, cytokine gene transcription was compared in peritoneal exudate cells obtained from infected newborn and adult mice. No significant differences in interleukin-1 beta, tumour necrosis factor-alpha and interleukin-6 gene expression were observed in peritoneal exudate cells obtained from newborn mice as compared with adult mice. In conclusion, cutaneous and intraperitoneal infection with herpes simplex virus-1 induces interleukin-1 beta, tumour necrosis factor-alpha and interleukin-6 gene transcription in epidermal and peritoneal exudate cells.


Relationship between HLA I surface expression and different cytopathic effects produced after herpes simplex virus infection in vitro. In the present study, we investigated the effects of herpes simplex virus (HSV) infection on the expression of HLA class I antigens and beta 2-microglobulin in human fibroblasts. The mRNA abundance for HLA class I was shown to be strongly reduced after infection with HSV strains either producing cell rounding or fusion from within (FFWI), however, HLA class I expression on the surface of cells is strongly reduced only after appearance of FFWI. Using a ts mutant (ts 78R) or CyA in combination with a fusion from without (FFWO) inducing strain of HSV, this loss of HLA class I antigens is assumed to be correlated to the rearrangement of the cell membrane during the fusion process itself as a late event of cytopathogenicity.


Phosphorylation of SV40 large T antigen at threonine residues results in conversion to a lower apparent molecular weight form. Simian virus 40 large T antigen (large T) was dephosphorylated with potato acid phosphatase at pH 5.5. Phosphate residues bound to serine were more susceptible to potato acid phosphatase than threonine-bound phosphate residues. Dephosphorylation of the threonine residues with potato acid phosphatase resulted in a reduced gel-electrophoretic mobility, while removal of the remaining phosphate groups had no effect on the mobility of large T. Pulse-chase experiments revealed a slower migrating form of newly synthesized large T which was converted by phosphorylation to a faster migrating form and had a half-life of approximately 1 h.


Subgroup I serotype 3 human rotavirus strains with long RNA pattern as a result of naturally occurring reassortment between members of the bovine and AU-1 genogroups. Two human rotavirus strains, PCP 5 and MZ 58, which possessed an unusual combination of subgroup (I), serotype (3) and RNA pattern (long) were examined by RNA-RNA hybridization to determine their genogroup. While these two strains did not belong to either the Wa or the DS-1 genogroup, PCP 5 and MZ 58 possessed seven gene segments that formed hybrids with bovine rotavirus strain NCDV and four gene segments that formed hybrids with human rotavirus strain AU-1. These results suggest that PCP 5 and MZ 58 were intergenogroup reassortants formed in nature between a member of the bovine rotavirus genogroup and a member of the AU-1 genogroup.


Passively acquired challenge immunity to enterotropic coronavirus in mice. Maternally-derived passive immunity of infant mice to challenge infection with enterotropic coronavirus mouse hepatitis virus strain Y (MHV-Y) was studied. Pups born to both naive and immune dams, but nursed by naive foster dams, were susceptible to infection, while naive or immune pups nursed by immune foster dams were protected. The MHV infectious dose was identical among naive pups inoculated at 1, 2, 3, or 4 weeks of age. Pups nursing immune dams resisted infection when inoculated at 1, 2, or 3 weeks of age. Three week old pups were protected only if they were allowed access to their immune dams. Pups born to MHV immune dams 4 in consecutive litters acquired equal MHV IgG titers in serum and whey and were all protected against challenge infection. Only pups actively ingesting immune whey at the time of or within two hours after virus inoculation were effectively protected. Pups born to dams immunized by oral inoculation with live MHV acquired both MHV-specific IgA and IgG in their whey, while pups born to dams immunized with killed virus acquired only IgG. Both IgA and IgG, but not IgG alone, were required for complete protection.


Analysis of the genetic diversity of genes 5 and 6 among group C rotaviruses using cDNA probes. Two partial cDNA clones of genes 5 (encoding the major inner capsid protein VP 6) and 6 (encoding a nonstructural protein) of the porcine group (Gp) C rotavirus (Cowden strain) were radiolabeled with 32P and used individually as probes in Northern and dot blot hybridization assays. The specificity of each probe was tested against genomic dsRNA from: (1) porcine Gp A, B, and C rotaviruses; (2) Gp C rotaviruses from different species; and (3) porcine Gp C rotavirus field strains with varying electropherotype patterns. Neither probe hybridized with ds RNA from the porcine Gp A and B strains under the stringency conditions employed in the study. However, the gene 5 probe hybridized with the corresponding gene from the homologous porcine and the heterologous human and bovine Gp C rotaviruses tested. The gene 6 probe hybridized with the corresponding gene from the homologous Cowden strain, but hybridized weakly with gene 6 from the human and bovine Gp C rotaviruses. Both probes recognized all six different porcine Gp C field strains, although with varying intensities. Our results demonstrate that the gene 5 and 6 probes used in this study are specific for Gp C rotaviruses. However, evidence for greater genetic variation in the gene 6 among porcine, bovine and human Gp C strains suggested that the gene 5 probe may prove more broadly reactive among Gp C strains from different species. cDNA probes used in our study should prove useful for the detection of Gp C rotaviruses in feces and facilitate epidemiologic studies.


[Diagnosis of papillary thyroid carcinoma]. The cells of papillary thyroid carcinoma are shown to have the following characteristic morphological features: oval or oval to roundish shape of nuclei, uneven sinuous, folded border of the nuclear membrane, nuclear fissure, intranuclear cytoplasmic inclusions, optically clean nuclei. None of these features is a marker, only combination of more than four of them represents diagnostic value.


Recognition of compartmentalized intracellular analogs of glycoprotein H of human cytomegalovirus. Infected cell proteins immunoprecipitated from human cytomegalovirus (HCMV)-infected fibroblasts with glycoprotein H (gH)-specific conformation-dependent monoclonal antibody (mab 14-4 b) were found to consist of three components of 86 kDa, 89 kDa, and 125 kDa (gp 86, 89, and 125). Affinity purified antibodies from human convalescent serum reactive with an NH2-terminal epitope of gH recognized three polypeptides of comparable size in immunoblots, suggesting antigenic relatedness of these three components of the gH-complex. Using subcellular fractions for immunoblotting, gp 86 was identified as an endoglycosidase H (endo H)-sensitive gH-form present in the nuclear fraction whereas gp 89 and gp 125 were endo H-resistant and present in the membrane fraction or in virions. Incomplete endo H-digestion suggested that four of six predicted N-glycosylation sites of the gH molecule were occupied by carbohydrate side chains. Analysis under nonreducing conditions revealed that the compartmentalized as well as virion-associated gH analogs form high molecular weight complexes. The relation of the recognized gH analogs to the processing pathway of gH is discussed.


Variations in dorsomedial hand innervation. Electrodiagnostic implications. Sensory potentials recorded from the dorsal cutaneous branch of the ulnar nerve (DCUN) help localize ulnar nerve lesions. This conduction technique was first described by Jabre in 1980. Abnormalities detected with this technique in control populations are commonly attributed to local trauma. Anatomic variations of cutaneous innervation to the dorsum of the hand, however, have been described. We recorded DCUN responses using both the normal DCUN stimulation point and a secondary site of stimulation at the radial aspect of the forearm in 44 patients. Eleven patients demonstrated a low-amplitude DCUN response. Four of the 11 patients demonstrated sensory potentials obtained from the routine recording site of the DCUN with radial stimulation. We conclude that an abnormal DCUN response may represent anomalous innervation. To avoid some errors in localization, radial forearm stimulation should be performed whenever the DCUN response is abnormal.


Viruses in human tumors--personal reflections. A personal view is presented reflecting some developments in human tumor virology during the past three decades. Since the discovery of the Epstein-Barr virus in 1964 members of additional viral groups have been linked to human tumors, e.g. hepatitis B virus, HTLV-1, and a number of different papillomavirus types. It became evident that the virus infection per se is not sufficient and that additional events modifying the host cell genome contribute to the development of virus-linked human cancers. At present approximately 15% of the worldwide cancer incidence can be linked to viral infections. The author describes his personal entry into the field and the research in his laboratory on human tumor viruses.


Virus disease as a consequence of viral pathogenicity and the anti-viral immune response. The brief description of two virus systems, influenza and infectious bursal disease, shows enigmatically how at least two requirements must be met to render a virus pathogenic: the array of the whole genome rather than the formation of a particular "pathogenicity gene" and the capacity of the host cell to provide the appropriate microenvironment for an optimal posttranslational processing of structural proteins. In the case of influenza viruses this relates particularly to the cleavability of the haemagglutinin. Efficient virus replication in cells of vital importance, however, does not necessarily result in the development of pathological conditions, as in Borna disease, where neural cells are loaded with virus, and the disease is mediated by a T cell immune response. Immunological stimuli against this virus do not induce neutralizing antibodies which could mount a protective immunity. Infection with influenza viruses is inhibited by neutralizing antibodies, but the course of the disease in an infected organism is largely influenced by virus-specific antibodies which block virus release. It is difficult, however, to evaluate the effectiveness of this type of mechanism directed against the infected cell besides antibody-dependent and cell-mediated cytolysis.


[Synthesis of nucleoside 5'-fluoroalkylphosphonate--compounds with potential antiviral properties]. Several 5'-fluoroalkylphosphonates of 3'-azido-3'-deoxythymidine and 3'-deoxy-2',3'-didehydrothymidine were synthesized as potential antiviral compounds. Fluoromethyl, difluoromethyl, fluorochloromethyl and vinyl phosphonates were used as phosphonylating components, with dicyclohexylcarbodiimide or triisopropylbenzenesulphonyl chloride as condensing reagents.


[Specific endonuclease BbvBI from Bacillus brevis]. A new restriction endonuclease BbvBI free from contaminating nonspecific nucleases and phosphatases was isolated from the Bacillus brevis cells. The enzyme was purified by fractionating the sonicated cell-free extract in a two-phase PEG/dextran system and subsequent chromatographies on DEAE-sepharose, blue sepharose and heparin sepharose. The endonuclease BbvBI displayed the maximal activity at 45 degrees C, pH between 8.0 and 8.5, MgCl2 concentration in the range of 5-10 mM and at the low ionic strength. It is shown that the enzyme cleaves the sequence G'GYPC'C, with the preferential cleavage of GGTACC and GGCACC sites as compared with GGTGCC and GGCGCC. Thus, the restriction endonuclease BbvBI is a true isoschizomer of nuclease BanI.


Marihuana attenuates the rise in plasma ethanol levels in human subjects. This study was conducted to determine if plasma ethanol levels are altered as a result of smoking marihuana. Fifteen healthy adult male volunteers who used ethanol and marihuana on a casual basis participated in this study. Subjects were randomly assigned to one of three groups: placebo, low-dose, or high-dose marihuana. The marihuana dose was held constant and each subject drank three different doses of ethanol on 3 separate days spaced at least 1 week apart. Subjects drank either placebo or ethanol at doses of 0.35 g/kg (7.60 mmol/kg) or 0.70 g/kg (15.19 mmol/kg). Thirty minutes after drinking they smoked either a placebo marihuana cigarette, or one containing either 1.26% or 2.53% delta 9-tetrahydrocannabinol. Plasma ethanol levels rose sharply after the 0.7 g/kg dose and peaked at 50 minutes after drinking began (78.25 +/- 4.95 mg/dl). When subjects smoked the high-dose marihuana cigarettes after the 0.7 g/kg dose of ethanol, peak plasma ethanols levels were only 54.80 +/- 8.32 mg/dl at 105 minutes after drinking began. These alterations in plasma ethanol levels paralleled a reduction in the duration of ethanol- and marihuana-induced subjective effects after high doses of both drugs. These data suggest that marihuana may alter ethanol bioavailability.


An NMR study of the conformation and thermodynamics of the circular dumbbell d [formula: see text] Slow exchange between two- and four-membered hairpin loops. The circular DNA decamer 5'-d [formula: see text] 3' is studied in solution by means of NMR spectroscopy. At low temperature the molecule adopts a dumbbell structure with three Watson-Crick C-G base pairs and two two-residue loops in opposite parts of the molecule. On raising the temperature another conformer appears, in which the closing C-G base pair in the 5'-GTTC-3' loop is disrupted, whereas the opposite 5'-CTTG-3' loop remains stable. The two conformers are in slow equilibrium over a limited temperature range.


Model for the porphyrin-DNA binding site: ENDOR investigations of Cu-porphyrins binding to DNA. Proton ENDOR has been observed from frozen solutions (ca. 38K degrees) of copper meso-(4-N-tetra-methylpyridyl)porphyrin (CuTMpyP(4)) complexed with Salmon sperm DNA in water and D2O. Lines from exchangeable protons of the DNA bases have been observed in these ENDOR spectra. Analyses of these ENDOR data show that the separations of these DNA protons from the copper atom are between 3.76 and 3.84 A with angles of 19.5 to 22.5 degrees between the Cu-H vectors and the gz axis. A distant ENDOR response has also been observed from phosphorous nuclei in the DNA backbone. We estimate that the phosphorous atoms producing this ENDOR signal are 7.5-10 A from the copper center of the porphyrin. These ENDOR data combined with results from an earlier NMR investigation have been used to construct a computer simulated model of the binding site in which the porphyrin is partially intercalated and extends into the major groove of DNA. The two GC base pairs at this site are slightly inequivalent. For each, the G imino proton and one of the C amino protons are at appropriate positions to account for the ENDOR signals arising from exchangeable protons. It is unlikely that this inequivalence would persist at room temperature where dynamic processes would give an apparently symmetric interaction. Although the model accounts for all reported experimental data involving tetracationic porphyrin species which have been suggested to be intercalators, it is not a unique solution.


3 Nsec molecular dynamics simulation of the protein ubiquitin and comparison with X-ray crystal and solution NMR structures. Mainly due to computational limitations, past protein molecular dynamics simulations have rarely been extended to 300 psec; we are not aware of any published results beyond 350 psec. The present work compares a 3000 psec simulation of the protein ubiquitin with the available x-ray crystallographic and solution NMR structures. Aside from experimental structure availability, ubiquitin was studied because of its relatively small size (76 amino acids) and lack of disulfide bridges. An implicit solvent model was used except for explicit treatment of waters of crystallization. We found that the simulated average structure retains most of the character of the starting x-ray crystal structure. In two highly surface accessible regions, the simulation was not in agreement with the x-ray structure. In addition, there are six backbone-backbone hydrogen bonds that are in conflict between the solution NMR and x-ray crystallographic structures; two are bonds that the NMR does not locate, and four are ones that the two methods disagree upon the donor. Concerning these six backbone-backbone hydrogen bonds, the present simulation agrees with the solution NMR structure in five out-of-the six cases, in that if a hydrogen bond is present in the x-ray structure and not in the NMR structure, the bond breaks within 700 psec. Of the two hydrogen bonds that are found in the NMR structure and not in the x-ray structure, one forms at 1400 psec and the other forms rarely. The present results suggest that relatively long molecular dynamics simulations, that use protein x-ray crystal coordinates for the starting structure and a computationally efficient solvent representation, may be used to gain an understanding of conformational and dynamic differences between the solid-crystal and dilute-solution states.


Computer modeling studies on the subsite interactions of ribonuclease T1. The modes of binding of pGp,ApG,CpG and UpG to the enzyme ribonuclease T1 were determined by computer modeling. Essentially two binding modes are possible for all the four ligands--one with the 3'-phosphate group occupying the phosphate binding site (substrate mode of binding) and the second with the 5'-phosphate group occupying the phosphate binding site (inhibitor mode of binding). The latter binding mode is energetically favoured over the former and in this mode the base (G) and the 5'-phosphate moieties occupy the same sites on the enzyme as 5'-GMP when bound to RNase T1. The ribose moiety of pGp adopts a C3'-endo pucker form when bound to the enzyme and the glycosyl torsion angle will be in -syn range as 5'-GMP in the RNase T1-5'-GMP complex. Based on these results, a mechanism for the release of the product subsequent to cleavage of the substrate by the enzyme has been proposed. The amino acid residues Asn98 and Tyr45 are shown to form the subsites for the phosphate and the base respectively on the 5'-side of the guanine occupying the primary binding site. These studies also provide a stereochemical explanation for the specificity of the 1N subsite for adenine.


The human "peripheral-type" benzodiazepine receptor: regional mapping of the gene and characterization of the receptor expressed from cDNA. A cDNA for the human "peripheral-type" benzodiazepine receptor (PBR) was isolated from a liver cDNA library. The 851-nucleotide probe hybridized with a approximately 1 kb mRNA in Northern blots of RNA extracted from various human tissues and cell lines. The human PBR probe was hybridized to DNA from a somatic cell hybrid mapping panel to determine that the gene maps to chromosome 22. With a regional mapping panel for chromosome 22, we localized the gene within band 22q13.31. The ligand-binding properties of the receptor expressed from the cDNA were examined in transient expression experiments and compared to the endogenous human PBR. The PBR ligand [3H]PK 11195 had high affinity for the expressed receptor in COS-1 cells, but the affinities of a pair of isoquinoline propanamide enantiomers differed remarkably in expressed and endogenous human PBR. These findings reveal that the host cell and/or post-translational modification may have an important influence on PBR function.


Clinical and immunological evaluation of isocyanate-exposed workers. Isocyanates are the most significant cause of occupational asthma in our country. To evaluate the prevalence of work-related respiratory symptoms and immunologic sensitization to it, we performed a questionnaire survey, allergy skin test, radioallergosorbent test (RAST) to toluene diisocyanate (TDI)-human serum albumin (HSA) conjugate and methacholine bronchial challenge test on 23 isocyanate-exposed employees and 9 unexposed controls working in a zipper factory. Six employees (26.1%) complained of work-related respiratory symptoms and three symptomatic workers showed significant bronchoconstrictions on TDI-bronchoprovocation test. Three (13%) asymptomatic workers had high specific IgE antibodies to TDI-HSA and none of the TDI-sensitive asthmatic workers had specific IgE antibody. One of the TDI-sensitive asthmatic workers showed a negative result on the initial methacholine bronchial challenge test, but bronchial hyperresponsiveness developed after the TDI challenge. It was suggested that TDI-sensitive asthma was noted in three (13%) of 23 exposed workers and that asymptomatic workers could have high specific IgE antibody. Measurement of the changes in bronchial hyperresponsiveness after the TDI challenge could be helpful to diagnose TDI-sensitive asthma.


Progressive atypical peripheral neuropathy following nephrectomy in a patient with renal cell carcinoma. Peripheral neuropathy or amyotropic lateral sclerosis can be associated with renal cell carcinoma. We report a 63-year-old male patient with renal cell carcinoma who developed an atypical, progressive neuropathy after nephrectomy.


Non-A, non-B hepatitis and dentistry: a status report for the American Journal of Dentistry. The recent identification of one of the causes of non-A, non-B hepatitis (NANBH) has led to an improved understanding of the epidemiology and possible clinical manifestations of this infectious disorder. NANBH is of some concern to dental personnel since the causative agents may be transmitted to health care workers and there is no effective immunization against NANBH. Current recommendations for cross infection control in the dental workplace must apply to NANBH.


Measurement of converting enzyme activity by antibody-trapping of generated angiotensin II. Comparison with two other methods. Activity of the angiotensin converting enzyme (ACE) is usually measured in vitro by estimation of products cleaved by the enzyme from synthetic substrates. These substrates have affinities for ACE different from the natural substrate angiotensin I, and insensitive detection systems necessitate millimolar substrate concentrations while physiological angiotensin I concentrations are in the picomolar range. A new assay for ACE activity measurement was developed which reliably quantitates femtomoles of generated angiotensin II in plasma from angiotensin I added at a 17 pmol/mL concentration. The production of high affinity monoclonal antibodies against angiotensin II (Kd = 7 x 10(-11) mol/L) allowed a quantitative trapping (and thus protection from degrading enzymes) of angiotensin II generated during the incubation step and subsequent radioimmunoassay by simple dilution with labelled angiotensin II. Using 40 microL plasma, the detection limit was 20 fmol/mL/min. Normal human plasma has an ACE activity of 335 +/- 83 fmol/mL/min (mean +/- SD). Precision was characterized by coefficients of variation of less than or equal to 11% both within-assay and between-assays. Accuracy of the new method was established by comparing ACE activity with the ratio of plasma angiotensin II/angiotensin I in plasma obtained from normal volunteers 0.5 to 24 h after oral administration of 20 mg enalapril. The percentage of ACE inhibition indicated by both methods was almost identical (r = 0.93, n = 60, P less than .001). Since the latter ratio appears to reflect in vivo ACE activity, these results indicate that accurate measurement in vitro of ACE activity in vivo has been achieved.


Enhanced responses to endothelin during perfusion of ischemic myocardium. Myocardial response to endothelin. Although endothelin perfusion at 50 pM did not reduce the coronary flow, at 200 pM, it reduced the coronary flow to a similar extent in both normal and ischemic hearts. Endothelin at 50 pM concentration enhanced the myocardial levels of inositol-1,4,5-triphosphate (IP3) and diacyl glycerol (DG) in ischemic, but not in normal, hearts. On the other hand, endothelin at 50 pM concentration enhanced the activities of membrane protein kinase C (PKC) and calmodulin dependent (CaM-PK) kinases in normal, but not in ischemic, hearts. The corresponding loss of cytosolic PKC, but not of CaM-PK, suggests a translocation of PKC and an activation of CaM-PK by endothelin. These results suggest that pM concentrations of endothelin activate membrane kinases in normal hearts and enhance PIP2, breakdown in ischemic hearts. The greater CK release and myocardial levels of nonesterified fatty acids by 200 pM concentrations of endothelin in ischemic hearts, as compared to control hearts, suggests that: (1) the myocardial cellular injury and phospholipid breakdown induced by pM concentrations of endothelin are enhanced during reperfusion; and (2) both IP3 and membrane kinase (PKC and CaM-PK) dependent biochemical cascades of reaction, rather than kinases alone, may be involved in the endothelin-induced myocardial cellular injury.


Serotonergic fibres degenerating in the aging rat brain or sprouting from grafted fetal neurons are not affected by the neurotrophic ACTH analogue Org 2766. The effects of chronic treatment with the purported neurotrophic factor ACTH(4-9) analogue Org 2766 were studied on age-related degeneration of serotonergic fibres and on gliosis in the rat hippocampus and caudate putamen complex. In addition, the potential growth-promoting effects of Org 2766 were investigated on fetal serotonergic cells implanted in a previously denervated hippocampus of young adult rats. Chronic treatment of rats from the age of 11 months to 17-18 months did not affect the incidence of aberrant serotonergic fibres in the caudate-putamen complex or the fibres densities in the hippocampus or the caudate-putamen complex. Gliosis was unaffected by Org 2766 treatment as indicated by increased number and staining intensity of glial fibrillary acidic protein-immunoreactive cell bodies in both brain areas. Grafting of fetal raphe cells in young adult rats caused a time-dependent reinnervation of the previously denervated hippocampus. The reinnervation was not affected by treatment of the rats with Org 2766 for 4 weeks following implantation.


Hydroxyl radical production during early reperfusion after different periods of ischemia in rat hearts and its effect on myocardial function. .OH in postischemic heart. To elucidate the significance of hydroxyl radical (.OH) in postischemic reperfusion injury, we measured the .OH production in the coronary effluent collected from isolated perfused rat hearts during reperfusion period of 15 minutes after various ischemic intervals ranging from 5 to 60 minutes. Salicylic acid was used as a probe for .OH formation, and its derivative, 2,5-dihydroxybenzoic acid (2,5-DHBA), was quantified using high performance liquid chromatography. A significant amount of 2,5-DHBA was detected from the hearts rendered ischemic for 10 minutes and longer. The peak of 2,5-DHBA was seen within 90 seconds after the onset of reperfusion in every group, and the accumulated amount of 2,5-DHBA was maximal in 15 minutes ischemia group (3.97 +/- 0.49 nmol/g/15 minutes reperfusion) in contrast to 1.22 +/- 0.30 nmol/g/15 minute in 60 minutes ischemia. This study demonstrated an ischemic time-dependent .OH production during reperfusion, and no direct effect of .OH was observed on the post-ischemic injury related to myocardial function.


The role of sodium-proton exchange in ischemic/reperfusion injury in the heart. Na(+)-H+ exchange and ischemic heart disease. Previous work has associated cardiac dysfunction and damage after ischemia/reperfusion with metabolic alterations in the heart or alterations in the myocardial ionic homeostasis. Unfortunately, neither mechanism on its own has been able to conclusively explain the pathology. Instead, recent data suggest that the two mechanisms may be interrelated. The low intracellular pH during ischemia (due to the accumulation of metabolic by-products) may stimulate the Na(+)-H+ exchange pathway during reperfusion to remove H+ from the cell in exchange for will lead to accelerated Ca2+ entry via Na+. The subsequent accumulation of Na+ in the cell Na(+)-Ca2+ exchange, which can ultimately result in intracellular Ca2+ overload, contractile dysfunction and damage. This hypothesis is supported by the known biochemical characteristics of the cardiac Na(+)-H+ exchanger. Pharmacological studies also support this hypothesis as a mechanism involved in ischemic/reperfusion damage. Dimethylamiloride, a blocker of Na(+)-H+ exchange, has provided significant protection against ischemic/reperfusion injury to the heart. A series of studies have indicated that the mechanism through which dimethylamiloride acts is via inhibition of the Na(+)-H+ exchange pathway. The data, therefore, are consistent with an important interaction between metabolism and ionic alterations, which includes a central role for Na(+)-H+ exchange in ischemic/reperfusion damage to the heart.


Intracellular sodium and potassium activities of skeletal muscle fibres of hypothyroid rats. In soleus muscle fibres of hypothyroid rats, the membrane potential (Vm) and the intracellular K+ activity (aKi) were significantly lower than in control muscles. These results are consistent with the well-documented decrease in the number of Na(+)-K+ pumps which occurs in hypothyroid muscles. aNai was unchanged in the hypothyroid muscles but this may reflect a change in passive Na+ fluxes which has been reported to occur in association with changes in the number of pump units.


Actions of the phorbol ester, PMA, on ATP-induced fluctuations in cytoplasmic calcium in single isolated bovine aortic endothelial cells. Agonist-evoked rises in [Ca2+]i were recorded from single bovine aortic endothelial cells in the middle of confluent monolayers by dual-wavelength microspectrofluorimetry. Low doses of ATP (1-5 microM) induced a transient rise in [Ca2+]i followed by a maintained plateau phase upon which were superimposed irregular fluctuations in [Ca2+]i. The mechanism underlying these fluctuations is not known. Addition of the phorbol ester, phorbol 12-myristate 13-acetate (PMA), to single cells displaying ATP-induced fluctuations reduced the amplitude and frequency of these fluctuations but the maintained plateau phase was unaffected. Elevation of intracellular cAMP by activation of adenylate cyclase with forskolin, or application of dibutyryl cAMP did not affect the ATP-induced fluctuations. These results suggest a possible role for the diacylglycerol limb of the phosphoinositide hydrolysis pathway, via activation of protein kinase C, but not cAMP, in the mechanism responsible for generating ATP-induced fluctuations in [Ca2+]i.


Nitric oxide controls oscillatory activity in thalamocortical neurons. Nitric oxide (NO) is considered a diffusible messenger involved in neuronal communication, although the post-synaptic target cells of NO action and the associated biological function in the CNS are still a matter of controversy. Within the discrete pattern of NO-synthesizing neurons in the brain, NO synthase is specifically colocalized with the cholinergic brain stem-thalamic system, which is thought to regulate the state-dependent activity of the thalamocortical circuit. Here we report evidence indicating that the release of NO onto thalamocortical neurons results in an alteration in voltage dependence of the hyperpolarization-activated cation conductance, probably mediated via the cGMP system. NO selectively dampens oscillatory neuronal activity, indicating a rapidly diffusing signaling mechanism that controls the functional state of the thalamocortical network.


Distinct agonist- and antagonist-binding sites on the glycine receptor. The distinction between receptor-binding sites for agonists and antagonists underpins the pharmacological differences between these two classes of ligands. In the glycine receptor, antagonist (strychnine) binding requires an interaction with residues Lys-200 and Tyr-202. We now demonstrate that the agonist-binding site of this receptor is located at the residue Thr-204. The agonist-binding site interaction is thus likely to be mediated by hydrogen bonding and not by ionic interactions. Our results demonstrate that, in contrast to other studies of ligand-gated ion channel receptors, agonist- and antagonist-binding sites are composed of distinct amino acid residues.


Inositol phosphate regulation of voltage-dependent calcium channels in cerebellar granule neurons. The effects of intracellularly applied inositol phosphates on voltage-dependent calcium channel currents were assessed in rat cerebellar neurons using the whole-cell recording configuration of the patch-clamp technique. Intraneuronal perfusion of 10 microM inositol 1,4,5-trisphosphate (IP3) increased the amplitude of currents elicited by depolarization from a holding potential of -40 mV. IP3 did not modify current activation, but shifted the steady-state inactivation curve toward more positive values. The dose-response curve indicated an EC50 of 0.5 microM for IP3. Inositol 1,3,4,5-tetrakisphosphate (IP4), but not inositol 4,5,-bisphosphate, mimicked the effect of IP3. The effect of IP3 persisted in the presence of 100 micrograms/ml heparin and did not depend on intracellular calcium mobilization, as similar responses were not produced by 10 mM caffeine or by intrapipette calcium buffering at pCa 6 instead of pCa 7.7. Preincubation with omega-conotoxin led to a 55% inhibition of barium current; however, inhibition was reversed by IP3, which reestablished the control current amplitude. These results imply that IP3 and IP4 can elicit calcium entry by modifying both the gating characteristics and the pharmacological properties of voltage-dependent calcium channels.


Suppression cloning of the cDNA for a candidate subunit of a presynaptic calcium channel. A novel strategy, termed suppression cloning, was used to identify a 7.4 kb cDNA encoding a putative subunit of the calcium channels that regulate transmitter release at nerve endings of Torpedo californica. The 585 nt open reading frame of this cDNA encodes a polypeptide of about 21.7 kd that is essential for the expression in frog oocytes of omega-conotoxin-sensitive, dihydropyridine-resistant, calcium channels. Sequence analysis reveals that this protein is closely related to two cloned cysteine string proteins of undertermined function that were recently localized to Drosophila nerve terminals using monoclonal antibodies.


Characterization of a plant SINE, p-SINE1, in rice genomes. We have previously found that a short interspersed element (SINE), named p-SINE1, is present in the Waxy gene of Oryza sativa in two copies. Here, we cloned five members of p-SINE1 located at other loci in O. sativa and determined their nucleotide sequences. These sequences had a T-rich pyrimidine tract at their defined 3' end and were flanked by direct repeats of a sequence of mostly 14-15 bp long like p-SINE1s in the Waxy gene. The consensus sequence derived from total seven members of p-SINE1 was 123 bp in length and had an internal promoter region for RNA polymerase III. The 5'-half region of the sequence was partially homologous to the tRNA-related block of rabbit C family, one of SINEs in the animal system. Two of the seven p-SINE1 members were not present in the corresponding loci in African rice, Oryza glaberrima, and may thus be available for classification of some rice strains. Comparison of the nucleotide sequences of the Waxy gene between O. sativa and O. glaberrima showed that base substitutions have frequently occurred in a p-SINE1 member (p-SINE1-r1) and a transposable element Tnr1 also present in the Waxy gene, suggesting that these elements, which appear as repetitive sequences in the rice chromosome, tend to acquire base substitutions at a higher frequency than do unique sequences.