Comprehensive restaging laparotomy in women with apparent early ovarian carcinoma. To determine the yield and morbidity of comprehensive restaging laparotomy in women with presumed early ovarian carcinoma who have undergone incomplete initial staging procedures. We conducted a retrospective review of 30 women with apparent early ovarian carcinoma who underwent a comprehensive restaging laparotomy including multiple random intraperitoneal biopsies and selective pelvic/para-aortic lymphadenectomy before receiving adjuvant therapy. Positive findings were compared with clinicopathologic features. Only 17% of patients had adequate skin incisions, 53% had pelvic washings, and 37% had omental biopsy at primary surgery. Complications of restaging laparotomy included 53% severe adhesions, 20% bowel complications, and 20% vascular complications requiring suture. Nine patients (30%) had disease upstaged and six (20%) had stage III disease established by comprehensive restaging laparotomy. Two-thirds of the upstaged patients had occult metastases identified only through cytology, random peritoneal biopsies, or selective lymphadenectomy. Women with poorly differentiated or papillary serous lesions were more likely to have disease upstaged than those with well- and moderately differentiated lesions or other histologic types (P < .05). Other clinicopathologic features did not predict upstaging. A comprehensive staging laparotomy is needed to detect occult metastatic disease in women with apparent early ovarian carcinoma.


Meta-analysis of cisplatin, doxorubicin, and cyclophosphamide versus cisplatin and cyclophosphamide chemotherapy of ovarian carcinoma. To compare the survival with cisplatin, doxorubicin (Adriamycin), and cyclophosphamide versus that of cisplatin and cyclophosphamide in women with advanced epithelial ovarian cancer, to evaluate the effect of dose intensity, and to evaluate meta-analysis methodology. Meta-analysis was done on 30 studies of 2060 women with stages III and IV epithelial ovarian cancer. All had 3-year survival data, adequate follow-up, no other chemotherapy, no radiation therapy, and had information for various prognostic variables (age, stage, grade, and residual disease). We used four different methods of meta-analysis: pooled published data and modified effect-size analyses of the entire group (30 studies), and pooled published data and effect-size analyses of the subset of five prospective randomized studies. Three-year survival for the entire group was 43% for cisplatin, doxorubicin, and cyclophosphamide versus 36% for cisplatin and cyclophosphamide; for the five prospective randomized studies, the rates were 46 and 35%, respectively. The survival advantage of cisplatin, doxorubicin, and cyclophosphamide was statistically significant when analyzed by the pooled published data and modified effect-size meta-analysis of the entire group and the pooled published data meta-analysis of the five prospective randomized studies. The effect-size meta-analysis of the five prospective studies did not reach statistical significance. Total dose intensity and doxorubicin dose intensity were not significantly associated with survival advantage in cisplatin, doxorubicin, and cyclophosphamide use. There seems to be a survival advantage to treatment with cisplatin, doxorubicin, and cyclophosphamide versus treatment with cisplatin and cyclophosphamide. We believe this to be due to the properties of multiagent chemotherapy (the addition of doxorubicin) rather than to increased dose intensity. In addition, we believe that physicians need to familiarize themselves with meta-analysis methodology.


Association of Epstein-Barr virus infection and pulmonary exacerbations in patients with cystic fibrosis. We observed severe pulmonary exacerbations during primary Epstein-Barr virus (EBV) infection in adolescent patients with cystic fibrosis. Since EBV is not a known respiratory tract pathogen in cystic fibrosis, we studied retrospectively all EBV-susceptible patients ages 6 to 18 years with chronic Pseudomonas respiratory tract colonization hospitalized for a pulmonary exacerbation during an 18-month period. Patients with serologic evidence of primary EBV infection (n = 5) were compared to control patients without EBV (n = 7). Before admission the groups had similar pulmonary function tests, clinical scores and frequency of hospitalization. On admission patients with EBV had significant weight loss, lower pulmonary function tests and lower clinical scores compared with controls. All remained significantly different 6 months after admission. Frequency of exacerbations requiring hospitalization increased after EBV infection but remained unchanged in controls. Primary EBV infection can be associated with severe pulmonary exacerbations and subsequent deterioration in clinical course in cystic fibrosis patients.


[Hepatic arteriography under temporary hepatic venous occlusion]. Hepatic arteriography with and without temporary segmental hepatic vein occlusion was performed in 10 patients, five of whom had chronic liver injury. Hepatic arteriograms obtained during hepatic venous obstruction demonstrated significantly more peripheral and definite arterial branches in the occluded area and fewer peripheral branches in the non-occluded segment. A prolonged, dense hepatogram (sinusoidogram) showing hepatofugal opacification of the portal vein was obtained in the occluded area. Only one case with a large veno-venous anastomosis did not show these findings. Hepatic arteriograms in two cases with hepatocellular carcinoma provided clear visualization of peripheral portal branches that could act as efferent tumor vessels during regional temporary hepatic vein occlusion. Temporary hepatic venous occlusion may cause a sudden increase of hepatic arterial flow in the occluded area and transsinusoidal arterioportal communication there. This method can be useful for the diagnosis and arterial infusion or embolization therapy of hepatic diseases.


[Percutaneous injection of cisplatin lipiodol suspension (CLS) in rabbit lung, aiming at intratumoral injection therapy for lung cancer]. Cisplatin lipiodol suspension (CLS: cisplatin 20 mg/ml) was percutaneously injected (cisplatin dose, 2, 4 or 6 mg/kg) in normal lungs of 10 rabbits (1.9-2.3 kg) to assess the safety and feasibility of intratumoral injection of CLS for lung cancer. Histological study revealed acute and chronic infiltrates with bronchiolitis and immature fibrosis at the injected lung tissue even at four weeks after injection. Intrathoracic leaks of CLS produced mild and focal fibrinous pleuritis. Intrabronchial leaks of CLS produced peripheral bronchiolitis with regenerative epithelia. However, no noxious parenchymal damage in the lung and surrounding tissues was noted. Neither oil embolism in brain nor renal toxicity was demonstrated. Seven of eight rabbits showed an increase in body weight. Concentration levels of plasma platinum were lower when compared with intravenous injection of cisplatin in the rabbit: highest at 30 minutes and unmeasurable one week after injection. Lipiodol accumulation in mediastinal lymph nodes was demonstrated in two of nine rabbits by X-ray examination, suggesting intralymphatic drainage of CLS. Intratumoral injection of CLS is safe even with CLS leaks in surrounding normal lung tissues and may be a potent therapy for controlling mediastinal lymph nodes metastasized from lung cancer as well as the primary tumor.


[Prospective randomized trials comparing hyperfractionated radiotherapy with conventional radiotherapy in stage III non-small cell lung cancer]. From September 1987 to August 1990, 36 patients with stage III non-small cell lung cancer were randomly assigned to 65 Gy in 26 fractions of 2.5 Gy each (conventional radiotherapy) or 71.5 Gy in 52 fractions of 1.375 Gy given twice a day (hyperfractionated radiotherapy). The overall time for both groups was 6.5 weeks. The groups were similar in terms of age, performance status, previous chemotherapy, stage IIIA and IIIB, and histopathology. The rate of complete response was 44.4% for hyperfractionation, and 16.7% for conventional fractionation. Overall survival rates at 24 months and 36 months were 50.0% and 21.8% for hyperfractionation, and 31.3% and 0% for conventional fractionation. Fever due to radiation pneumonitis occurred in seven of 18 patients with hyperfractionation (38.9%) and four patients with conventional fractionation (22.2%). No severe late toxicity has been observed in either group. We conclude that hyperfractionated radiotherapy in non-small cell lung cancer may improve survival without increasing severe toxicity.


Antithrombin III activity and diabetes mellitus in the Ivory Coast population. Antithrombin III activity was determined in 100 patients with diabetes mellitus (24 type I and 76 type II) residing on the Ivory Coast. Results showed a significant decrease of antithrombin III in the diabetic subjects as compared to a normal Ivorian population and an inverse correlation was found between antithrombin III activity and hyperglycemia.


Hirschsprung's disease: a viral etiology? This study investigates the hypothesis that Hirschsprung's disease (HD), congenital rectal aganglionosis, may be etiologically linked to antenatal cytomegalovirus (CMV) infection. Bowel specimens from 72 HD patients, 144 control I infants (Hirschsprung-like symptoms, normal histology), and 36 control II infants (deaths from nongastrointestinal causes) were analyzed for CMV genomes by polymerase chain reaction. Positive results were obtained in 6 HD patients (8.8%) and none of the controls. Our findings suggest that antenatal CMV infection, a potentially preventable condition, may be one of the etiological factors in HD.


Picroliv and its components kutkoside and picroside I protect liver against galactosamine-induced damage in rats. D-Galactosamine (800 mg/kg, intraperitoneally) caused significant decrease in the activities of 5'-nucleotidase, glucose-6-phosphatase and cytochrome P450 and increase in activities of gamma-glutamyl transpeptidase, succinate dehydrogenase, acid phosphatase and acid ribonuclease in liver after 24 hr. The levels of RNA, protein and glycogen decreased while total lipids, phospholipids, cholesterol and lipid peroxides increased. It also increased the serum levels of transaminases, alkaline phosphatase and bilirubin while protein concentration decreased significantly. Oral administration of Picroliv (12 mg/kg/day for 7 days), a standardised iridoid glycoside fraction of Picrorhiza kurroa, significantly prevented the biochemical changes in liver and serum of galactosamine-toxicated rats. Kutkoside (12 mg/kg/day for 7 days) also protected against changes in most of the hepatic and serum constituents studied. Another iridoid glycoside from Picroliv, Picroside I, at the same dose level could only prevent toxicant-induced changes in acid phosphatase, phospholipids and lipid peroxides in liver and alkaline phosphatase in serum. Mixture of Picroside I and Kutkoside in the ratio of 1:1.5 at 12 mg/kg dose elicited lesser response than Picroliv.


Nucleolar organizer regions in congenital mesoblastic nephroma. A review of 78 renal tumors in patients under 6 months of age revealed 35 congenital mesoblastic nephromas (CMNs). Based on cellular criteria, 14 were classified as classical, 4 as partly cellular, and 17 as cellular CMN. The mean ages were 24, 11, and 70 days, respectively. There were 13 intrarenal tumors (stage I) but 9 classical, 3 partly cellular, and 5 cellular CMNs extended to the perirenal fat (stage II) and 5 cellular tumors ruptured (stage III). In order to assess cellular proliferative activity, silver staining of nucleolar organizer region (Ag-NOR) proteins was performed on 19 CMNs. The number of Ag-NOR dots per cell was significantly lower in classical and partly cellular CMN than in cellular CMN, whatever the stage (P < .01). Within the cellular CMNs, the mean number of Ag-NOR dots was statistically higher in the single case that recurred with fatal outcome (P < .02). Counting of Ag-NOR dots appears to be a reproducible method for evaluating the biologic potential of CMNs. The number of Ag-NOR dots, DNA content measurements, the histological subclassification, and the presence or absence of tumor at the surgical margins may be useful features in selecting those patients who will benefit from further treatment after nephrectomy.


Forskolin but not cholera toxin bypasses flufenamate-induced inhibition of cAMP production in anterior pituitaries. The pharmacological activators of adenylyl cyclase (cholera toxin and forskolin) were utilized in the present study to determine whether they could bypass the inhibitory effects of flufenamate on cAMP production in rat hemipituitaries. During 2 hr incubations, 10 microM flufenamate inhibited gonadotropin-releasing hormone (GnRH)-stimulated (25 nM) cAMP production. Flufenamate did not affect GnRH-receptor interactions as evidenced by its inability to significantly affect either the binding affinity or the binding capacity for GnRH. Additionally, flufenamate inhibited the cholera toxin-stimulated cAMP production, but was ineffective against forskolin-induced activation of adenylyl cyclase. These results indicate that forskolin can be used to restore cAMP production in the presence of flufenamate. Since GnRH and cholera toxin stimulate cAMP production via the GnRH receptor and the Gs protein respectively, and forskolin exerts its stimulatory effects via the catalytic component, the data are consistent with the possibility that flufenamate exerts its inhibitory effect at the level of the Gs protein.


The use of flufenamate and forskolin to evaluate the role of cAMP in gonadotropin-releasing hormone-stimulated luteinizing hormone secretion from pituitaries of ovariectomized rats. Flufenamate which inhibited gonadotropin-releasing hormone (GnRH)-stimulated cAMP production in pituitaries from ovariectomized (72 hr) rats, was used to determine whether ovariectomy induces a change in the role of cAMP as a mediator of luteinizing hormone (LH) secretion. Additionally, the study evaluated the practicability of utilizing forskolin to restore intracellular cAMP concentrations in the presence of flufenamate. Infusions of flufenamate to perifused pituitary tissue blocks did not affect the protein synthesis-independent component of GnRH-stimulated LH secretion, but completely inhibited the protein synthesis-dependent component. Dibutyryl cAMP (dbcAMP) and forskolin potentiated the GnRH-stimulated responses, and restored the LH secretion inhibited by flufenamate, even though these agents were ineffective secretagogues when administered singly. The LH responses affected by forskolin were dependent on protein synthesis, while dbcAMP affected both the protein synthesis-dependent and -independent components of GnRH-stimulated LH secretion. Since the effects of dbcAMP on the protein synthesis-independent component of LH secretion might be due to interactions with GnRH receptors, the results suggest that forskolin might be a better choice for restoring intracellular cAMP levels in the presence of flufenamate when assessing the role of cAMP in gonadotropes. The study also indicates that ovariectomy does not result in a change in the role of cAMP, which appears to be a pivotal, but indirect mediator of the protein synthesis-dependent component of GnRH-stimulated LH secretion.


Solitary pancreatic metastasis of malignant fibrous histiocytoma treated by distal pancreatectomy. A tumor in the body of the pancreas was detected in a 31-year-old man who had undergone a resection of a malignant fibrous histiocytoma (MFH) of the left distal femur 2 years before. The patient underwent a distal pancreatectomy with regional lymph node dissection. The surgical specimen revealed MFH metastatic to the pancreas. He is alive without recurrence or metastasis at 1 year after pancreatectomy. This case seems to be the first report of successfully resected pancreatic metastasis of a malignant fibrous histiocytoma in the published literature, as far as we can determine.


Branched-chain amino acids increase the seizure threshold to picrotoxin in rats. During infusion of branched-chain amino acids (BCAAs) in humans, changes in ventilatory drive, appetite, and sleep have been reported. The mechanism by which BCAAs exert their effects on CNS remains unclear. Picrotoxin is a proconvulsant drug, acting as an antagonist on the GABA-benzodiazepine receptor complex. Twenty rats were randomized to receive either an IP injection with 4% BCAAs (300 mg/kg; 8 ml/kg) (n = 10) or placebo (saline 8 ml/kg) (n = 10). The mean latency time from injection to onset of seizures was recorded as an indication of the seizure threshold. Latency time was significantly longer for BCAAs than for placebo, 11.2 (+/- 1.9) vs. 8.3 (+/- 1.8) min. Thus, a BCAA injection increased the seizure threshold to picrotoxin (p < 0.03). This suggests that BCAA infusion may exert effects on the GABA-benzodiazepine receptor complex.


Effect of various training procedures on performance in an elevated plus-maze: possible relation with brain regional levels of benzodiazepine-like molecules. Rats submitted to one, two, or seven sessions of exploration to a new environment (habituation) or exposed to an inhibitory avoidance training showed different degrees of anxiety, evaluated by the elevated plus-maze test. Also, the brain regional levels of benzodiazepine (BDZ)-like molecules in rats submitted to one, two, or seven sessions of habituation were differentially decreased with respect to nontrained rats. The percentage of time spent in the open arms of the elevated plus-maze for each group correlates with the data of decrease in the BDZ-like immunoreactivity in amygdala (r = 0.77, p < 0.0005), hippocampus (r = 0.68, p < 0.0005), and septum (r = 0.57, p < 0.005). These results suggest that the limbic system responds to anxiogenic experiences by changing the BDZ-like molecule levels in relation to the degree of anxiety and/or stress that accompany these experiences.


Mechanistic investigation of the stimulus properties of 1-(3-trifluoromethylphenyl)piperazine. Using a standard two-lever operant procedure with rats trained to discriminate 1-(3-trifluoromethylphenyl)piperazine (TFMPP) (0.5 mg/kg) from saline, tests of stimulus antagonism and stimulus generalization were performed to better understand the stimulus properties of this agent. The agents examined for ability to antagonize the TFMPP stimulus were prazosin, quipazine, zacopride, buspirone, 8-hydroxy-2-(di-N-propylamino) tetralin (8-OH-DPAT), 1-(2-methoxyphenol)-4-[4-(2-phthalimido)butyl]-piperazine (NAN-190), haloperidol, and 1-(2-pyrimidinyl)piperazine (1-PP); only buspirone attenuated the response to TF-MPP. In separate experiments, the lowest nondisrupting dose of buspirone (1.2 mg/kg) caused a rightward shift of the TFMPP dose-response curve (TFMPP alone, ED50 = 0.19 mg/kg; TFMPP + buspirone, ED50 = 0.43 mg/kg). In addition, 3-(1,2,5,6-tetrahydropyrid-4-yl)pyrrolo[3,2-b]pyrid-5-one (CP 93, 129), 7-trifluoromethyl-4-(4-methyl-1-piperazinyl)pyrolo[1,2-a]quinox ali ne (CGS 12066B), 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI), 3-chlorophenylbiguanide (mCPBG), NAN-190, nisoxetine, zacopride, 1-PP, (+)-N-allylnormetazocine ((+)-NANM), and N-methyl-1-(3,4-methylenedioxyphenyl)-2-aminopropane (MDMA) were analyzed in tests of stimulus generalization. The TFMPP stimulus generalized only to CGS 12066B (ED50 = 4.2 mg/kg) and (+)-NANM (ED50 = 8.8 mg/kg). Tests with DOI and MDMA resulted in partial generalization. Up to doses that disrupted behavior, all other agents had little effect on TFMPP-appropriate responding. The results of these and other published studies suggest roles for 5-hydroxytryptamine 1B (5-HT1B), 5-HT1C, and, possibly, sigma-receptors in the mediation of the TFMPP stimulus and indicate a lack of involvement of 5-HT1A, 5-HT2, dopaminergic, and adrenergic mechanisms in this behavior.


Diprenorphine as a stimulus in drug discrimination learning. Using the conditioned taste aversion baseline of drug discrimination learning, animals were trained to discriminate diprenorphine from distilled water. In subsequent generalization tests, the opiate antagonists naltrexone and naloxone and the mixed opiate agonist/antagonist nalorphine substituted for the diprenorphine stimulus in a dose-dependent manner, while the opiate agonist morphine and the nonopiate pentobarbital failed to substitute even at the highest doses tested. That a range of opiate antagonists substituted for the diprenorphine stimulus (and an opiate agonist and a nonopiate failed to substitute) suggest that diprenorphine's antagonist properties may mediate the discrimination, presumably by blocking endogenous opiate activity. The ability of these drugs to substitute for the diprenorphine stimulus may also be a function of this receptor activity. The differences in the specific generalization patterns reported in the present assessment and those of earlier reports were discussed.


Bombesin-induced hypothermia: a dose-response and receptor antagonist study. Bombesin infusion into the preoptic area (POA) has previously been shown to induce hypothermia in rats that are food deprived or made hypoglycemic with insulin. The present study evaluated the potency and receptor specificity of this response. Bombesin was microinfused into the POA of food-deprived rats (n = 7) and insulin-pretreated rats (n = 7) at doses of 0, 5, 12, 25, and 50 ng/0.5 microliters. Changes in core body temperature (rectal) were assessed at 1 h. Hypothermia was observed under both conditions with doses as low as 5 ng (3.1 pM) as compared to vehicle (0 ng). In a separate study, infusion of the reduced peptide bond analog (Psi13,14 Leu14)bombesin (2.5 micrograms) prior to bombesin injection (25 ng) was found to prevent the hypothermic response observed in the bombesin control condition. These data suggest that bombesin is a potent hypothermic agent that interacts with gastrin-releasing peptide receptors localized within the POA region to impact thermoregulation.


Comparison of nedocromil sodium at two dosage frequencies with placebo in the management of chronic asthma. Nedocromil sodium (4 mg b.d. or q.i.d.) was added to the therapy of 76 chronic asthmatic patients in a four-centre, double-blind cross-over, placebo-controlled trial. Patients had troublesome symptoms uncontrolled by high doses of inhaled corticosteroids (mean 1450 micrograms). In 54 patients who completed the study, nedocromil sodium was significantly more efficacious than placebo (P < 0.01) in relieving morning chest-tightness and cough, in reducing total diary card score and nocturnal bronchodilator usage, and in increasing morning and evening peak flow. Asthma severity at clinic visits decreased significantly (P = 0.001) following treatment with nedocromil sodium, which was globally rated more effective than placebo (P < 0.01). Treatment differences favored q.i.d. over b.d. dosage but without statistical significance. There were no serious adverse effects. Although the pulmonary function changes were small, these findings suggest that the addition of nedocromil sodium may benefit asthmatic patients who are inadequately controlled by high doses of inhaled corticosteroids.


Transposon mutagenesis in Legionella pneumophila. I.--Persistence of suicide and broad host-range plasmids. Two of three highly virulent strains of Legionella pneumophila could act as recipients at high frequencies in conjugation experiments with Escherichia coli donor strains carrying broad host-range plasmids belonging to incompatibility groups N, P and W. All broad host-range and most transposon-delivery plasmids persisted within transconjugants with high stability. Only one (pSUP1021) of several vehicles designed for the delivery of transposons into the chromosome of Gram-negative bacteria was found to yield transposon mutants of Legionella at a detectable frequency.


Transposon mutagenesis in Legionella pneumophila. II.--Mutants exhibiting impaired intracellular growth within cultured macrophages and reduced virulence in vivo. Transposon Tn5 mutants of L. pneumophila were isolated and screened for loss of virulence-associated characteristics. Three mutants were found with normal ability to produce putative pathogenicity determinants and to be endocytosed by guinea pig alveolar macrophages in vitro but with a greatly reduced ability to multiply within them. These mutants showed considerable loss of virulence in an authentic animal model of the pneumonia.


[Cerebral metastasis disclosing primary bronchogenic cancers]. We report the cases of 37 patients with carcinoma of the lung revealed by brain metastases. The most frequent clinical manifestation was focal neurological symptoms associated with headache and vomiting in 50% of the cases. X-ray films of the chest were abnormal in 34 patients. At the time of diagnosis 11 patients also presented with extra-cerebral metastases. The histological type of the primary lung tumor was obtained by examination of the thorax in 32 cases and in 5 cases from brain or lymph node metastases: 11 patients had small-cell lung carcinoma and 26 had non small-cell lung carcinoma. The overall actuarial median survival was 4.5 months, irrespective of the histological type. The group of 20 patients who underwent neurosurgery had a longer median survival (10 months versus 4.5, p < 0.05), and in the subgroup where brain and lung resections were combined the median survival was even longer (13 months). Cerebral relapses occurred in 12 patients: in 7 out of 15 patients with brain surgery but without adjuvant brain radiotherapy, and in 5 out of 16 patients with brain radiotherapy without neurosurgery. No cerebral relapse was observed in the group of 5 patients who had complete resection followed by radiotherapy of the brain. This demonstrated a clear benefit from postoperative radiotherapy. Conventional chemotherapy induced objective responses only in the small-cell carcinoma group and could be too toxic when combined with simultaneous radiotherapy, but it proved a useful adjuvant treatment in patients with radiotherapy of the brain.


Mutational analysis of the enzyme IIIGlc of the phosphoenolpyruvate phosphotransferase system in Escherichia coli. The phosphoenolpyruvate phosphotransferase system (PTS) component EIIIGlc is responsible for transport and phosphorylation of glucose via EIIGlc. It also regulates the catabolism of other carbon sources, such as lactose and maltose, by modulating both the intracellular concentrations of the corresponding inducers and of cAMP. Mutational analysis of EIIIGlc was performed in order to identify crucial residues mediating the interactions between EIIIGlc and its target proteins. Such mutations were isolated by in vitro hydroxylamine mutagenesis of the cloned EIIIGlc gene, crr. Five mutated EIIIGlc impaired in the function of inducer exclusion were obtained. However, these mutations did not abolish the function of EIIIGlc in the transport and phosphorylation of glucose, nor in activation of adenylate cyclase. A single amino acid change was found for each mutation, which is located in a restricted area of the polypeptide chain: Gly47-->Ser47 for the HA2 and HA5 mutations, Ala76-->Thr76 for HA4 mutation and Ser78-->Phe78 for HA3 mutation, indicative of quaternary interactions between the corresponding region of EIIIGlc and its target protein(s).


[Male-type scleroderma: the role of occupational exposure]. A number of occupational exposure risks have been suspected as potential causes of scleroderma (systemic sclerosis), particularly in males. These include silica dust and numerous organic chemicals. With particular reference to the type of employment, we have reviewed the charts of male scleroderma patients receiving treatment in our Rheumatology Department since 1976. Of 39 patients, 25 could be included in the present study. Among these, 14 (56%) had an occupational activity which put them at risk: 11 were exposed to silica dust, one to organic chemicals and two to both silica dust and chemicals. Mean exposure time was 14.5 years (range 4-33 years); and the interval between the beginning of exposure and the onset of scleroderma averaged 24.4 years (range 4-45 years). Considering these 14 cases as silica- or chemically-induced scleroderma, we found that, except for the absence of muscular involvement, the clinical features and prognosis were similar to those of "idiopathic" scleroderma patients. Antinuclear antibodies and autoantibodies directed against Scl-70 were also found in both occupationally induced and idiopathic scleroderma. Our results emphasize the unusually high frequency of occupational exposure in scleroderma in males. Hence, a detailed professional history should be taken for all scleroderma patients. Our results also suggest that, in certain cases, scleroderma should be classified as an occupational disease.


Surgical complications after nephrectomy for Wilms' tumor. We reviewed the charts of 1,910 children enrolled in the Third National Wilms' Tumor Study who underwent primary nephrectomy. Four hundred and ninety-five surgical complications occurred in 379 children (19.8 percent). Patients with inoperable tumors, bilateral renal tumors, peroperative therapy and those who refused treatment were excluded from this review. The most common complication was intestinal obstruction, which occurred in 132 patients (6.9 percent). This was followed by extensive intraoperative hemorrhage (112 patients), defined as blood loss exceeding 50 milliliters per kilogram of body weight. Intraoperative injuries to other visceral organs (including intestine, liver and spleen) occurred in 21 children and extensive vascular injuries were reported in 27 patients. There were nine deaths attributed to surgical complications (0.5 percent), only one of which was intraoperative. Survival of patients with complications was similar to patients without complications when stratified by histologic study and stage. Factors associated with the development of surgical complications included advanced local tumor stage at diagnosis, intravascular tumor extension and resection of other visceral organs at the time of nephrectomy. Complete removal of the tumor is important, but not at the expense of radical removal of adjacent structures, because of gross appearances at operation. They are often not invaded by the tumor, but rather are compressed, distorted or adherent without tumor infiltration. Identification of these groups will aid the surgeon in choosing the appropriate management for these patients at high risk. When initial exploration and precise surgical staging indicate that only a formidable operation will achieve total excision, shrinkage of the tumor with selective use of chemotherapy or radiotherapy, or both, may facilitate removal and decrease surgical morbidity. Preoperative therapy may also be the preferred approach for children with extensive intravascular tumor.


Hybridization of transposon Tn501 for detection of mercury resistance sequences in a marine environment. Total genomic DNA isolated by concentrating seawater and mercury-resistant bacteria were hybridized with a mer probe to detect the presence of homologous DNA sequences in marine coastal waters of the Bay of Bengal, India. Coastal water extracts induced with mercury hybridized with the mer operon of transposon Tn501. Most of the mercury-resistant bacteria that volatilized mercury also contained homologous DNA sequences to the mer probe.


Serum osteocalcin as a marker for vitamin K-status in pregnant women and their newborn babies. Osteocalcin (bone Gla-protein) is a vitamin K-dependent protein synthesized by osteoblasts. Its hydroxylapatite binding capacity (HBC) is generally used to estimate the Gla-content of circulating osteocalcin. Here we have used the HBC of serum osteocalcin as a marker for the vitamin K-status in pregnant women and their offspring. For all cases investigated the HBC values in the cord samples were substantially lower than in the corresponding maternal ones. Babies from mothers who had been treated with vitamin K during the last 6 weeks prior to delivery, had significantly higher HBC values than those from a placebo group. The results presented in this paper are indicative for a generally occurring vitamin K deficiency in newborns. At delivery the HBC in untreated women was low as well. In both the placebo- and the vitamin K-group a good correlation was found between the HBC values in paired samples from mother and child. Whether the maternal HBC value may be used as a prenatal marker for estimating the fetal vitamin K-status remains to be seen.


Coagulation activation following estrogen administration to postmenopausal women. We investigated coagulation system activation following estrogen treatment in 29 healthy postmenopausal women. Study participants received conjugated estrogens at 0.625 and 1.25 mg per day, and placebo for 3-month periods in a randomized crossover protocol. Blood samples were obtained on two consecutive days at the end of each treatment period for immunoassays of F1+2 and fibrinopeptide A (FPA), markers of factor Xa action on prothrombin and thrombin action on fibrinogen in vivo, respectively. Treatment with estrogens at a dose of 0.625 or 1.25 mg resulted in significant increases in mean F1+2 levels of 40 and 98%, respectively, and in mean FPA levels of 37 and 71%, respectively. The measurements of F1+2 were significantly higher in women receiving 1.25 mg of estrogen than 0.625 mg. We also observed significant declines in the levels of antithrombin III and total protein S antigen. Immunologic levels of protein C increased modestly at only the 1.25 mg estrogen dose level. These data indicate that low doses of oral estrogens (< or = 1.25 mg per day) frequently increase the amount of thrombin generated in vivo. Our observations may help to explain the increased thrombotic risk that has been observed with higher doses of this medication (> or = 2.5 mg).


Fluctuations in plasma levels of thrombomodulin in patients with DIC. Plasma thrombomodulin (TM) has attracted considerable attention as a marker of endothelial cell membrane injury. We examined fluctuations in plasma TM levels in patients receiving therapy for the disseminated intravascular coagulation syndrome (DIC) using an enzyme immunoassay. Sixty healthy controls and 18 patients with DIC were studied. The mean +/- SD of the TM values initially measured immediately after the onset of DIC was 42.00 +/- 20.85 ng/ml, which was markedly increased as compared with the control value of 15.36 +/- 4.85 ng/ml (p < 0.001). Fluctuations in the TM levels over time were studied after dividing the patients according to the presence or absence of improvement in the underlying disease and improvement or lack thereof in the coagulation findings. Group I showed improvement in both categories, Group II showed improvement only in the latter, and Group III showed no improvement in either category. In Group I, the mean +/- SD of initial measured TM levels was 37.02 +/- 10.12 ng/ml and the mean of final values decreased to 58.9% of the initial value. This decrease was significant by paired Student's t-test (p < 0.01). The initial value in Group II was 45.86 +/- 18.86 ng/ml and the final values increased to 117.0% of the initial values, this difference was not significant. The initial value in Group III was 44.48 +/- 21.53 ng/ml and the final values increased to 143.4% of the former. This increase was significant by paired Student's t-test (p < 0.05). The difference in % fluctuations between Group I and Group III was significant by Wilcoxon's test (p < 0.01). These results suggest that the measurement of plasma TM can be useful in the management of DIC.


Neuroendocrine stress response after minimally invasive surgery in pigs. Minimally invasive operations such as laparoscopic cholecystectomy appear to result in more rapid recovery of normal function, less physiological disturbance, and presumably less stress to the organism than open operation counterparts. The purpose of this study was to determine the stress response associated with minimally invasive surgery compared to conventional laparotomy. Three groups of pigs underwent general endotracheal anesthesia. The first group had laparoscopic cholecystectomy, the second open cholecystectomy, and the last group (controls) had only general anesthesia. The neuroendocrine serum stress markers adrenocorticotropic hormone (ACTH), cortisol, insulin, and glucagon were measured prior to anesthesia and for the first 3 postoperative days. Analysis of the data showed significant elevations of both ACTH and cortisol for laparoscopic operations as well as for open operation (cortisol only) in the immediate postoperative period. No differences were found for the other serum stress markers. We conclude that minimally invasive surgery in this porcine model confers no advantage, as measured by four neuroendocrine stress hormones, over conventional surgery. Further study is required to determine the clinical implication of these findings.


Pertussis toxin sensitive G-protein coupling of HDL receptor to phospholipase C in human platelets. Initially we established that, in human platelets, low concentrations of HDL3 stimulate phosphatidylcholine (PC) hydrolysis and a transient increase in 1,2-diacylglycerol (DAG). In (3H) PC prelabelled platelets, phosphocholine is released into the medium during HDL3 induced PC turnover with a 1.5 to 2 fold increment, indicating that HDL3 stimulated DAG generation in platelets is likely due to phospholipase C (PLC). GTP or GTP-gamma-S augments, and pertussis toxin inhibits HDL3 stimulated DAG production. Treatment of platelet membranes with HDL3 or with proteoliposome containing apo A-I or A-II substantially prevents 41 kDa protein ADP-ribosylation that was induced by pertussis toxin, with apo A-II having an inhibitory potency greater than apo A-I. These data provide strong evidence that the pertussis sensible G protein (Go or Gi) is directly involved in coupling PLC to HDL3 receptor in platelets.


Binding of recombinant variants of human tissue-type plasminogen activator (t-PA) to human umbilical vein endothelial cells. Endothelial cells synthesize and secrete hemostatic components like tissue-type plasminogen activator (t-PA) which is thought to be the major determinant of fibrinolytic activity in the blood. Most recently, a receptor protein for t-PA on human umbilical vein endothelial cells (HUVEC) in culture has been described (1); there are, however, in addition low affinity binding sites for t-PA on HUVEC. The sites of binding are of particular interest, because they are potential regulators of t-PA activity and clearance. We analysed the low affinity binding of recombinant t-PA (rt-PA) to normal diploid HUVEC and to the permanent human cell lines Jurkat, Daudi, HL 60 and K562 by flow cytometry applying t-PA specific monoclonal antibodies. Using this test system binding of both recombinant glycosylated human t-PA produced in Chinese hamster ovary cells (CHO-t-PA) and of nonglycosylated t-PA, produced in E. coli (BM 06.021) was investigated. Analysis of the binding pattern to HUVEC and other cell lines revealed that deglycosylation of full length rt-PA increases non-specific binding. Additionally, we investigated the binding properties of an unglycosylated t-PA deletion variant which comprises the kringle 2 and the protease domains (BM 06.022). Data obtained show that deletion of these domains most drastically reduces non-specific binding to HUVEC and other human cell lines.


Heparin Study in Internal Medicine (HESIM): design and preliminary results. The Heparin Study in Internal Medicine (HESIM) compares the efficacy and safety of an unfractionated (UF) heparin with a low molecular weight (LMW) heparin (CY 216 D) for prevention of proximal deep vein thrombosis (DVT) and pulmonary embolism (PE) in medical inpatients with a high risk for development of thromboembolism. Patients are randomized and receive three times daily 5000 IU UF heparin or once daily 3100 IU LMW heparin and two placebo injections subcutaneously for 10 days. All patients are screened for the presence of proximal DVT at day 1 and 10 by real-time B-mode compression sonography and for PE by repeated clinical examinations. Perfusion scintigraphy is used for confirmation of the clinical diagnosis of PE. The study protocol includes a stratified randomization of patients on admission to the hospital according to one of the following main diagnoses: malignant disease, cardiovascular disease, bronchopulmonary disease, neurologic disease, and other diseases. The present study may serve as a model for further clinical trials in medical inpatients using the biometric approach of statistical analysis for proving equivalence of drug efficacy, and to adopt less sensitive but noninvasive methods for the detection of primary endpoints.


The inhibition of thrombin and chymotrypsin by heparin-cofactor II. Heparin cofactor II (HC II) is known as a bifunctional inhibitor inactivating trypsin- and chymotrypsin type proteases. Its inhibitory activity increases in the presence of heparin, dermatan sulfate and chondroitin E. In the present study the inhibitory activity of HC II was investigated as function of various dermatan sulfate fractions and its stability was tested against oxidation reagents similar to thus secreted by activated leucocytes. High affinity dermatan sulfate (DS) increased the antithrombin inhibition activity of HC II about 1000-fold in contrast to about 100-fold in the case of low affinity DS. Oxidation of HC II carbohydrate side chains with sodium periodate showed less inactivation effects than oxidation by chloramine T or ammonium peroxodisulfate.


[Sorption immobilization of glycerol on disperse silica and its interaction with the surface of bovine reproductive cells, seminal plasma and sperm]. The binding of hydroxylated disperse silica (I) surface and surface, modified by glycerol (II), with components of above-membrane matrix of bovine reproductive cell and seminal plasma was investigated. It was found that structure of inorganic support defined the character of binding SiO2 with proteins and glycoproteins, including those with hexose and N-acetylneuraminic acid as terminal residue. Value of binding depends on concentration of disperse silica contacting with biomaterial.


[Ouabain sensitivity of Na+,K+-ATPase in neuronal membranes]. Na+, K(+)-ATPase preparations of the rat and bovine brain and kidney were studied for ouabain sensitivity. Differences in apparent affinities to inhibitor of alpha(+)- and alpha-isozymes of Na+, K(+)-ATPase catalytic subunit were detected only in rat tissues but not in bovine ones. It is concluded that glycoside-sensitive and glycoside-resistant enzymic forms are not fully identical to alpha(+)- and alpha-subunit forms of Na+, K(+)-ATPase.


Sequence, genomic organization of the EcoRI-A fragment of Autographa californica nuclear polyhedrosis virus, and identification of a viral-encoded protein resembling the outer capsid protein VP8 of rotavirus. We present the sequence and genomic organization of the EcoRI-A fragment of the Autographa californica multicapsid nuclear polyhedrosis virus, which represents 11% of the AcMNPV genome. Fifteen putative open reading frames and their respective amino acid sequences are described. One open reading frame is similar to the VP8 protein of rotavirus.


Signet ring cell carcinoma of the stomach: a morphometric, ultrastructural, and DNA cytometric study. The present study was undertaken to determine whether or not signet ring cell (diffuse, isolated cell) gastric carcinomas display a specific profile at the ultrastructural, morphometric, and DNA cytometric levels. Thirty-two cases of gastric carcinoma and 8 cases of peptic ulcer (control group) were studied with electron microscopy, morphometry, flow cytometry, and image cytometry. Despite the ultrastructural cellular heterogeneity of signet ring cell carcinomas, the neoplastic cells display fairly constant morphometric features: The cellular and nuclear volumes are significantly smaller than those of the other types of gastric carcinomas and closely resemble those of normal foveolar cells. The relatively small size of signet ring cell carcinoma nuclei fits with the high percentage of the cases of this type of gastric carcinoma that are either diploid or nearly diploid. There is a relationship between the infiltrative pattern of growth of gastric carcinoma (regardless of histologic subtype and ultrastructural cell differentiation) and the small size of neoplastic cells and their nuclei.


Transdominant inhibition of herpes simplex virus growth in transgenic mice. A mutant allele (X25) of an essential regulatory protein, ICP4, encoded by herpes simplex virus (HSV) has been shown to have a transdominant, negative effect on the activity of the wild-type protein, resulting in the inhibition of virus growth in vitro. The X25 protein appears to exert its transdominant effect by sequestering functional ICP4 monomers into nonfunctional, heterodimeric complexes (A. Shepard, P. Tolentino, and N. A. DeLuca, 1990, J. Virol. 64, 3916-3926). In order to assess the antiviral potential of X25 in vivo, four transgenic mouse lines were generated bearing 1 to 10 copies of a DNA fragment encoding the mutant allele. Monolayers of embryonic cells prepared from each of the lines expressed the transgenic X25 protein. When challenged via the eye, every line exhibited at least some enhanced resistance to HSV infection. In the best line, transgenic animals exhibited a statistically significant (> 95% confidence) 5- to 13-fold lower eye swab titer relative to their nontransgenic littermates at Day 1 postinfection. A similar reduction in titer was observed in the trigeminal ganglia at Day 3 postinfection. These results indicate that the X25 protein is able to exert a significant antiviral effect in vivo.


Purification and crystallization of intact human rhinovirus complexed with a neutralizing Fab. We report the first crystallization of an intact virion, human rhinovirus 14, complexed with the Fab fragment from a neutralizing antibody. These crystals diffract to at least 6.0A resolution. It has been suggested that Fab's and mAb's can induce large conformational changes in the capsid upon binding. The structure of this complex should enable us to detect the existence and role of such changes.


A common integration locus in type B retrovirus-induced thymic lymphomas. Type-B leukemogenic retrovirus (TBLV) is a replication-competent type-B thymotropic retrovirus which lacks a transforming gene and whose genome is > 98% homologous to that of type-B mouse mammary tumor virus (MMTV). In contrast to MMTV, which induces mammary adenocarcinomas, TBLV induces a high incidence of T-cell thymic lymphomas in mice after a very short latent period. To investigate the molecular mechanisms by which TBLV induces T-cell lymphomas, we screened TBLV-induced tumor DNA for the frequent disruption of a particular cellular locus by TBLV proviral copies. In approximately 20% of the 55 primary tumors screened, the presence of proviruses in a common integration site was detected. This locus spans at least 53 kb of genomic DNA and maps to the mouse X chromosome. The presence of a functional gene at this locus is suggested by the conservation of nucleotide sequences from this locus among diverse animal species and by the expression of these sequences as mRNA in normal mouse tissues and tumors. The majority (17/18) of TBLV-induced primary tumors examined have elevated levels of this expressed mRNA.


Identification and transcriptional mapping of genes encoded at the IR/Us junction of equine herpesvirus type 1. Two open reading frames (ORFs) encoded at the inverted repeat unique short (Us) junction of the Short (S) region of the equine herpesvirus type 1 genome were identified by DNA sequencing of a 2876 base pair (bp) genomic segment, and transcripts encoding these ORFs were characterized by Northern blot, S1 nuclease, and primer extension analyses. These studies also established the size of each inverted repeat to be 12,768 nucleotides (nts). The IR6 ORF (816 bp), mapping at nts 12,317-11,502 of the S region, is the last gene completely encoded within each inverted repeat and encodes a predicted 30.1-kDa protein of 272 amino acids, which does not exhibit homology to other alphaherpesvirus proteins. IR6 is expressed as an early transcript of 1.2 kb which is detected initially at 1.5 hr p.i. and up to 12 hr p.i. The transcription initiation and termination sites of IR6 were mapped by primer extension and S1 nuclease analyses to nts 12,465 and 11,408, respectively. The first ORF encoded within the Us segment (909 bp; EUS1), mapping at nts 13,397-12,489, encodes a predicted 33.5-kDa protein of 303 amino acids that exhibits 29% identity to the US2 protein of herpes simplex virus 1. EUS1 is expressed as a 2.3-kb mRNA of the gamma-1 class, as its synthesis begins prior to viral DNA replication at 4 hr p.i. but is retarded by phosphonoacetic acid, an inhibitor of viral DNA replication. The Tci and Tct sites of EUS1 were mapped by S1 nuclease analyses to nts 13,637 and 11,408, respectively. Interestingly, this termination site is also utilized by three late mRNAs of 5.8, 3.8, and 1.7 kb which originate within the Us and overlap the IR6 mRNA encoded in the terminal inverted repeat (TR) of the prototype genomic isomer. EUS1 is 3' coterminal with IR6 in the inverted repeat, whereas, the 5.8, 3.8, and 1.7 kb transcripts are 3' coterminal with IR6 of the TR.


Intracellular localization of poliovirus RNA by in situ hybridization at the ultrastructural level using single-stranded riboprobes. Polioviral RNA was localized by electron microscopic in situ hybridization on sections of poliovirus-infected HEp-2 cells. Viral plus-strand RNA was found accumulated in the close surroundings of the membrane-bound replication complex. Two different regions of the viral genome were detected with the same frequency, which indicates the predominant presence of full-length genomic RNA. Viral proteins of the P2 and the P3 genomic region were detected mainly over the core of the replication complex, whereas the hybridization signal was present rather at the peripheral parts of the complex. A more than 100-fold excess of viral plus- over minus-strand RNA was found by strand-specific hybridization to RNA extracted from isolated replication complexes. These findings support the idea of a pool of viral plus-strand RNA set free from the replication complex and accumulating in the close vicinity of the replication complex possibly before encapsidation.


The rotavirus nonstructural protein, NS35, possesses RNA-binding activity in vitro and in vivo. Toward the goal of identifying and characterizing rotavirus RNA-binding proteins, we have used a gel retardation assay and protein-RNA cross-linking by ultraviolet (uv) light to examine cytoplasmic lysates prepared from SA11-infected cells for the presence of RNA-binding proteins. Analysis of band shifts produced in the gel retardation assay indicated that infected cells contained significant amounts of a viral protein which had affinity for both single-stranded and double-stranded RNA but lacked sequence specificity. Cross-linking of this protein to radiolabeled RNA in vitro followed by RNase treatment and immunoprecipitation with an anti-NS35 monoclonal antibody revealed that the RNA-binding activity was associated with NS35. Moreover, sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the protein-RNA complex isolated from native gels revealed that NS35 was the only viral protein component of the complex. Since NS35 expressed by translation in rabbit reticulocyte lysates exhibited affinity for poly(U)-Sepharose, NS35 must possess intrinsic RNA-binding activity that is able to function in the absence of other viral proteins. Immunoprecipitation of RNase-treated cross-links formed in intact cells following exposure to uv light confirmed that NS35 was intimately associated with ssRNA in the infected cell. On the basis of its ability to bind RNA and given that previous studies have shown that NS35 localizes to the viroplasm in infected cells, is essential for RNA replication, and is a component of replicase particles, we propose that NS35 functions to concentrate viral mRNAs in the viroplasm and that NS35-mRNA complexes serve as substrates for genome assortment and replication.


Polyoma virus middle T is essential for virus replication and persistence as well as for tumor induction in mice. A mutant strain of polyoma virus encoding a truncated middle T protein has been studied for its ability to replicate and induce tumors following inoculation into newborn mice. Virus replication in the acute period prior to expected onset of tumors as well as persistence of virus in older animals were followed. The mutant virus proved to be defective in replication and persistence and failed to induce tumors. These results demonstrated that middle T plays an essential role in productive viral infection in the animal. Since the mutant virus encodes normal large and small T proteins, the results also indicate that functions associated with these T antigens, including large T binding of the retinoblastoma tumor suppressor gene product and the ability to immortalize, are insufficient to cause development of tumors in this system.


Polyoma tumorigenesis in mice: evidence for dominant resistance and dominant susceptibility genes of the host. We have determined the tumor responses of nine inbred mouse strains and of two F1 hybrids following inoculation with polyoma virus. The results showed wide variations in the frequencies of mice developing tumors. Correlation with H-2 haplotype were evident. C3H/BiDa mice of H-2k type were fully susceptible, while C57BL/6 mice of H-2b type were completely resistant. DBA/2 and BALB/c mice, both H-2d, were of low susceptibility. Contributions of the major histocompatibility complex were demonstrated using pairs of H-2 congenic mice. Substitution of H-2k on either a BALB/c or a C57BL/10 background resulted in an increase in the frequencies and kinds of tumors induced. Susceptibilities of BALB and C57BL mice bearing the H-2k haplotype were still well below those of C3H/BiDa mice, however. Crosses of C3H/BiDa with BALB/c or BALB.K (H-2k) produced F1s that were of moderate and full susceptibility, respectively. These results indicate two distinct classes of host genes, one at the H-2 locus where different alleles confer varying degrees of resistance and the other a dominant susceptibility gene(s) carried in C3H/BiDa mice.


Lymphotropic papovavirus transforms hamster cells without altering the amount or stability of p53. Expression of the early regions of several primate polyomaviruses (SV40, BKV, JCV, and LPV) in hamster cells induces transformation, manifested by the ability to grow in soft agar. Hamster cells transformed by SV40 contain complexes between the SV40 T antigen and the cellular tumor suppressor protein p53. We detected analogous complexes between p53 and the BKV T antigen in hamster cells transformed by the BKV early region, where the half life of p53 increased 16-fold. However, neither a LPV-transformed hamster fibroblast cell line [LPV-HE (F); K. K. Takemoto and T. Kanda, 1984, J. Virol. 50, 100-105] nor BHK-21 cells transformed by the LPV early region contained detectable complexes between the LPV T antigen and p53, nor was the stability of p53 in LPV transformed BHK-21 cells altered. Association between hamster p53 and the LPV T antigen expressed as glutathione S-transferase fusion protein could not be detected in vitro. These data indicate that alteration of the amount or stability of p53 is not required for transformation of hamster cells by LPV. However, as viruses such as SV40 and BKV whose T antigens bind p53 are oncogenic in hamsters, whereas LPV is not, the alteration of p53 amount or stability may be required for tumorigenesis.


The interleukin-10 homolog encoded by Epstein-Barr virus enhances the reactivation of virus-specific cytotoxic T cell and HLA-unrestricted killer cell responses. We determined what influence the Epstein-Barr virus (EBV)-encoded homologue of IL-10 (viral or vIL-10) had on immune responses important for the control of EBV infection. We produced recombinant vIL-10 in a B cell line. A 17-kDa recombinant protein was secreted and had the same molecular weight as vIL-10 secreted by EBV-infected B cells. Functional activity of recombinant vIL-10 was shown by the inhibition of interferon-gamma production by activated leukocytes. Cytotoxic T cells and HLA-unrestricted activated killer cells are both important arms of the immune response to EBV. vIL-10, either expressed by B cell stimulators or added exogenously, enhanced the in vitro reactivation of allo- and EBV-specific cytotoxic T cells. vIL-10 also enhanced the activation of HLA-unrestricted killer cells by EBV-transformed B cells. In contrast, the interleukin-2-mediated activation of these killers was unaffected. Since vIL-10 is expressed during the lytic cycle of EBV, we conclude that the expression of vIL-10 may enhance immune responses to EBV-infected cells during periods of virus replication in vivo. In this way, the virus may limit its own replication and maintain the apathogenic virus carrier state that is characteristic of EBV.


Identification and functional analysis of the fowlpox virus homolog of the vaccinia virus p37K major envelope antigen gene. A fowlpox virus (FPV) gene with homology to the vaccinia virus p37K major envelope antigen gene was identified and sequenced. The predicted product has a molecular weight of 43,018 Da (p43K). The FPV p43K gene has 37.5% identity with its vaccinia counterpart and higher homology with a molluscum contagiosum virus gene (42.6% identity). Based on upstream sequences, p43K appears to be regulated as a late gene. Recombinant FPV were generated in which a large portion of p43K was replaced by the Escherichia coli lacZ gene. These recombinants failed to produce visible plaques under standard conditions. After prolonged incubation the microplaques developed into small macroscopic plaques. Plaques were purified on the basis of lacZ expression. Single-cycle growth curves comparing the p43K-deleted recombinant (designated fJd43Z) with parental FPV showed that the two viruses produce identical amounts of intracellular virions, but that fJd43Z released 20-fold fewer infectious particles into the medium. CsCl gradient centrifugation of [3H]thymidine-labeled virus was employed to examine differences in the production of physical particles. The two viruses produced equivalent levels of intracellular virions, but fJd43Z failed to produce detectable levels of released particles. FPV p43K is therefore involved in the release of virions from infected cells.


Conserved tertiary structure elements in the 5' untranslated region of human enteroviruses and rhinoviruses. A combination of comparative sequence analysis and thermodynamic methods reveals the conservation of tertiary structure elements in the 5' untranslated region (UTR) of human enteroviruses and rhinoviruses. The predicted common structural elements occur in the 3' end of a segment that is critical for internal ribosome binding, termed "ribosome landing pad" (RLP), of polioviruses. Base pairings between highly conserved 17-nucleotide (nt) and 21-nt sequences in the 5' UTR of human enteroviruses and rhinoviruses constitute a predicted pseudoknot that is significantly more stable than those that can be formed from a large set of randomly shuffled sequences. A conserved single-stranded polypyrimidine tract is located between two conserved tertiary elements. R. Nicholson, J. Pelletier, S.-Y. Le, and N. Sonenberg (1991, J. Virol. 65, 5886-5894) demonstrated that the point mutations of 3-nt UUU out of an essential 4-nt pyrimidine stretch sequence UUUC abolished translation. Structural analysis of the mutant sequence indicates that small point mutations within the short polypyrimidine sequence would destroy the tertiary interaction in the predicted, highly ordered structure. The proposed common tertiary structure can offer experimentalists a model upon which to extend the interpretations for currently available data. Based on these structural features possible base-pairing models between human enteroviruses and 18 S rRNA and between human rhinoviruses and 18 S rRNA are proposed. The proposed common structure implicates a biological function for these sequences in translational initiation.


Molecular cloning and nucleotide sequence of a pestivirus genome, noncytopathic bovine viral diarrhea virus strain SD-1. Genomic RNA of noncytopathic (NCP) bovine viral diarrhea virus (BVDV) strain SD-1 was extracted directly from serum obtained from a persistently infected animal. cDNA was synthesized and amplified by polymerase chain reaction (PCR) before cloning. The complete genomic nucleotide sequence was determined by sequencing at least two different clones from independent PCR reactions. The 5' and 3' end sequences of the SD-1 genome was determined from 5'-3' ligation clones. The complete genome sequence was comprised of 12,308 nucleotides containing one large open reading frame which encodes an amino acid sequence of 3898 residues with a calculated molecular weight of 438 kDa. In contrast to cytopathic (CP) BVDV strain NADL, which contains a cellular RNA insert of 270 nucleotides and CP BVDV strain Osloss, which has an inserted ubiquitin RNA sequence of 228 nucleotides, the NCP strain SD-1 had no insertion along the genome. Sequence comparison with other pestiviruses revealed that the overall nucleotide sequence homologies of SD-1 are 88.6% with NADL, 78.3% with Osloss, 67.1% with HoCV Alfort, and 67.2% with HoCV Brescia. The overall deduced amino acid sequence homologies of SD-1 are 92.7% with NADL, 86.2% with Osloss, 72.5% with HoCV Alfort, and 71.2% with HoCV Brescia. The most conserved nucleotide and amino acid sequences are located in the 5' untranslated region (5'UTR) and nonstructural protein p80 region, respectively. The viral glycoproteins, particularly gp53, and nonstructural proteins p54 and p58 have the lowest homology comparing both nucleotide and amino acid sequences between SD-1 and other pestiviruses. Extensive analyses of amino acid sequences for the viral structural proteins and nonstructural protein p54 regions from five pestiviruses led to the identification of four conserved domains (designated as C1, C2, C3, C4) and three highly variable domains (designated as V1, V2, V3) within this region. The C1, C2, and C3 domains are located in the capsid protein p14, glycoprotein gp48, and gp25, respectively. The C4 domain is located in the junction between gp53 and p54. Interestingly, out of three variable domains, two (V1, V2) are located in the same glycoprotein gp53. The third variable domain is located in the nonstructural protein p54.


Identification of a sequence element in the alphavirus core protein which mediates interaction of cores with ribosomes and the disassembly of cores. Early in infection core protein is transferred from alphavirus cores to ribosomes (Wengler and Wengler, 1984, Virology 134, 435-442) and it has been suggested that ribosome binding is a property of alphavirus core protein which is involved in core disassembly. Here we describe in vitro analyses of this transfer. Sindbis virus cores, incubated with ribosomes either in a reticulocyte lysate or in buffer, are disassembled with a concomitant transfer of core protein to the large ribosomal subunit. Preincubation of ribosomes with core protein blocks disassembly. Limited proteolysis of Sindbis virus core releases the carboxy-terminal core protein domain as a soluble fragment (Strong and Harrison, 1990, J. Virol. 64, 3992-3994). Trypsin- or proteinase Lys-C-released fragments contain the amino-terminal residue met (106) or gln (94), respectively. The fragment generated by proteinase Lys-C binds to ribosomes and interferes with core disassembly whereas the slightly shorter tryptic fragment has none of these activities. These and further analyses indicate that a conserved sequence element which surrounds amino acid met (106) of SIN CP, the so-called RBSc element, leads to binding of core protein to ribosomes and thereby to core disassembly. Implications of the experiments for regulation of assembly of alphavirus cores and for the core protein-induced resistance to viral multiplication observed in plant virus systems are discussed.


The virulence-determining genomic BamHI fragment 4 of pseudorabies virus contains genes corresponding to the UL15 (partial), UL18, UL19, UL20, and UL21 genes of herpes simplex virus and a putative origin of replication. The genomic BamHI-fragment 4 of pseudorabies virus (PrV) has previously been shown to encode functions necessary for expression of PrV neurovirulence (B. Lomniczi, S. Watanabe, T. Ben-Porat, and A. S. Kaplan, 1984, J. Virol. 52, 198-205). To identify proteins that might be involved in the neurotropism of PrV we sequenced the complete 9382-bp fragment BamHI-4, the longest contiguous sequence determined in the UL region of PrV so far, and analyzed its coding capacity. In an arrangement similar to that found in herpes simplex virus type 1 we identified complete open reading frames encoding proteins with strong homology to the UL18 (50% homology), UL19 (60% homology), UL20 (33% homology), and UL21 (36% homology) polypeptides and the 3'-part of a gene homologous to UL15 (67% homology) of HSV-1. In addition, a consensus sequence for an alphaherpesviral origin of replication was found at the left terminus of the fragment.


Sequence characterization and expression of the matrix protein gene of human parainfluenza virus type 1. The nucleotide sequence of the M gene of human parainfluenza virus type 1 (hPIV1) was determined from genomic RNA and cDNA copies of the entire gene. The M gene contained 1173 nucleotides. It had one large open reading frame capable of encoding a protein of 348 amino acids (M(r) = 38,404). The predicted amino acid sequence of the hPIV1 M protein is highly basic (+20 at neutral pH). A pGEM-1 expression vector containing the M gene was used for cell-free transcription and translation. The resultant protein was confirmed to be M by electrophoretic mobility and immunoprecipitation. Among other paramyxoviridae the hPIV1 M amino acid sequence was most closely related to the Sendai virus M sequence (87% identity). The pattern of M gene relatedness observed from the alignment of 16 paramyxoviridae M protein amino acid sequences was not predicted by the viruses' taxonomic classification.


Human papillomavirus type 16 expresses a variety of alternatively spliced mRNAs putatively encoding the E2 protein. The full-length E2 protein of human papillomavirus type 16 is believed to act as a trans-repressor of the viral p97 promoter. Previous reports have provided evidence that transcripts with the potential to encode the E2 protein contain the 880/2708 splice junction. We have further analyzed the structure of the E2-encoding transcripts. Employing the RNA polymerase chain reaction (PCR) technique and analyses of the RNA PCR products by Southern blot hybridization and DNA sequencing, we revealed the existence of a variety of alternatively spliced mRNAs, with the capacity to encode the full-length E2 protein. Two novel splice junctions were identified at nucleotides 880/2581 and 226/2708. E2 mRNAs characterized by the 880/2581 splice junction contain sequences from the E1 orf predicted to encode a truncated E1 polypeptide consisting mainly of the C terminal amino acids. Transcripts with the 226/2708 splice junction could encode a novel E6 protein, designated E6IV, containing C terminal amino acids derived from an out-of-frame region of the E1 ORF. Three different E6-E7 exons were identified in mRNAs containing the 880/2708 and the 880/2581 splice junctions, namely, E6-E7, E6I-E7, E6II-E7. The E6I-E7 mRNAs are the most abundant. Expression of the various E2 mRNAs was detected in human keratinocytes immortalized by HPV16, in cervical tumors, and in carcinoma cell lines.


Properties of bovine papillomavirus E1 mutants. Ostensibly comparable mutants of bovine papillomavirus type 1 (BPV-1) affecting the E1 open reading frame that were constructed in several laboratories have been reported to exhibit either reduced or increased transformation efficiencies in established mouse cell lines relative to wild-type BPV-1 DNA. To resolve these discrepancies, we have reexamined many of the mutants in mouse C127 cells by using focus formation assays. Our primary conclusions are that all E1 mutants tested consistently generated reduced numbers of transformants and that the reduced transformation was not due to cell toxicity associated with E1 mutations, as had been proposed. Our results can best be explained by the inability of the E1 mutants to replicate extrachromosomally, therefore leading to a rapid loss of the BPV-1 DNA and consequently, reduced transformation. In support of this hypothesis, we demonstrated that the human papillomavirus type 11 E1 protein was able to suppress BPV-1 transformation, probably because of interference with BPV-1 replication. Therefore, we attribute the phenotypic disparities reported by the various laboratories to still undefined differences in assay conditions.


Identification of a nuclear localization sequence in the polyomavirus capsid protein VP2. A nuclear localization signal (NLS) has been identified in the C-terminal (Glu307-Glu-Asp-Gly-Pro-Gln-Lys-Lys-Lys-Arg-Arg-Leu318) amino acid sequence of the polyomavirus minor capsid protein VP2. The importance of this amino acid sequence for nuclear transport of newly synthesized VP2 was demonstrated by a genetic "subtractive" study using the constructs pSG5VP2 (expressing full-length VP2) and pSG5 delta 3VP2 (expressing truncated VP2, lacking amino acids Glu307-Leu318). These constructs were transfected into COS-7 cells, and the intracellular localization of the VP2 protein was determined by indirect immunofluorescence. These studies revealed that the full-length VP2 was localized in the nucleus, while the truncated VP2 protein was localized in the cytoplasm and not transported to the nucleus. A biochemical "additive" approach was also used to determine whether this sequence could target nonnuclear proteins to the nucleus. A synthetic peptide identical to VP2 amino acids Glu307-Leu318 was cross-linked to the nonnuclear proteins bovine serum albumin (BSA) or immunoglobulin G (IgG). The conjugates were then labeled with fluorescein isothiocyanate and microinjected into the cytoplasm of NIH 3T6 cells. Both conjugates localized in the nucleus of the microinjected cells, whereas unconjugated BSA and IgG remained in the cytoplasm. Taken together, these genetic subtractive and biochemical additive approaches have identified the C-terminal sequence of polyoma-virus VP2 (containing amino acids Glu307-Leu318) as the NLS of this protein.


The latency-related gene of bovine herpesvirus 1 inhibits the activity of immediate-early transcription unit 1. Bovine herpesvirus 1 (BHV-1) establishes a latent infection in sensory neurons of infected animals. Only one virus-encoded latency-related (LR) gene is expressed during a latent infection. The LR transcript overlaps immediate-early transcription unit 1 (IEtu1) and is anti-sense with respect to IEtu1. The transcriptional start site of the LR RNA was mapped to position 724 of the LR gene, downstream from two putative TATA elements. When LR promoter sequences were deleted from a plasmid containing IEtu1 and the LR gene, the resulting construct trans-activated the HSV-1 thymidine kinase (TK) promoter more efficiently than IEtu1 plus the LR gene. Cotransfection of a plasmid containing the intact LR gene with IEtu1 inhibited the ability of IEtu1 to trans-activate the TK promoter. These results imply that a LR gene product(s) repressed the trans-acting capacity of IEtu1.


Plasma levels of the lipid mediators, leukotriene B4 and lyso platelet-activating factor, in intraoperative salvaged blood. It has been shown that white cells and platelets release their granules into the plasma of salvaged blood. Whether this release is due to destruction alone or a combination of destruction and activation is not known. Lipid mediators, platelet-activating factor and leukotriene B4, are produced by activated white cells and platelets and have effects on the cardiovascular, respiratory and immune systems and the microcirculation. The aim was to determine if white cells and platelets are activated in salvaged blood by measuring the levels of these lipid mediators. Ten patients undergoing aortic surgery, where intraoperative salvage was used, were studied. Blood samples were taken from the patient's circulation and the salvaged blood before and after washing. The levels of leukotriene (LTB4) and lyso platelet-activating factor (PAF, the stable degradation product of PAF) were measured in the samples by a radioimmunoassay and a bioassay, respectively. The levels of both these substances increased in the unwashed salvaged blood (mean patient levels: LTB4 27 +/- 4.3 ng/ml and L-PAF 73 +/- 8.5 ng/ml; mean unwashed blood levels: LTB4 95 +/- 12.2 ng/ml and L-PAF 172.9 +/- 26.4 ng/ml) and were reduced by washing of the collected blood (mean washed blood levels of LTB4, 23.9 +/- 4.8 ng/ml, and L-PAF 18 +/- 5 ng/ml). The increase of the lipid mediators in the unwashed salvaged blood indicates that white cells and platelets are activated and releasing lipid mediators. Washing of the collected blood is effective in removing the lipid mediators.


Risk factors for hepatitis C virus antibody positivity in blood donors in a low-risk country. Demographic variables, sexual risk behavior and prevalence of parenteral risk factors were studied in 305 randomly selected donors seronegative for hepatitis C virus, in 170 randomly selected donors reactive on solely enzyme-linked immunosorbent assay (ELISA C-100), in 71 consecutive donors reacting indeterminately according to the second-generation recombinant immunoblot assay (RIBA II) and in 46 consecutive donors found to be positive using the RIBA II. Donors who were positive by RIBA II had significantly more often a risk factor, for example use of intravenous drugs or previous blood transfusion, than donors reacting indeterminately (34 out of 46) (73.9%) versus 14 out of 71 (19.7%, p = 0.0000). Donors reacting indeterminately by RIBA II had one of those risk factors significantly more often than seronegative donors (14 out of 71) (19.7%) versus 23 out of 280 (7.8%, p < 0.005). When donors either positive or indeterminate by RIBA II were compared with donors negative for hepatitis C antibodies, the odds ratio for a possible parenteral source of infection was 7.6 (p = 0.0000). Subjects who had received a poor education (odds ratio 0.3, p < 0.001) or who lived in southern Finland (odds ratio 2.3, p < 0.05) were also at higher risk for being positive or indeterminate in RIBA II. First-time donors were also prone to having antibodies according to RIBA II (odds ratio 2.2, p = 0.1), whereas sexual risk behavior, gender, age, occupational class and type of residential area were not risk factors for hepatitis C antibodies in RIBA.(ABSTRACT TRUNCATED AT 250 WORDS)


Confirmation of hepatitis C virus positive blood donors by immunoblotting and polymerase chain reaction. In a series of 385 sera obtained from volunteer blood donors positive for the first-generation hepatitis C virus assay (Ortho), the viral genome was detected by polymerase chain reaction (PCR) in 89 sera (23%). Most PCR-positive sera were found positive with the c100-3 neutralisation assay (Abbott) and by two second-generation enzyme immunoassays (Abbott, Ortho). However overall specificity of these assays was rather low. By immunoblotting (Innogenetics and Chiron/Ortho) the specificity could be considerably improved and the best correlation with carrier state was obtained when analysing the results for lane-specific reaction: all 89 viral carriers and only 9 other donors had antibodies against structural 'core' epitopes. From the present data we can conclude that in screening a volunteer blood donor population the confirmation of antibodies against 'core' epitopes by immunoblotting is strongly associated with viral carriage.


Antibody responses to hepatitis C virus by second-generation immunoassays in a cohort of patients with bleeding disorders. The antibody responses and the prevalence patterns of antibodies to hepatitis C virus (anti-HCV) in a cohort of patients (n = 210) with bleeding disorders were studied using a first-generation and a second-generation enzyme immunoassays (EIA-1, EIA-2) as well as a second-generation recombinant immunoblot assay (RIBA-2). The anti-HCV prevalence as determined by EIA-1 and EIA-2 was 183/210 (87.1%) and 197/210 (93.8%), respectively (p = 0.0026). None of the 17 EIA-2(+)/EIA-1(-) samples was scored nonreactive by RIBA-2. At follow-up, samples of 123 patients were tested. Twenty-nine out of 111 patients reactive by EIA-1 seroreverted according to EIA-1 while the seroreversion rate with EIA-2 was 0 out of the 121 (p < 10(-8)). The anti-HCV prevalence by EIA-2 was 150/154 (97.4%) in anti-HIV-1-positive individuals and 47/56 (83.9%) in the anti-HIV-1-negative ones (p = 0.001). However, high assay signals (OD 492 nm > 2.0) were observed in 94/150 (62.7%) and 45/47 (95.7%) of the anti-HIV-1-positive and -negative patients, respectively (p = 10(-5)). The decreasing anti-HCV reactivity among anti-HIV-1-positive individuals was mainly due to diminishing c33c reactivity. Seroconversion to anti-HCV was observed in 3/7 (42.9%) cases with acute icteric non-A, non-B hepatitis by both EIA-1 and EIA-2, while the remaining 4 cases had detectable levels of anti-HCV 1-18 months before the acute episode.


[The characteristics of the dispensary observation and treatment of patients with duodenal peptic ulcer following organ-preserving operations]. An analysis of results of organ saving operations in combination with selective proximal vagotomy in 234 patients with duodenal ulcer revealed that one of the causes of unfavourable treatment outcomes is the development of erosive-ulcerative lesions of the stomach. The leading etiological factors for distal gastritis in duodenogastric reflux, for proximal gastritis-ischemia of the gastric wall in the devascularization zone. Individualized treatment with consideration of the etiopathogenesis of postoperative gastritis allows to reduce the frequency of development of postvagotomy disorders by 19.8%.


[Experimental studies of the inhibition of fibrogenesis]. Colchicine as well HOE 077 were found to be effective in the inhibition of hepatic collagen accumulation after bile duct ligation in rats. The effect was additive and was truly reflected by changes in the serum PIIIP and 7s-collagen (IV) concentration. Extrahepatic effects on collagen synthesis were not detected when HOE 077 was used. Thus further studies on the potential clinical usefulness of these compounds are clearly indicated.


Antigonadotropic activity of hop extract. Biological activity of water soluble fractions F-1 and F-2, which were extracted from hop, was studied and its action mechanism was speculated using the immature female SD rats. Administration of the substance significantly inhibited the effects of pregnant mare serum gonadotropin (PMSG) on 22-day-old female rats. Thus, PMSG-induced increases in ovarian weight, estrogen secretion, number of ovulated egg, progesterone production, uterine thymidine kinase activity, and plasma LH level were suppressed significantly. Furthermore, addition of the substance to incubated ovarian cells of the second day after PMSG injection resulted in suppression of FSH-induced estradiol secretion in vitro, probably via cAMP-dependent mechanism. But addition of the substance to incubated pituitary cells from ovariectomized rats did not change in LH secretion.


Innervator NS 252, a new, constant current and programmable peripheral nerve stimulator. A peripheral nerve stimulator should be able to deliver a constant current to provide supramaximal nerve stimulation even under conditions of increasing resistance. We tested a new programmable peripheral nerve stimulator, the Innervator NS 252. It was able to maintain a constant current of 80 mA up to a resistance of 3.9 kohm. The pulse appearance was correct and there were only small variations in stimulation time intervals. The different type of double burst stimulation and the lack of synchronization with the previously applied stimulus pattern may be regarded as slight disadvantages.


Lectin binding patterns in normal liver, chronic active hepatitis, liver cirrhosis, and hepatocellular carcinoma. An immunohistochemical and immunoelectron microscopic study. We studied the binding patterns of 14 lectins in human normal and cirrhotic liver (LC) tissues and hepatocellular carcinomas (HCC) using the ABC method. Lectins were divided into 4 groups according to their binding patterns in normal tissues: (A) PHA, MPA, LcH, RCA-I, and WGA, which bound to hepatocytes and all three types of sinusoidal cells; (B) BPA, GS-I, PNA, and SBA, which bound to Kupffer cells and endothelia of interlobular arteries and veins and bile duct epithelia in the portal tract, but not to hepatocytes; (C) UEA-I, which bound only to endothelia of interlobular arteries and veins and bile duct epithelia in the portal tract; (D) LBA, Lotus, LPA, and SJA, which showed no binding. Thus group B lectins may be useful markers of Kupffer cells. Only electron microscopic examination revealed the precise binding sites of lectins in sinusoidal cells and hepatocytes. Hepatocyte cell surface polarities demonstrated by lectin binding in LC and HCC were different from those in the normal liver. The binding pattern of PHA to LC hepatocytes changed from a membranous to both a membranous and a cytoplasmic pattern, and that of LcH to HCC cells changed to dot-like staining in the cytoplasm. These changes of polarities in LC and HCC might be caused by changes in the distribution of lectin-binding carbohydrates or by the altered glycosylation of glycoconjugates.


Altered stoichiometry of the Na,K-ATPase. With inside-out membrane vesicles derived from human red cells and incubated at pH > or = 6.6, an altered sodium pump stoichiometry (1Na+:2K+) associated with altered charge transfer is observed when the cytoplasmic Na+ concentration is reduced to very low levels (0.2 mM). With increased proton concentration (approximately pH 6.0), protons can substitute for Na+ or K+ ions such that the Na,K-ATPase can effect either electroneutral Na+/H+ exchange (K+ absent), H+/K+ exchange (Na+ absent), or H(+)-plus-Na+ cotransport in exchange for K+ (low Na+ concentration). Evidence that the stoichiometries of these exchanges are 3Na+/3H+, 2H+/2K+ and 1H(+)-plus-1Na+/2K+, respectively, is presented.


Fine needle aspiration cytology of rare malignant tumors of the breast. This report describes the fine needle aspiration (FNA) cytologic findings in 17 rare malignant breast tumors. The series consisted of invasive cribriform carcinoma, papillary carcinoma, apocrine carcinoma, carcinoma with pseudosarcomatous metaplasia, carcinosarcoma, fibrosarcoma, malignant phyllodes tumors, primary malignant lymphomas, plasmocytoma, metastatic melanoma and metastatic renal clear cell carcinoma. Besides cytomorphology, the results of immunostaining in eight cases are presented, as is a review of the literature. It is important for rare primary malignancies, as well as for metastatic tumors, to be diagnosed, or at least have the diagnosis suggested, preoperatively by FNA and immunocytochemistry, permitting better therapy planning.


Charge movements via the cardiac Na,K-ATPase. The voltage dependence of transient and steady-state pump currents was examined in guinea pig ventricular myocytes to investigate mechanisms of charge translocation by the Na,K-ATPase. Na/K pump current was determined at approximately 36 degrees C as strophanthidin-sensitive whole-cell current in myocytes voltage clamped and internally dialyzed via wide tipped pipettes containing a pipette perfusion device. External Na ions diminished stationary pump current during forward Na/K cycling in a voltage dependent manner, the inhibition becoming stronger upon hyperpolarization. When Na,K-ATPase activity was restricted to Na translocation steps, stationary pump current was prevented but voltage pulses still elicited large transient pump currents which could be abolished by oligomycin B (> or = 2 micrograms/ml). The transients arose instantaneously on stepping the voltage, and decayed with voltage-dependent approximately single exponential time courses. The decay rates, and their high temperature sensitivity (approximately 200 s-1 at 0 mV at 36 degrees C; approximately 40 s-1 at 20 degrees C), suggest that the charge movements were limited by a conformational change associated with Na deocclusion. Those rates varied asymmetrically with voltage, changing little at positive voltages but increasing roughly exponentially with hyperpolarization (e-fold/approximately 80 mV). Lowering the extracellular [Na] ([Na]o) slowed the relaxation of charge movement at negative potentials but had little effect at positive potentials, and so shifted the rate constant-voltage curve to the left. The implied dependence on [Na]o of the backward rate constant governing pump charge movement accounts satisfactorily for the observed [Na]o sensitivity of stationary outward Na/K pump current, and indicates that the voltage-dependent step somehow involves the release of Na ions to the external medium. However, no strophanthidin-sensitive current was seen, at saturating external [K], when Na,K-ATPase activity was limited to K translocation steps by complete withdrawal of Na ions. But, at very low [Na]o, a weak negative slope appeared in the stationary pump current-voltage relationship at subsaturating, but not at saturating, external [K], indicating an increased apparent affinity for external K at more negative potentials. The results support the existence of a high field access channel through which extracellular Na and K ions must pass before interacting with their binding sites deep within the Na,K-ATPase molecule.


Isoforms of the alpha subunit of Na,K-ATPase and their significance. Recent studies of the Na,K-ATPase have demonstrated that multiple isoforms of both the alpha and beta subunits exist and that these are expressed in a tissue and developmental specific manner. In the case of the alpha subunit, there are three known isoforms, alpha 1, alpha 2 and alpha 3. We have examined adult human heart for the presence of these isoforms and found that all three exist in approximately equal amounts. This is in contrast to the adult rat heart which contains only alpha 1 and alpha 2 isoforms. The difference in abundance of various isoforms in various tissues could result from the necessity to express Na,K-ATPase with different properties at various developmental stages or in specific cell types. For example, enzymes with differences in Na+ or K+ affinity or the ability to respond to various effector molecules may be required. Alternatively, the presence of three isoforms may simply result from the triplication of the alpha subunit gene and the divergence of expression of these genes during evolution. In this case the isozymes would not confer a specific function to the Na,K-ATPase. In order to provide information with respect to these two alternatives, cell lines producing rat alpha 1, alpha 2 and alpha 3 were developed and the enzymatic properties of the resulting enzyme determined. The results indicate that Na,K-ATPase carrying the alpha 1 are alpha 2 isoforms are fairly similar while enzyme with the alpha 3 isoform differs in its apparent affinity for sodium. The K0.5 for Na+ is approximately three fold lower for this isoform.(ABSTRACT TRUNCATED AT 250 WORDS)


Subunit assembly and posttranslational processing of Na(+)-pumps. Assembly, posttranslational processing and intracellular transport of Na,K-ATPase subunits are closely linked processes. Only coexpression of alpha- and beta-subunits in Xenopus oocytes permits the structural maturation and the ER exit of the two subunits. A putative beta 3-isoform of Na,K-ATPase and the beta-subunit of H,K-ATPase can support similar functions to those of beta 1-subunits of Na,K-ATPase in allowing the functional expression of alpha-beta complexes with Na,K-pump transport properties in the plasma membrane. These data indicate that a primary role of the beta subunit is to impose a conformational change on the newly synthesized alpha-subunits probably involved in the acquisition of transport competence and functional maturity of the catalytic alpha-subunit.


Cytochrome oxidase as a redox-linked proton pump. Some general principles for redox-linked proton pumps are described and applied to cytochrome oxidase. A reaction scheme obeying these principles is described. The scheme includes electron transfer between the redox centers of the oxidase, the mechanism of dioxygen reduction, protonation and deprotonation steps for both scalar and vectorial protons, as well as input-output transitions for electrons and protons. It is consistent with a large amount of kinetic information.


Ionics and conformational transitions of Na,K-ATPase. Equilibrium binding studies have been carried out by spectrofluorometric precision titrations on FITC-Na,K-ATPase and employing the styryl dye RH-421 to obtain equilibrium constants and stoichiometric coefficients together with information related to competition between different binding equilibria. A new interpretation concerning the assignment of spectral properties and cation complex formation equilibria, as well as the involvement of conformational transitions, is suggested, based on a differentiation between selective and unselective alkali ion binding. The kinetics of K+ binding to the FITC-enzyme have been studied by employing a new microvolume technique consisting of flash photolysis of caged-K+.


Heterogeneity of Na,K-ATPase from kidney. Na,K-ATPase was purified from pig and mink kidney. Enzyme batches of high specific activity apparently contained only alpha- and beta-peptides, judging from the SDS-gel-electrophoretic pattern. Careful determinations of the ouabain-binding capacity and binding kinetics were carried out. The question whether the functional unit is an alpha beta-protomer, a diprotomer or even a polymer was analysed from the binding stoichiometry and the binding pattern at nonsaturating ouabain concentrations. The binding capacity exceeded half of the theoretical maximum value for exclusively alpha beta-promoters. A half-of-the-sites reactivity of a diprotomer thus seems less likely, though nearly half of the protein is not accounted for in its absence. When ouabain binding took place in the presence of Na+, at least two binding components were noticed. Most probably the two components are not of equal size, thus excluding a co-operative diprotomer construction of the active ouabain-binding units. If the co-operativity model has to be abandoned, the phenomenon could be due to two or more isozymes exhibiting different Na(+)-affinities. Isoforms of the hydrolytic alpha-peptide were analysed by an ELISA technique and on blots of Na,K-ATPase peptides utilizing commercial isoform-specific antibodies. Though not absolutely specific, the individual antibodies were assumed to be monospecific with respect to the single isoforms of the alpha-peptide. In that case, data were consistent with a significant contribution of alpha 2- and alpha 3-isoforms (about 15%), in addition to the predominant alpha 1-isoform in kidney Na,K-ATPase.


Mitochondrial F-type ATPases: the glycine-rich loop of the beta-subunit is a pyrophosphate binding domain. The beta-subunit of the mitochondrial ATP synthase complex comprises the bulk, if not all, of the catalytic nucleotide binding site on the enzyme. A region of homologous sequence rich in glycines (G) and containing a basic lysine (K) and a threonine (T) is found in the beta-subunit as well as many other purine nucleotide binding proteins. The consensus sequence of this region is Gx4GKT, where x represents any amino acid, and is called the A region or glycine-rich loop. The related function of these proteins implies that the glycine-rich loop is directly involved in nucleotide binding. Here we directly test the involvement of the beta-subunit's glycine-rich region in adenine nucleotide binding using two independent approaches. A synthetic fifty amino acid peptide, PP-50, containing the glycine-rich region and the surrounding sequence was assessed for secondary structure and interaction with potential ligands. Circular dichroism spectropolarimetry indicates that PP-50 assumes a predominantly beta-sheet conformation in solution. Significantly, the peptide precipitates from solution when ATP, ADP, GTP, ITP, and pyrophosphate are added, but not when AMP or phosphate are included. Magnesium is not required for the interaction with the purine nucleotides. Complimentary to these studies, the sequence around the Gx4GKT motif was deleted from a recombinant rat liver beta-subunit overexpressed in E. coli. While the wild type beta-subunit showed specificity for the tri- and diphosphonucleotides, the deletion mutant bound tri-, di-, and monophosphate nucleotides with equal affinity.(ABSTRACT TRUNCATED AT 250 WORDS)


The influence of membrane charge on the kinetic properties of Na,K-ATPase. Lipophilic ions modify the affinity of the cation binding sites of the membrane-bound Na,K-ATPase. We studied the effect of the lipophilic ions tetraphenyl-phosphonium (TPP+) and tetraphenylboron (TPB-) on the binding of Na+ and K+ to the cation site(s) that are exhibited by the enzyme during the catalytic cycle: the high-affinity (inside) Na-binding site, site I, the low-affinity (outside) Na-leaving site, site II, and the high-affinity (outside) K-site, site III. Site I: In the presence of TPP+ (positive charge added to the lipid environment) a higher Na(+)-concentration was needed to obtain phosphorylation of the enzyme, whereas in the presence of TPB- (negative charge added to the lipid environment) phosphorylation was obtained at a lower Na(+)-concentration, but the change in apparent K0.5 for Na+ was small, (K0.5Na,TPP = 0.180 mM and K0.5Na,TPB = 0.07 mM), indicating only a minor influence of membrane charge on Na+ binding to site I. Site II: Compared to control conditions, more Na+ was required to inhibit ATP-hydrolysis and to increase the steady-state level of ADP-sensitive phosphoenzyme when TPP+ was present, and the opposite was observed with TPB-, indicating a strong influence of membrane charge on the Na+ occupancy of site II. Site III: TPP+ induced a significant decrease both in the rate of K-dependent dephosphorylation of preformed E32P and in the K+ affinity. The effect of TPP+ on the ATP hydrolysis rate strongly resembled the effect of decreasing [KCl]. The results indicated a pronounced effect of adding positive charge to the lipid environment of site III.(ABSTRACT TRUNCATED AT 250 WORDS)


A continuous flow technique for analysis of the stoichiometry of the gastric H,K-ATPase. A continuous flow method was developed for determining the stoichiometry of the gastric proton pump H,K-ATPase in its hydrolysis of ATP, translocation of H+ and the K+ congener 86Rb+. H,K-ATPase-containing vesicles which had been isolated from pig gastric mucosa were incubated at 37 degrees C for 2 h in 150 mM 86RbCl, 0.5 mM EGTA and 3 mM Mes-buffer adjusted to pH 6.1 with Tris, and then applied to a 0.45 micron pore size filter. The immobilized vesicles were superfused with 0.15 mM Mes/Tris buffer, pH 6.1, containing 150 mM choline-Cl and 0.2 mM MgCl2. After the medium was changed to one containing 0.1 mM ATP, the amounts and rates of H+ uptake, 86Rb+ efflux, and ATP hydrolysis were measured in fractions collected after the filter. The initial ratio of transported Rb+ to hydrolysed ATP gave values of 0.96 +/- 0.26 (mean +/- SD, n = 28). The initial ratio of ATP-dependent Rb+ efflux to H+ uptake gave values of 0.92 +/- 0.28 (mean +/- SD, n = 28). The MgATPase activity was measured in vesicles which had been incubated with choline-Cl instead of RbCl. In the initial fractions used for calculation of the stoichiometry, the MgATPase activity was 15.8% +/- 8.7 (mean +/- S.D.) of the maximal ATPase activity obtained with Rb(+)-loaded vesicles. The MgATPase may be an intrinsic activity of the H,K-ATPase. However, whether corrections were made for the MgATPase or not, it had only marginal effects on the calculations of the stoichiometry of the pump.(ABSTRACT TRUNCATED AT 250 WORDS)


A K+ competitive, conformational probe of the H,K-ATPase. The H,K-ATPase was noncovalently labelled with a fluorescent quinoline derivative, 1-(2-methylphenyl)-4-methylamino-6-methyl-2,3-dihydropyrrolo [3,2-c]quinoline, (MDPQ). MDPQ competitively inhibited the K+ stimulated ATP hydrolysis with a Ki of 0.22 microM but did not inhibit the MgATP-dependent phosphoenzyme to an extent greater than 10% of control. Inhibitor binding to the H,K-ATPase enhanced MDPQ fluorescence. This fluorescence was quenched by lumenal K+ with a K0.5 of 1.8 mM. MDPQ binding to the H,K-ATPase shifted the fluorescence Ex/Em maxima from 342/478 nm to 342/453 nm. Phosphorylation of the H,K-ATPase by MgATP further enhanced fluorescence with a difference spectra [MgATP-(MgATP+KCl)] emission peak at 446 nm. Trypsin dependent proteolysis of the H,K-ATPase stabilized within the E2K conformation eliminated the phosphoenzyme response, but enhanced the K+ specific dephosphoenzyme response. These observations show that MDPQ is a fluorescent, competitive inhibitor of the H,K-ATPase that interacts with a lumenal cation binding site. Under specific conditions, both the cation and MDPQ binding sites remain intact within trypsin produced cleavage peptides of the H,K-ATPase.


The membrane sector of vacuolar H(+)-ATPase by itself is impermeable to protons. The sensitivity for vanadate of proton uptake activities of isolated chromaffin granules and yeast vacuoles was investigated. About 0.5 mM vanadate caused 50% inhibition of ATP-dependent proton uptake activity in both membranes. The proton conductivity across chromaffin granule membranes and yeast vacuoles was assayed in the presence and absence of the catalytic sectors of their respective V-ATPases. Removal of the catalytic sectors by cold treatment in the presence of MgATP did not change the proton conductivity of the membranes. Similar results were obtained with yeast vacuoles of mutants that did not assemble the catalytic sector of the enzyme. These results are in contrast to the effect of removing the catalytic sectors of F-ATPases from various sources. The mechanistic and biological significance of the difference in the behavior of the two families of proton pumps is discussed.