Insulin-like growth factor II acts through an endogenous growth pathway regulated by imprinting in early mouse embryos. We present evidence that insulin-like growth factor II (IGF-II) mediates growth in early mouse embryos and forms a pathway in which imprinted genes influence development during preimplantation stages. mRNA and protein for IGF-II were expressed in preimplantation mouse embryos, but the related factors IGF-I and insulin were not. IGF-I and insulin receptors and the IGF-II/mannose-6-phosphate receptor were expressed. Exogenous IGF-II or IGF-I increased the cell number in cultured blastocysts, but a mutant form of IGF-II that strongly binds only the IGF-II receptor did not. Reduction of IGF-II expression by antisense IGF-II oligonucleotides decreased the rate of progression to the blastocyst stage and decreased the cell number in blastocysts. Preimplantation parthenogenetic mouse embryos expressed mRNA for the IGF-II receptor but not for either IGF-II ligand or the IGF-I receptor, indicating that the latter genes are not expressed when inherited maternally. These data imply that some growth factors and receptors, regulated by genomic imprinting, may control cell proliferation from the earliest stages of embryonic development.


Protection by salvianolic acid A against adriamycin toxicity on rat heart mitochondria. It was found that salvianolic acid A (Sai A) has potent antioxidant activity. The effects of Sai A on adriamycin-induced heart mitochondrial toxicity of rats in vitro and on adriamycin antitumor activity are investigated in this article. Malondialdehyde (MDA) formation and membrane rigidification of rat heart mitochondria intoxicated with adriamycin were significantly reduced by Sai A. In the electron spin resonance (ESR) studies, Sai A has no significant effect on the formation of adriamycin semiquinone radicals (AQ.), while hydroxyl radicals generated by electron transfer from AQ. to H2O2 were scavenged by Sai A dose-dependently. On the other hand, Sai A was shown to have no effects on the antitumor activity of adriamycin in cultured L1210 ascitic tumor cells and in mice with P388 ascite tumor. These results indicate that Sai A protects against adriamycin induced heart mitochondrial toxicity of rats, while Sai A has no antagonizing effect on the antitumor activity of adriamycin.


Radiation-induced generation of chlorine derivatives in N2O-saturated phosphate buffered saline: toxic effects on Escherichia coli cells. The radiolysis of aqueous chloride solutions has been investigated using pulse and steady-state methods. We have found a correlation between the yields of Cl2- and HOCl formed in pulse-irradiated N2O-saturated solutions. The yields increased with the increasing concentrations of Cl- and phosphate. Phosphate enhanced the yield of Cl2- in neutral solutions because of a proton transfer from H2PO4- to HOCl- with a rate constant of (2.6 +/- 0.5) x 10(8) M-1s-1. HOCl could not be detected in pulse-irradiated He or air-saturated, phosphate-buffered saline (PBS) solutions or in gamma-irradiated N2O, He, or air-saturated PBS solutions. The results are discussed in light of previously suggested mechanisms for the formation and decay of Cl2-. Pulse-irradiated N2O-saturated PBS solutions have a lethal effect on Escherichia coli cells, which is proportional to the amount of HOCl in the solutions. Gamma-irradiation of cells in N2O-saturated PBS solution also raises the radiosensitivity of the cells, although HOCl does not accumulate in this system. The effects of the radiation-induced toxic products on E. coli cells are similar to the effects of NaOCl. The cell membrane is probably the site of physiological injury induced by the radiation products.


The inhibitory effect of extracts of cigarette tar on electron transport of mitochondria and submitochondrial particles. Acetonitrile extracts of cigarette tar inhibit state 3 and state 4 respiration of intact mitochondria. Exposure of respiring submitochondrial particles to acetonitrile extracts of cigarette tar results in a dose-dependent inhibition of oxygen consumption and reduced nicotinamide adenine dinucleotide (NADH) oxidation. This inhibition was not due to a solvent effect since acetonitrile alone did not alter oxygen consumption or NADH oxidation. Intact mitochondria are less sensitive to extracts of tar than submitochondrial particles. The NADH-ubiquinone (Q) reductase complex is more sensitive to inhibition by tar extract than the succinate-Q reductase and cytochrome complexes. Nicotine or catechol did not inhibit respiration of intact mitochondria. Treatment of submitochondrial particles with cigarette tar results in the formation of hydroxyl radicals, detected by electron spin resonance (ESR) spin trapping. The ESR signal attributable to the hydroxyl radical spin adduct requires the presence of NADH and is completely abolished by catalase and to a lesser extent superoxide dismutase (SOD). Catalase and SOD did not protect the mitochondrial respiratory chain from inhibition by tar extract, indicating that the radicals detected by ESR spin trapping are not responsible for the inhibition of the electron transport. We propose that tar causes at least two effects: (1) Tar components interact with the electron transport chain and inhibit electron flow, and (2) tar components interact with the electron transport chain, ultimately to form hydroxyl radicals.


Desferrioxamine enhances the reactivity of vanadium (IV) and vanadium (V) toward ferri- and ferrocytochrome c. Ligands, especially desferrioxamine, affect the rate at which vanadium reduces or oxidizes cytochrome c. Whether reduction or oxidation occurs, and how fast, depends on the nature of the ligand, the state of reduction of the vanadium, the pH (6.0, 7.0, or 7.4), and the availability of oxygen. In general, oxidation of ferrocytochrome c was favored by (1) low pH, (2) an oxidized state of the vanadium, (3) the presence of oxygen, and (4) more strongly binding ligands (desferrioxamine much greater than histidine = ATP greater than EDTA greater than albumin greater than aquo). Thus, at pH 6.0, desferrioxamine accelerated the V(V)-catalyzed ferrocytochrome c oxidation 160-fold aerobically, and 3500-fold anaerobically. In general, strongly binding ligands slowed oxidations, especially at higher pH. Desferrioxamine was unique among the five ligands in that it not only accelerated oxidation of ferrocytochrome c at pH 6.0, but at pH 7.4 the redox balance shifted to the point where it paradoxically reduced ferricytochrome c. V(V) is an improbable electron donor, but desferrioxamine will reduce cytochrome c, and V(V) accelerates this process. Oxidation of cytochrome c by V(V):desferrioxamine was faster anaerobically, and reduction by V(IV):desferrioxamine was faster aerobically. Although V(V) did not oxidize ferrocytochrome c at pH 7.4, V(IV) did, provided oxygen and desferrioxamine were both present. V(IV):desferrioxamine almost completely reduced ferricytochrome c, and this reduction was followed by a slow, progressive oxidation. This latter oxidation of cytochrome c is mediated by active species generated in the reaction between V(IV):desferrioxamine and oxygen, because none of these reagents alone can induce oxidation at a comparable rate. The mediating species were transient, and generated in reactions with oxygen.(ABSTRACT TRUNCATED AT 250 WORDS)


tert-Butyl hydroperoxide-induced radical production in rat liver mitochondria. When rat liver mitochondria are treated with tert-butyl hydroperoxide (TBHP) in the presence of the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO), electron paramagnetic resonance (EPR) signals are detected attributable to spin adducts resulting from the trapping of methyl, tert-butoxyl, and tert-butylperoxyl radicals. The addition of respiratory substrate results in a 3- to 7.5-fold increase in the signal intensity of the DMPO/methyl adduct, no change in the signal intensity of the DMPO/tert-butoxyl adduct, and complete loss of the DMPO/tert-butylperoxyl adduct signal. The magnitude of increase of methyl radical production in the presence of respiratory substrate is related to the respiratory control ratio (RCR) of the mitochondrial preparation. In the presence of antimycin A, which blocks electron flow between cytochromes b and c1, no stimulation of methyl radical production is detected with respiratory substrate. Stimulation of methyl radical production by the addition of respiratory substrate is detected in cytochrome c-depleted mitochondria. A similar increase in methyl radical production is detected when ferrous cytochrome c is treated with TBHP in the presence of DMPO (as compared to when ferricytochrome c is used). These results indicate that TBHP is reduced directly by either cytochrome c1, cytochrome c, or by both of these electron transport chain components in mitochondria undergoing state 4 respiration.


Protein damage by photoproducts of merocyanine 540. Exposure of certain photoactive dyes to light prior to their use in biological systems (preactivation) has been shown to result in formation of long-lived cytotoxic photoproducts. The cytotoxic species responsible for the biological activity of preactivated merocyanine 540 (pMC540) appears to be a hydroperoxide generated by oxidation of ground-state dye by singlet molecular oxygen, formed via energy transfer from triplet excited-state dye to oxygen. A positive correlation (r = .93) exists between the levels of hydroperoxides and percent of tumor cells killed upon exposure to pMC540. Exposure of bovine serum albumin (BSA) (0.5 mg/mL) to pMC540 (0.2 mg/mL-1 mg/mL) results in loss of tryptophan fluorescence and 345 nm emission, suggesting a probable role of either hydroxyl (.OH) or .OH + superoxide (O2-). Polyacrylamide gel electrophoresis indicates fragmentation of treated BSA. Aggregation of pMC540-treated BSA is not detected. Bityrosine production is not observed. A dose-dependent decrease in BSA solubility is observed in treated samples, suggesting an increase in hydrophobicity. Amino acid analysis of BSA treated with pMC540 shows loss of some amino acids residues. The data presented here suggest that photoproducts of MC540 derived via the process of preactivation may mediate their effect (at least in part) by reactive oxygen species.


Ferritin as a source of iron for oxidative damage. The generation of deleterious activated oxygen species capable of damaging DNA, lipids, and proteins requires a catalyst such as iron. Once released, ferritin iron is capable of catalyzing these reactions. Thus, agents that promote iron release may lead to increased oxidative damage. The superoxide anion formed enzymatically, radiolytically, via metal-catalyzed oxidations, or by redox cycling xenobiotics reductively mobilizes ferritin iron and promotes oxidative damage. In addition, a growing list of compounds capable of undergoing single electron oxidation/reduction reactions exemplified by paraquat, adriamycin, and alloxan have been reported to release iron from ferritin. Because the rapid removal of iron from ferritin requires reduction of the iron core, it is not surprising that the reduction potential of a compound is a primary factor that determines whether a compound will mobilize ferritin iron. The reduction potential does not, however, predict the rate of iron release. Therefore, ferritin-dependent oxidative damage may be involved in the pathogenesis of diseases where increased superoxide formation occurs and the toxicity of chemicals that increase superoxide production or have an adequate reduction potential to mobilize ferritin iron.


Reduction of infarct size by cell-permeable oxygen metabolite scavengers. The timely restoration of blood flow to severely ischemic myocardium limits myocardial infarct size. However, experimental studies demonstrate that the myocardial salvage achieved is suboptimal because of additional injury that occurs during reperfusion, due in part to the generation of reactive oxygen metabolites. Initially, superoxide (O2-) was considered to be the central mediator of reperfusion injury. While there are several potential pathways of O2- generation in reperfused myocardium, O2- is poorly reactive toward tissue biomolecules. However, O2-, in the presence of redox-active metals such as iron, generates .OH or hydroxyl-like species that are highly reactive with cell constituents. Thus, while O2- may initiate reaction sequences leading to myocardial injury, it may not be the actual injurious agent. In vitro studies suggest that oxygen metabolite injury occurs at intracellular sites and involves iron-catalyzed processes. Consistent with this mechanism, extracellular oxygen metabolite scavengers have not convincingly reduced infarct size. However, treatment around the time of reperfusion, after ischemia is well established, with cell-permeable scavengers of .OH reduce infarct size. Results with these cell-permeable agents suggest that in the intact animal during regional ischemia and reperfusion, oxygen metabolite injury also occurs at intracellular sites. Cell-permeable scavenger agents are a promising class of drugs for potential clinical use, though further experimental and toxicologic studies are required.


Preoperative radiation therapy in clinical stage II endometrial carcinoma. From 1980 to 1987, 30 patients with FIGO clinical Stage II carcinoma of the endometrium were treated with 5000 cGy preoperative pelvic radiation therapy at Thomas Jefferson University Hospital. Patients with gross cervical disease received additional intracavitary irradiation with a tandem and ovoids for a combined total dose of 7000 cGy to point A. All patients then underwent exploratory laparotomy, total abdominal hysterectomy, and bilateral salpingo-oophorectomy (TAH/BSO). The 5-year actuarial survival for the entire group was 69%. The 5-year actuarial survival for the 8 patients with papillary serous, clear cell, and undifferentiated small cell carcinoma was 38%, with most patients failing in the upper abdomen. The 5-year actuarial survival for the remaining 22 patients was 82%. The only local failure occurred in the patient with an undifferentiated small cell carcinoma. Although preoperative pelvic radiation therapy together with TAH/BSO appears to offer excellent local control in all patients with Stage II endometrial carcinoma, additional treatment options should be considered for patients with papillary serous and clear cell histologies because of the poor survival and high failure rate in the upper abdomen.


[Primary breast cancer: conservation therapy]. A series of 200 patients with primary breast cancer, treated with breast conservation between 1981 and 1988, was analyzed retrospectively. The mean age was 54 +/- 14 years and mean follow-up 36 months. 58 patients (29%) were in pathological Stage I, 85 (42.5%) in II A, 34 (17%) in II B, and 7 (3.5%) in III. 16 (8%) were in clinical Stages I-II; 40 (20%) had multifocal tumors and 40 intraductal components. 45% of the lesions were excised with good margins, 13.5% with close margins (0.5 cm), 9% with microscopic residual, 4% with macroscopic residual and in 57 (28.5%) margins could not be determined. Level II axillary lymph node dissection was performed in 193 (96.5%). Adjuvant therapy (combined chemotherapy and/or hormones) was given to 105 (52.5%). Radiotherapy usually consisted of 50 GY tangential photon irradiation to the whole breast in 25 fractions. Electron or photon boost to the tumor bed was given to 112 (59%). Most patients received 20 GY, and most node-positive patients 50 GY to the lymphatic drainage. 69% tolerated combined radiotherapy-chemotherapy well and radiotherapy did not have to be interrupted. A year after completion of radiotherapy the cosmetic result was rated as good in 166/173 (96%) and fair in 7 (4%); there were no poor ratings. 11 (5.5%) developed breast recurrence and 5 (2.5%) supraclavicular or axillary lymph node metastases. 1 (0.5%) developed local recurrence and supraclavicular lymph node metastases simultaneously. 50 (10%) developed distant metastases, of whom 2 developed loco-regional recurrences simultaneously.(ABSTRACT TRUNCATED AT 250 WORDS)


[Regulation of GABA receptors by intracellular factors]. gamma-Aminobutyric acid (GABA) directly inhibits the postsynaptic membrane through GABAA receptor Cl- channel complexes. This inhibition is promoted or blocked by various intra- and extracellular substances at the site of either receptor or channel. Recent studies focused on the intracellular inhibitory mechanisms of the GABAergic response in neurons: one is the inhibition of GABA receptors by the increase in intracellular Ca2+ and the other is inhibition through ATP receptors triggered by the decrease in intracellular ATP level. In addition, the spontaneous inhibitory postsynaptic currents (IPSCs) induced by GABA released from the nerve terminals were suppressed by activation of the GABAB receptor, which acts as a negative autoreceptor in the nerve ending. The intracellular mechanism of the suppression will be discussed.


[Regulation by chloride ion of astroglial cell functions and morphological transformation]. Recently, several lines of evidence have indicated the important roles of glial cells, especially astrocytes, in the regulation of neuronal functions. The neuron-glia interaction is one of the most important issues in neuroscience, including neuropharmacology. I reviewed the present status and perspectives on the physiologic and pathologic functions of astrocytes in relation to the roles of intracellular Cl-. Astrocytes have different types of Cl- transport systems, such as voltage-sensitive and ligand-gated channels; HCO3(-)-Cl- exchange; and Na+, K+, Cl- cotransport systems. Anion exchange and cotransport systems are responsible for intracellular pH regulation and astrocytic volume regulation, respectively. Especially, astrocytic volume regulation is physiologically important for reducing the concentrations of K+ and glutamate in the extracellular space by their uptake systems. Disturbance of astrocytic volume regulation is expressed as astrocytic swelling, which is usually observed in various brain pathologic states including ischemia. Experimentally, glutamate caused a typical swelling of astrocytes in culture by Cl- and Ca(++)-dependent processes. Glutamate-induced swelling is qualitatively different from reversible swelling induced by hypoosmotic medium. Recently, we found that Cl- is intracellular factor for modulating the receptor-adenylate cyclase system in brain slices. Similarly, the receptor- and forskolin-stimulated adenylate cyclase of astrocytes showed a clear Cl- dependence. This was functionally confirmed by astrocytic morphological transformation induced by the cyclic AMP system.


[Comparison of bidirectional collagenase perfusion with one way perfusion in the isolation of periportal or perivenous hepatocytes in rats]. To selectively isolate hepatocytes from the periportal (PP) and perivenous (PV) regions of rat liver acinus, we compared two different perfusion methods with collagenase: the bidirectional perfusion method (2-P) and the one way perfusion method (1-P). We determined the optimal conditions for each method on the basis of the zonal selectivity of isolated hepatocytes with a hematoxylin-eosin stained liver specimen. By both methods, hepatocytes were selectively isolated from the PP and PV regions. Comparing cell yield and cell viability after the two perfusion methods, 1-P was found to be better than 2-P. Density gradient centrifugation with Percoll was found to be an effective procedure for removing the damaged hepatocytes. We concluded that 1-P could isolate viable PP and PV hepatocytes with a normal glucagon-cyclic AMP response and ultrafine structure in high yield from rat liver.


[Genetic analysis of adenomatous polyposis coli: analysis of alterations of oncogenes and tumor suppressor genes in colorectal tumors]. Adenomatous polyposis coli (APC) is a genetic disorder transmitted as an autosomal dominant trait. This syndrome is characterized by the development of numerous polyps during the first 20-30 years of life and classified into two phenotypes according to the number of polyps: the profuse and sparse types. If left untreated, most or all affected individuals are at a high risk of developing adenocarcinoma by as early as 40 years of age. Therefore, comparison of APC adenocarcinomas with non-polyposis colorectal carcinomas (NPCC) was thought to be useful for understanding genetically determined carcinogenesis. I investigated gene alterations in specimens obtained from 53 APC patients, of which 16 represented the profuse type and the others the sparse type, and from 15 NPCC patients. The results are summarized as follows: 1) K-ras gene mutations were detected more frequently in the profuse-type adenomas (43%) than in the sparse ones (14%) (p less than 0.05). 2) Loss of heterozygosity on the long arm of chromosome 5(5q), 18(18q) and the short arm of chromosome 17(17p) in the profuse-type adenomas was observed more frequently (22%) than in the sparse ones (7.3%) (p less than 0.05). 3) No significant differences were observed between APC adenocarcinomas and NPCCs regarding the allelic deletions on 5q, 17p and 18q in these tumors. 4) The alteration of the DCC gene, which is known to be involved in the formation of NPCC, was frequently detected in the APC adenocarcinomas, suggesting that similar genetic events are involved in the oncogenesis of adenocarcinomas from APC and NPCC.


Hepatitis C viremia in patients with hepatitis C virus infection. Sera from 103 patients were tested for hepatitis C virus RNA by nested polymerase chain reaction assay. Using primers from the highly conserved 5'-untranslated region, we detected hepatitis C virus RNA in 67 (88.2%) of 76 patients positive for antibody to hepatitis C virus by both second-generation and neutralization enzyme immunoassays. Hepatitis C virus RNA was detected in 93% of patients who had been infected for 10 yr or less and in 89% of those who had been infected for longer than 10 yr. Hepatitis C virus RNA was detected in all patients with chronic hepatitis, active cirrhosis or hepatocellular carcinoma and in 50% of those with nonspecific reactive hepatitis or inactive cirrhosis. Hepatitis C virus RNA was not detected in sera from 22 patients negative for antibody to hepatitis C virus or in 5 patients positive for antibody to hepatitis C virus by second-generation but not by neutralization enzyme immunoassay. Using primers from the less conserved nonstructural region 4, we detected hepatitis C virus RNA at a lower frequency, in 66% of patients who were positive for antibody to hepatitis C virus by both second-generation and neutralization enzyme immunoassays. The detection rate was higher in patients with frequent parenteral exposure. Our study showed that hepatitis C viremia can be detected in most patients with hepatitis C virus infection, including those with long-standing infection or advanced liver disease.


Long-term follow-up of patients with chronic hepatitis C treated with alpha-interferon. We reanalyzed the results of a pilot study of recombinant alpha-interferon therapy for chronic non-A, non-B hepatitis in light of the recent discovery of the hepatitis C virus and the development of diagnostic assays for this agent. Stored serum samples from 10 patients treated between 1984 and 1986 were tested for antibody to hepatitis C virus and hepatitis C virus RNA before, during and after therapy. In addition, the current clinical, serum biochemical and virological statuses of these patients were evaluated to determine the long-term effects of interferon therapy. All patients had evidence of hepatitis C virus infection, with hepatitis C viral RNA, antibody to hepatitis C virus or both markers detectable in serum. Serum hepatitis C virus RNA was found to disappear in seven of eight patients whose aminotransferase levels became normal with interferon therapy but remained present in two patients who did not respond to therapy. Levels of hepatitis C virus RNA decreased and disappeared when serum aminotransferases fell to normal levels but rose with subsequent elevation of aminotransferase levels in two patients who had relapses in disease when interferon was stopped. During a follow-up of 3 to 6 yr, hepatitis C virus RNA remained undetectable in the six patients whose serum aminotransferase levels remained normal after interferon therapy. However, neither initial titers of hepatitis C virus RNA nor disappearance of viral RNA from serum during treatment predicted a sustained response. Thus long-term beneficial responses to alpha-interferon can occur in patients with chronic hepatitis C and are associated with sustained loss of hepatitis C virus RNA from serum.


Modification of the amount of cholesterol in hepatic steatosis induced in susceptible and resistant mice infected with MHV3: a biochemical and ultrastructural study. A mouse hepatitis virus-3 strain subcultured in our laboratory is a unique experimental model in which to study virus-induced liver steatosis. This strain produces massive lipid deposition not only in sensitive adult BALB/c mice but also (though less extensive) in virus-resistant adult A/J mice. Biochemical determinations have shown that this steatosis is characterized by an increased amount of neutral lipids (sterols and triglycerides) in infected livers of BALB/c mice and by a smaller increase in those of A/J mice. However, the relative percentage of cholesterol and triglycerides is similar in both strains. Liver phospholipid content was significantly decreased in both strains of mice. To discriminate between cytoplasmic and membrane cholesterol content in different types of liver cells, an ultrastructural study was performed with filipin, a specific cholesterol marker. This study shows on one hand an important increase in the cholesterol in the hepatocytes of BALB/c mice and a smaller increase in those of A/J mice, in agreement with biochemical data. However, marked cholesterol decrease and abnormal cholesterol distribution were observed in the endothelial liver cells of infected BALB/c mice. This decreased cholesterol content probably led to higher fluidity of these membranes, which could be related to the important drop in the number of endothelial cell fenestrae observed after mouse hepatitis virus-3 infection. Because in A/J infected mice neither a decrease in the amount and distribution of cholesterol nor decreased fenestration were observed in endothelial liver cells, these findings could be correlated with the resistance of these mice to the infection.


Effect of galactosamine on hepatic carbohydrate metabolism: protective role of fructose 1,6-bisphosphate. Intraperitoneal administration of galactosamine (400 mg/kg body wt) to rats results in reversible liver cell injury that is related to a dose-dependent depletion of uridine phosphates by formation of UDP-sugar derivatives. This damage was monitored through changes in serum enzymatic activities that increased after the first 6 hr of drug administration. Glycemia and serum albumin remained stable during liver injury, whereas cholesterol and triglycerides decreased. To maintain plasma glucose concentration, the hepatic carbohydrate metabolism was greatly altered. Glycogen dropped during the first hours, remaining low for up to 48 hr. Fructose 2,6-bisphosphate and ATP levels decreased even faster than glycogen, with lactate following a similar diminution and being restored in parallel with both metabolites. The reduction in fructose 2,6-bisphosphate can be explained by changes in the substrates or modulators of the 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase, because neither the cyclic AMP levels nor the activity ratio of the enzyme were modified. Simultaneous administration of galactosamine and fructose 1,6-bisphosphate (2 gm/kg) prevented liver cell death, as monitored by serum enzyme activities. Furthermore, the bisphosphorylated metabolite had protective effects on the changes in liver calcium content and ATP and fructose 2,6-bisphosphate concentrations. In contrast, fructose, fructose-1-phosphate and fructose-6-phosphate had no significant protection. Fructose 1,6-bisphosphate might decrease galactosamine toxicity by increasing fructose 2,6-bisphosphate and ATP levels, the changes in both metabolites probably being related. The significance of these findings with respect to the mechanism of galactosamine-induced liver injury is also discussed.


Time course of hepatitis A virus antibody titer after active and passive immunization. To investigate the antibody titer necessary to prevent hepatitis A virus infection, either 15 or 7.5 mg/kg of immune serum globulin was injected into 10 antihepatitis A virus negative volunteers and their serum antihepatitis A virus titers were observed for 28 wk. In addition, antibody titers were observed for 96 wk in a phase 1 clinical trial of a hepatitis A vaccine. The two studies were then compared to assess the immunogenicity of the vaccine and the persistence of the antibody. Serum-neutralizing antibody titers that were greater than or equal to 4 (considered as positive) persisted for 18 wk and 14 wk after the injection of 15 and 7.5 mg/kg of globulin, respectively. Hepatitis A virus vaccine recipients showed adequate neutralizing antibody titers, with the groups receiving 1, 0.5 and 0.25 micrograms/dose showing titers of 4(5.5), 4(4.7) and 4(4), respectively, at 18 mo after the third inoculation. These findings suggested that effective blood antibody titers were likely to be retained in the 1.0 micrograms or 0.5 micrograms/dose groups for at least several years. Moreover, the serum antihepatitis A virus titers demonstrated by a modified radioimmunoassay changed in parallel with the neutralizing antibody titers in the volunteers injected with globulin.


Significance of IgM antibody to hepatitis C virus in patients with chronic hepatitis C. We assessed the correlation between the positivity for serum IgM antibody to hepatitis C virus and the activity of liver disease in patients with chronic hepatitis C virus infection. Serum samples were taken from 10 antibody to hepatitis C virus-positive asymptomatic patients with normal serum ALT levels, from 14 untreated patients with clinically and histologically proven chronic hepatitis C and from 26 patients with clinically and histologically proven chronic hepatitis C assigned to receive recombinant interferon alpha-2a (6 million IU three times a week for 6 mo). Each serum specimen was tested for IgM antibody to hepatitis C virus-associated C100-3 antigen by enzyme-linked immunosorbent assay. Patients were observed for at least 12 mo. All 10 patients with normal ALT values tested negative for IgM antibody to hepatitis C virus. In contrast, 33 of 40 (82%) patients with chronic hepatitis C had IgM antibody to hepatitis C virus, and a positive correlation was seen between the ALT level and the level of IgM antibody to hepatitis C virus (r = 0.803, p less than 0.001). During interferon treatment, ALT levels declined into the normal range in 18 of 26 treated patients (69%) and remained normal after stopping treatment in 8 patients (31%). In untreated patients, in treated patients who did not respond to interferon treatment and in responder patients who relapsed, no significant changes in IgM antibody to hepatitis C virus levels were seen during the study period. In contrast, IgM antibody to hepatitis C virus became undetectable by the end of interferon treatment in seven of eight patients with a sustained response.(ABSTRACT TRUNCATED AT 250 WORDS)


Vascular supply in adenomatous hyperplasia of the liver and hepatocellular carcinoma: a morphometric study. Vascular supply of adenomatous hyperplasia (AH) of the liver, a preneoplastic or early neoplastic lesion of hepatocellular carcinoma (HCC), and that of HCC were morphometrically examined. Seventy-three nodules of AH were divided into 43 ordinary and 30 atypical AHs. The latter showed a variety of hepatocellular atypias that were, however, insufficient to make a diagnosis of HCC, while the former lacked such atypias. Arteries were slightly more numerous and portal veins were slightly less frequent in both ordinary and atypical AHs compared with the surrounding liver. In ordinary AHs, cumulative areas of arterial lumen and portovenous lumen were almost equal to or less than those in the surrounding liver in two thirds of our cases. The cumulative area of arterial lumen was equal to, and that of portovenous lumen was less than, the cumulative area in the surrounding liver in the remaining one third of our cases. In a majority of atypical AHs, the cumulative area of arterial lumen was equal to, and that of portovenous lumen was less than, the cumulative area in the surrounding liver. In most HCC nodules, the number and cumulative luminal area of arteries were much more, and those of portal veins were much less, than the number and cumulative area in the surrounding liver. The relative number and cumulative luminal area of abnormal arteries compared with all arteries showed a stepwise increase in the following order: ordinary AH (20.7% and 17.5%), atypical AH (46.8% and 52.5%), and HCC (93.6% and 92.0%). These data suggest that ordinary AH, atypical AH, and HCC are different in vascular supply, and that these differences may reflect sequential changes in the hemodynamic state during hepatocarcinogenesis.


Low-grade angiosarcoma of the skin of the breast: a complication of lumpectomy and radiation therapy for breast carcinoma. A case of low-grade angiosarcoma arising in the skin of a breast previously irradiated for breast carcinoma is reported. Initially, an asymptomatic breast mass was detected. Excisional biopsy and axillary lymph node dissection revealed a 1.5-cm infiltrating ductal carcinoma with 21 negative lymph nodes. The neoplasm was staged as T1, N0, M0. The patient was entered in a research protocol and was treated with high-dose external beam (4,860 rad) and iridium implant (1,860 rad) irradiation. Seven years later the patient developed low-grade angiosarcoma of the breast skin. The lesion recurred following excision and eventually was treated by simple mastectomy. The patient never had evidence of lymphedema. Cutaneous angiosarcomas occurring as a complication of lumpectomy and radiation therapy for breast carcinoma are rare. In some reported cases the patients have had lymphedema, a known factor predisposing to angiosarcoma. Furthermore, almost all cases previously reported have been high grade. This case suggests that radiation therapy for breast carcinoma may also be complicated by low-grade angiosarcoma.


Nucleotide sequence of the cDNA and the derived amino acid sequence for the major antigenic protein of foot and mouth disease virus, type Asia 1 63/72. A 0.9 kb cDNA for the foot and mouth disease virus (FMDV) type Asia 1 63/72, cloned in the plasmid pUR222 by dC/dG tailing method, was expressed into a protein which was immunogenic in guinea pigs and cattle. The protein purified to homogeneity was found to be basic and of 38 kDa. A sequence of 879 nucleotides of the inserted cDNA was obtained. The nucleotide sequence was 65% GC-rich and was homologous to the gene for VPI of FMDV types A5, OIK and C3 to the extent of 35-40%. From the nucleotide sequence, a sequence of 293 amino acids was derived which contained 43 arginine, 4 lysine, 7 glutamic acid and 18 aspartic acid residues making the protein highly basic. The molecular weight was calculated to be 31.6 kDa. The 38 kDa protein produced by the cloned cDNA is a fused protein composed of the 293 amino acids; 5 and 55 amino acids of the alpha-complementation protein of the beta-galactosidase at the N and C terminal, respectively, and 5 amino acid coded by the dG/dC tails used for cloning the cDNA.


Interactions of praseodymium and neodymium with nucleosides and nucleotides: absorption difference and comparative absorption spectral study. The interactions of praseodymium(III) and neodymium(III) with nucleosides and nucleotides have been studied in different stoichiometry in water and water-DMF mixtures by employing absorption difference and comparative absorption spectrophotometry. The 4f-4f bands were analysed by linear curve analysis followed by gaussian curve analysis, and various spectral parameters were computed, using partial and multiple regression method. The magnitude of changes in both energy interaction and intensity were used to explore the degree of outer and inner sphere coordination, incidence of covalency and the extent of metal 4f-orbital involvement in chemical bonding. Crystalline complexes of the type [Ln(nucleotide)2(H2O)2]- (where nucleotide--GMP or IMP) were characterized by IR, 1H NMR, 31P NMR data. These studies indicated that the binding of the nucleotide is through phosphate oxygen in a bidentate manner and the complexes undergo substantial ionisation in aqueous medium, thereby supporting the observed weak 4f-4f bands and lower values for nephelauxetic effect (1-beta), bonding (b) and covalency (delta) parameters derived from coulombic and spin orbit interaction parameters.


Interleukin-6 regulates the cytotoxic effect of tumour necrosis factor on U937 cells. In this study, we demonstrate that low but not high concentrations of interleukin-6 (IL-6) potentiate the cytotoxic effect of tumour necrosis factor-alpha (TNF-alpha) on U937 cells, in a dose-dependent manner. Killing of U937 cells by 100 U/ml of TNF-alpha, was maximally potentiated by 50 U/ml of IL-6. No potentiation of cell killing was observed when the concentration of IL-6 was increased to 4000 U/ml. At a concentration of 50 U/ml, IL-6 up-regulated TNF receptor expression but no change in TNF receptor number was observed when the concentration of IL-6 was increased to 4000 U/ml. Low concentrations of IL-6 can also induce sub-cytotoxic doses of TNF-alpha (0.1 and 0.33 U/ml) to kill U937 cells. Up-regulation of TNF receptors by IL-6 is dependent on de novo protein synthesis since receptor induction is abolished in the presence of cycloheximide. Taken together the data suggest that the potentiation of cell killing observed by a combination of these lymphokines is mediated in part by IL-6-induced changes in TNF receptor expression.


FMLP is a potent activator of guinea-pig eosinophils but its activity is dependent on the prior overnight in vitro culture of the cells (facilitation). The activation of guinea-pig eosinophils was studied by measuring the production of superoxide anion (O2-) and the secretion of eosinophil peroxidase (EPO). Phorbol myristate acetate (PMA), calcium ionophore, plasma-activated zymosan, concanavalin A and recombinant human anaphylatoxin C5a induced the release of varying amounts of EPO. Some of these same activators, as well as platelet-activating factor, and aggregated homologous IgG, either by themselves or after a brief priming of the cells with low concentrations of PMA, also caused the formation of O2-. Formyl-methionyl-leucyl-phenylalanine (FMLP) failed to induce either of these reactions in freshly isolated cells. It was found serendipitously, however, that cells which had been maintained in culture overnight secreted EPO upon challenge with FMLP, and, if they were primed with PMA, they also produced O2-. The conversion from unresponsive to responsive cells ('facilitation') depended on the presence of mononuclear cells or mononuclear cell-conditioned medium in the overnight cultures. Although there also was a shift in the density of the majority of the eosinophils after overnight culture to a density lower than 1.085, this shift was not dependent on the inclusion of monocytes or of monocyte-conditioned medium (MCM) in the cultures and thus was not sufficient to impart responsiveness to FMLP. Responses of eosinophils to other activators were qualitatively unchanged after overnight facilitation. Binding studies using radiolabelled FMLP revealed that, during facilitation, binding of FMLP to guinea-pig eosinophils increased about sixfold overall and suggested the expression of a high affinity receptor. This change may explain the basis for the facilitation phenomenon.


Increased vascular formation of angiotensin II in one-kidney, one clip hypertension. To assess the role of the vascular angiotensin II-generating system in one-kidney, one clip hypertension, we determined the angiotensin converting enzyme activity in plasma and vascular tissues and examined the pressor response to angiotensin II, angiotensin I, and tetradecapeptide renin substrate in isolated mesenteric arteries from one-kidney, one clip hypertensive rats 7 and 30 days after clipping the renal artery and in mesenteric arteries from age-matched normotensive rats. Angiotensin converting enzyme activity, determined in aortic and mesenteric tissues, was significantly augmented in the hypertensive (30 days after clipping) group, whereas plasma activity was normal. The vasoconstrictor responses elicited by angiotensin I and tetradecapeptide in arteries from hypertensive rats were found to be significantly potentiated 30 days after clipping, whereas the angiotensin II responses were basically unchanged. Saralasin completely blocked the vasoconstrictor responses, whereas captopril blocked only the responses to angiotensin I without affecting the responses elicited by angiotensin II and tetradecapeptide. Enalapril, an angiotensin converting enzyme inhibitor given intravenously to unanesthetized rats, significantly lowered the blood pressure of hypertensive rats. The pressor responses elicited by angiotensin II, angiotensin I, and tetradecapeptide were completely inhibited by saralasin, whereas enalapril blocked only the responses of angiotensin I but not those elicited by angiotensin II and tetradecapeptide. These results indicate that local formation of angiotensin II is increased in arteries of one-kidney, one clip hypertensive rats. The data obtained with tetradecapeptide renin substrate suggest an important role for nonrenin proteases in vascular angiotensin II formation.


In vivo metabolism of angiotensin I by neutral endopeptidase (EC 3.4.24.11) in spontaneously hypertensive rats. We investigated the processing enzymes involved in the formation of circulating angiotensin-(1-7) after intravenous administration of angiotensin I to conscious spontaneously hypertensive and Wistar-Kyoto rats. Immunoreactive products, including angiotensin I, angiotensin II, and angiotensin-(1-7), were measured in arterial blood by three specific radioimmunoassays. Angiotensin I infusion (2 nmol) induced a rapid increase in immunoreactive angiotensin II and angiotensin-(1-7). Pretreatment with the angiotensin converting enzyme inhibitor enalaprilat (2 mg/kg) eliminated angiotensin II formation and augmented circulating levels of angiotensin I and angiotensin-(1-7) in spontaneously hypertensive and Wistar-Kyoto rats. The elevated levels of angiotensin-(1-7) in enalaprilat-treated rats were blocked by concurrent treatment with the neutral endopeptidase (EC 3.4.24.11) inhibitor SCH 39,370 (15 mg/kg) in both strains. Administration of SCH 39,370 alone decreased angiotensin-(1-7) levels in spontaneously hypertensive rats, whereas angiotensin II levels increased in both strains (p less than 0.01). Comparisons of the metabolism of angiotensin I in the two rat strains showed increased formation of angiotensin-(1-7) in spontaneously hypertensive rats not given any of the enzyme inhibitors. In addition, levels of angiotensin I were higher after administration of SCH 39,370 in hypertensive rats. These novel findings reveal that neutral endopeptidase EC 3.4.24.11 participates in the conversion of angiotensin I to angiotensin-(1-7) and in the metabolism of angiotensin II in the circulation of both spontaneously hypertensive and Wistar-Kyoto rats. Our results suggest that neutral endopeptidase EC 3.4.24.11 is a major enzymatic constituent of the circulating renin-angiotensin system.


Effects of a nonpeptide vasopressin antagonist (OPC-21268) on cytosolic Ca2+ concentration in vascular and cardiac myocytes. A selective V1 antagonist, 1-(1-[4(3-acetylaminopropoxy)benzoyl]-4-piperidyl)-3,4-dihydro-2(1 H)- quinolinone (OPC-21268), which is nonpeptide and orally effective, has been recently synthesized. We studied the effects of vasopressin and OPC-21268 on cell contraction with a video motion detector and cytosolic Ca2+ concentration ([Ca2+]i) by using indo-1 in cultured rat vascular smooth muscle cells and cultured chick embryo ventricular myocytes. Exposure of cultured vascular smooth muscle cells to vasopressin (1-100 nM) dose-dependently produced an initial transient increase (from control level [Ca2+]i of 133.6 +/- 10.9 nM to peak [Ca2+]i of 842.7 +/- 172.8 nM at 100 nM vasopressin, p less than 0.01) and then a small sustained increase in [Ca2+]i. After pretreatment of vascular smooth muscle cells with 1 microM OPC-21268, the effects of 100 nM vasopressin on [Ca2+]i were abolished. Exposure of ventricular myocytes to 100 nM vasopressin slightly but significantly decreased peak systolic cell position (-8.7 +/- 3.7%, p less than 0.05) and also produced reductions in peak systolic [Ca2+]i (from 962.2 +/- 76.4 to 751.2 +/- 70.5 nM, p less than 0.01) within 30 seconds. Pretreatment of ventricular myocytes with OPC-21268 (1 microM) completely suppressed vasopressin-induced changes in peak systolic cell position and [Ca2+]i. These results suggest that vasopressin may increase vascular tone and may also cause a direct negative inotropic effect via V1 receptors and that this orally active V1 antagonist (OPC-21268) may have potential clinical usefulness.


Alpha-adrenergic receptors and 45Ca2+ efflux in arteries from deoxycorticosterone acetate hypertensive rats. Increased vascular sensitivity to catecholamines characterizes mineralocorticoid hypertension. The present study investigated three possible sites that may account for this abnormality: agonist affinity, Ca2+ release from intracellular stores, and Ca2+ sensitivity of the contractile proteins. Adult male Sprague-Dawley rats underwent uninephrectomy and were implanted subcutaneously with deoxycorticosterone acetate (DOCA; 200 mg/kg, 1% NaCl:0.2% KCl drinking water, 4-6 weeks). Control rats were sham treated. Helical strips of mesenteric arteries were placed in muscle baths for measurement of isometric force development. Although the ED50 for norepinephrine was significantly lower in arteries from DOCA rats (pD2, 8.21 +/- 0.15) than in those from sham controls (pD2, 7.24 +/- 0.11), agonist affinity, determined by partial blockade with phenoxybenzamine, did not differ between the two groups. In contrast, norepinephrine-stimulated 45Ca2+ efflux in the absence of extracellular Ca2+ was significantly greater in arteries from DOCA rats than in those from sham rats. In the presence of ryanodine to deplete intracellular Ca2+ stores, force development to Ca2+ was not different in saponin-permeabilized vessels from DOCA rats, indicating that the Ca2+ sensitivity of the contractile proteins was not altered in DOCA hypertension. We conclude that increased vascular sensitivity to norepinephrine in mineralocorticoid hypertension is related to increased release of Ca2+ from a subcellular store and not to changes in agonist affinity or to the contractile protein interaction. Based on previous reports, it is likely that this abnormality reflects a postreceptor change in signal transduction, but there is also evidence to suggest that an increase in the number of alpha-adrenergic receptors may be involved.


Calcium, phosphoinositide, and 1,2-diacylglycerol responses of blood vessels of deoxycorticosterone acetate-salt hypertensive rats to endothelin-1. In previous studies a decreased responsiveness to endothelin-1 (ET-1) of conduit arteries and resistance vessels of deoxycorticosterone acetate (DOCA)-salt hypertensive rats was found in comparison with uninephrectomized controls. Decreased isometric force, number of receptors, and inositol phosphate accumulation were reported in the DOCA-salt animals. In the present study effects of ET-1 on cytosolic free calcium, inositol phosphates, and 1,2-diacylglycerol were investigated in blood vessels of DOCA-salt hypertensive rats. Basal cytosolic free calcium, measured with the fluorescent dye fura-2, was 201 +/- 41 nmol/l in mesenteric arteries of DOCA-salt rats and 45 +/- 9 nmol/l in uninephrectomized controls (p less than 0.01). The maximal response of cytosolic free calcium (to 30 nmol/l ET-1) was 176 +/- 22% of the basal value for DOCA-salt and 242 +/- 6% for uninephrectomized rats (p less than 0.05). The concentration giving 50% of the maximum response was 9.0 and 6.5 nmol/l for DOCA-salt rats and controls, respectively. Inositol phosphate production after stimulation with 100 nmol/l ET-1 in the presence of LiCl was lower by at least 30% (p less than 0.01) in both aorta and mesenteric arteries of DOCA-salt hypertensive versus control rats. Basal levels of diacylglycerol in aorta were similar in DOCA-salt rats and in controls and did not respond to a 100 nmol/l ET-1 stimulation in the DOCA-salt rats, in contrast to the increase found in the control uninephrectomized rats (p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)


Nitric oxide alters renal function and guanosine 3',5'-cyclic monophosphate. Endothelium-derived relaxing factor (EDRF) activates soluble guanylate cyclase, resulting in an increase in vascular smooth muscle guanosine 3',5'-cyclic monophosphate (cGMP) levels, which correlates with its relaxing effect. Using a microdialysis technique, we investigated changes in right and left renal interstitial fluid cGMP levels in response to right intrarenal administration of an EDRF inhibitor, NG-monomethyl-L-arginine (L-NMMA). Studies were conducted in anesthetized dogs (n = 5) in metabolic balance at a sodium intake of 40 meq/day. Urine was collected directly from the right and left ureters individually. Changes in the right and left urinary cGMP excretion and renal function in response to cumulative doses of L-NMMA were studied. In the right kidney, 20-100 micrograms/kg/min L-NMMA caused 1) a dose-dependent decrease in renal interstitial fluid and urinary cGMP levels (p less than 0.0001 and p less than 0.001, respectively), 2) antinatriuresis (p less than 0.01), 3) antidiuresis (p less than 0.01), 4) a decrease in renal blood flow (p less than 0.01) and glomerular filtration rate (p less than 0.01), and 5) a decrease in fractional sodium excretion (p less than 0.01). No changes in left renal interstitial fluid and urinary cGMP levels or excretory and hemodynamic function were observed during right intrarenal administration of L-NMMA at 20 and 60 micrograms/kg/min.(ABSTRACT TRUNCATED AT 250 WORDS)


Neutrophils from asthmatics exhibit diminished responsiveness to 2-chloroadenosine which is reversed by theophylline. Evidence for a cyclic-AMP-independent pathway on human neutrophils. We have shown previously that neutrophils (PMNs) from patients with asthma have a more potent stimulated respiratory burst than normals and that their respiratory burst is significantly less suppressed with exposure to 2-chloroadenosine (2-CADO). The present studies investigated the basis of this defect in responsiveness to 2-CADO. PMNs obtained from asthmatics either not on theophylline (minus theophylline) or taking theophylline (plus theophylline) generated significantly more superoxide in response to 2 x 10(-8) M FMLP (2.08 +/- 0.36 nmol/5 x 10(5) PMNs (minus theophylline) (P less than 0.01 compared to controls) vs. 2.16 +/- 0.44 (plus theophylline) (P less than 0.01) as compared to controls (1.05 +/- 0.17 nmol). In the presence of FMLP (2 x 10(-8) M), PMNs from the minus theophylline cohort had less 2-CADO (10(-6) M)-mediated suppression of superoxide generation as compared to controls (38.3 +/- 3.8% vs. 67.1 +/- 3.8%; (P less than 0.001). The plus theophylline group exhibited suppression values similar to controls (64.5 +/- 7.2%). Theophylline, in the presence of a physiological concentration of 2-CADO (0.1 microM) accentuated the suppression of the respiratory burst in normals (74.1 +/- 5.9%, 80.1 +/- 4.9% (P less than 0.02) and 84.7 +/- 3.8% (P less than 0.02) at 0, 10, and 100 microM, respectively). PMNs from asthmatics not taking theophylline demonstrated suppression values of 46.2 +/- 6%, 53.8 +/- 6.6% (P = NS), and 63.2 +/- 7.1% (P less than 0.01), respectively. Resting PMNs from normal controls generated 0.97 +/- 0.20 pmol cAMP/10(7) cells compared to 2.83 +/- 0.75 pmol in the presence of 0.1 microM 2-CADO. The combination of 2-CADO and theophylline (10-100 microM) produced cAMP concentrations not significantly different from that observed with 2-CADO alone. These findings support the existence of a novel cAMP-independent adenosine receptor in PMNs. The specific binding of 10(-8)M 3H-labeled 2-CADO (in delta cpm) was 10,358 +/- 1502 (P less than 0.001 compared to controls), 5468 +/- 843 (NS compared to controls), and 3751 +/- 477 in the plus theophylline group, minus theophylline group, and controls, respectively. Such up-regulation of specific binding may represent the effects of theophylline as shown by the specific binding of [3H]2-CADO in PMNs from normal controls exposed to 10 microM theophylline for 30 min (6013 +/- 969) compared to unexposed PMNs (3768 +/- 656; P less than 0.05).(ABSTRACT TRUNCATED AT 400 WORDS)


Bovine neutrophils recruited by endotoxin to a teat cistern continuously produce oxygen radicals and show increased phagocytosis and extracellular chemiluminescence. Bovine neutrophils were harvested from a teat cistern following endotoxin infusion and were compared with blood neutrophils by measurements of chemiluminescent and phagocytic activity towards C3- and IgG-opsonized and unopsonized yeast particles. Both phagocytosis and luminol-dependent chemiluminescence elicited by all three particles were enhanced in the teat cells. The increase in the luminol-dependent chemiluminescence towards C3- and IgG-opsonized particles was due to an enhanced extracellular release of myeloperoxidase. The observed increase in phagocytosis of unopsonized yeast was shown to reflect the interaction between up-regulated CR3 receptors on the surface of the teat neutrophils and the yeast particles. A high chemiluminescent activity of the teat neutrophils in both the luminol- and lucigenin-dependent systems in the absence of a phagocytic prey indicated that the NADPH oxidase was permanently active and that myeloperoxidase was continuously released by the cells. Treatment of neutrophils with cytochalasin B showed that the chemiluminescence and phagocytosis of teat neutrophils were less sensitive to this drug than that of blood neutrophils. These results indicate that the teat neutrophils have up-regulated their receptors for IgG- and C3-opsonized and unopsonized yeast on the cell surface by the action of actin. The cells also have a permanently active NADPH oxidase dependent on the association with actin and show a higher tendency than blood neutrophils to secrete the content of their primary granules during phagocytosis.


Neutrophil responses to intravascular pneumococcal sonicate. Leukopenia and pulmonary leukostasis are prominent features in patients succumbing to pneumococcal (PNC) infections. We examined mechanisms involved in recruitment of polymorphonuclear neutrophils (PMNs) into pulmonary capillaries and alveolae after PNC sonicate injection. We showed that by 15 min postinjection, PMN chemotactic activity was found in bronchoalveolar lavage (BAL) fluids and increased with time until the end point of the study at 90 min. Accompanying the increased chemotactic activity in BAL fluids was a decrease in circulating PMNs more pronounced in the femoral artery (FA) than the pulmonary artery (PA). Superoxide anion (O2-) production by peripheral PMNs was depressed following PNC sonicate injection, and comparison of FA and PA showed that FA PMNs produced less O2- than PA PMNs. PA PMNs also showed enhanced random migration when compared to the depressed random migration of FA PMNs. This study demonstrated that an intravascular challenge of PNC sonicate was associated with increased chemotactic activity for PMNs in BAL fluid. Fewer PMNs and altered PMN function resulted from passage through the pulmonary microvasculature after PNC sonicate injection.


Modulation of neutrophil functions by activated platelet release factors. Platelets activated with physiological agonists, such as thrombin, ADP, or collagen, released products able to modulate neutrophil functions. In particular, platelet supernatant contained an inhibitor of superoxide anion generation induced by phorbol ester and a chemotactic factor for human neutrophils. The proteolytic digestion of platelet supernatant completely abrogated chemotactic activity without interfering with the inhibitory effect, indicating the presence of different molecules involved in the modulation of different neutrophil functions. This was further confirmed by the pretreatment of platelets with aromatic benzamidine which abolished chemotactic activity, but did not affect superoxide inhibition of neutrophils. This report provides evidence for interaction of platelets and inflammatory cells, suggesting that platelets are able to induce accumulation of neutrophils and control their respiratory burst, which also has a critical role in tissue damaging in inflammation.


A collaborative case-control study of nutrient intake and pancreatic cancer within the search programme. Case-control studies of pancreatic cancer were conducted in 5 populations with moderate to high rates and differing dietary practices, using a common protocol and questionnaire. Comprehensive diet histories were completed for a total of 802 cases and 1669 controls identified in Adelaide (Australia), Montreal and Toronto (Canada), Utrecht (The Netherlands) and Opole (Poland). Positive associations were observed with intake of carbohydrates and cholesterol, and inverse associations with dietary fiber and vitamin C. These relationships were generally consistent among the 5 studies, and showed statistically significant and generally monotonic dose-response relationships. The relative risks for highest vs. lowest quintile of intake were estimated for carbohydrates to be 2.57 (95% confidence interval 1.64-4.03), cholesterol 2.68 (1.72-4.17), dietary fiber 0.45 (0.30-0.63), and vitamin C 0.53 (0.38-0.76). The consistency, strength, and specificity of these associations provides evidence for the hypothesis that some or all of these dietary factors may alter the risk of pancreatic cancer.


Induction of growth suppression and modification of gene expression in multi-drug-resistant human glioblastoma multiforme cells by recombinant human fibroblast and immune interferon. The combination of recombinant human fibroblast (IFN-beta) and immune (IFN-gamma) interferon induces enhanced growth suppression and modifies the antigenic phenotype in parental and multi-drug-resistant (MDR) human glioblastoma multiforme (GBM) cells. The present study was conducted to explore the mechanism underlying this cooperative interaction between interferons in inducing growth suppression in MDR-GBM cells. For this analysis we have utilized 2 MDR-GBM cell lines which display a differential sensitivity to growth suppression when exposed to IFN-beta or IFN-gamma. GBM-18-B3 (MDR) cells are more sensitive to growth inhibition by IFN-gamma than by IFN-beta, whereas GBM-18-A3 (MDR) cells are inhibited to a greater degree by IFN-beta than by IFN-gamma. In both cell types, however, growth is suppressed to a greater degree by the combination of interferons than by equivalent concentrations of either type of interferon used alone. Growth suppression induced by the interferons, alone or in combination, was not associated with comparable changes in the steady-state level of MDRI mRNA. In addition, the anti-proliferative effect of interferon was similar in GBM-18 (MDR) cells grown in the presence or absence of colchicine. GBM-18-A3 and GBM-18-B3 cells differed in their de novo and interferon-inducible expression levels of IFN-beta-responsive genes, isg-15 and isg-54. In contrast, both cell types responded in a similar manner with respect to expression of the IFN-gamma-responsive gene, HLA Class II (HLA-DR beta), and HLA Class I, fibronectin and ICAM-I. No further increase in expression of any of the genes was observed which was unique to the combination of interferons.


Capacity of adipose tissue to promote growth and metastasis of a murine mammary carcinoma: effect of estrogen and progesterone. Previously we have shown that a murine mammary carcinoma cell line, designated SPI, grows and metastasizes more efficiently in the mammary gland than in the subcutis. In this report, we examine the tissue specificity of this phenomenon. Our results show that SPI cells grow best in the mesenteric and ovarian fat pads and well in the mammary gland, but very poorly in the subcutis or peritoneal cavity. Massive dissemination of tumors from the ovarian and mesenteric sites occurs to the liver, spleen and diaphragm. In contrast, metastases from the mammary site occur primarily in the lung. Co-transplantation of a threshold number of SPI cells with mammary or ovarian fat fragments into the subcutis results in increased tumor growth, whereas very few tumors form in sham controls receiving no fat fragments. Removal of the ovaries of donor and recipient mice abrogates tumor growth in adipose tissue transplants. Estrogen can stimulate growth of SPI in adipose tissue sites, whereas progesterone inhibits growth. In contrast, in vivo growth of a stable metastatic variant selected from SPI cells was not inhibited by progesterone. SPI cells growing in ovarian and mesenteric fat pads showed increased expression of estrogen receptors and progesterone receptors, as well as detectable levels of epidermal-growth-factor receptors, whereas receptor levels decreased to baseline on tumors in the subcutis. The levels of estrogen-receptor mRNA reflect the corresponding functional expression of receptors; this finding suggests that the regulation of estrogen-receptor expression in this system is, at least in part, at the mRNA level. Our results are consistent with the model that adipose tissue exerts an estrogen-dependent positive regulatory effect on primary SPI tumor growth, and promotes the formation of metastases.


Expression of integrins and other adhesion molecules in Epstein-Barr virus-transformed B lymphoblastoid cells and Burkitt's lymphoma cells. Lymphocytes adhere to cells or extracellular matrices to perform functions relating to cytotoxicity, extravasation and tissue localization, as well as modulation of lymphocyte growth and maturation. This adherence is mainly mediated by 3 families of cell-surface adhesion molecules: integrins, immunoglobulin-related molecules and selectins. Since variations in the degree of adherence may affect the pathophysiology of lymphoproliferative disorders, the expression of a large number of adhesion molecules was analysed on Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCLs), and on EBV-positive or EBV-negative Burkitt's lymphoma (BL) lines, by immunofluorescence flow cytometry and immunoprecipitation with monoclonal antibodies. With regard to the beta 1, beta 2 and beta 3 integrin subfamilies, LCLs strongly expressed CD49d/CD29 (VLA-4), CD11a/CD18 (Leu-CAMa, LFA-1) and CD51/CD61 (vitronectin receptor). These cells also abundantly expressed CD54 (ICAM-1) and CD58 (LFA-3) as well as the "homing receptors" L-selectin (LECAM-1) and CD44. BL lines had considerably lower amounts of VLA-4 than LCLs, and ICAM-1 was expressed only by some of the tumor lines. All other adhesion molecules were absent or minimally expressed in the BL cells.


Impact of hospital discharge planning on meeting patient needs after returning home. This study examines the contribution of hospital discharge planning in meeting the needs of patients for care after their return home. A random sample of 919 admissions (age 60 and over) to five hospitals was studied to obtain information on characteristics of discharge planning during the patients' hospital stay. Specifically, information was obtained on the involvement of a designated professional for managing and coordinating the discharge plan, and the extent to which the planning was interdisciplinary. Patient interviews conducted two weeks after discharge provided information on needs for care related to: (1) treatment, (2) activity limitations, and (3) other self-sufficiency limitations. Patients were asked about their need for care in these three areas and about whether or not these needs were being met. Overall, 97 percent reported one or more needs for care and 33 percent reported that at least one of these needs was not being met. Findings show that the involvement of a discharge planning case manager is related to a significant reduction in unmet treatment needs, but not to reductions in activity limitation, other self-sufficiency needs, or overall needs. No significant effects of interdisciplinary planning were identified. These findings suggest that treatment-related benefits result when a case manager has specific responsibility for the discharge planning of elderly patients returning home after hospitalization. These results provide insights into what is being achieved through current discharge planning practices. The meeting of specific patient needs through enhanced discharge planning may save future costs by reducing the rates of complications and hospital readmissions in an era of prospective payment, thus potentially offsetting the increased costs involved in planning and coordinating postdischarge care for older adults.


Synthesis and antibacterial activity of O-methylazithromycin derivatives. A series of O-methylazithromycin derivatives have been synthesized and their antibacterial activities were compared with those of azithromycin (1). O-Methylation of 1 proceeded stepwise by the two main pathways beginning at the C-6 and C-11 hydroxyl groups, individually. Among O-methyl derivatives, 6-O-methylazithromycin A (11) was slightly less active than 1. The methylation of the secondary hydroxyl group at the C-11 position resulted surprisingly in an increase of their in vitro activity. The antibacterial activities of novel azalides decreased with increasing the number of the methyl groups introduced.


Comparative in-vitro activity of four peptide antibiotics against penicillin-resistant Streptococcus pneumoniae isolated from cerebrospinal fluid (CSF). The in-vitro activity of four peptide antibiotics against 43 penicillin-resistant Streptococcus pneumoniae isolated from cerebrospinal fluid (CSF) was evaluated. The activity of SKF104662 was slightly greater to that of vancomycin, teicoplanin and daptomycin (MICs for 90% of the isolates tested 0.06, 0.25, 0.12 and 0.25 mg/L, respectively) and superior to the other 15 drugs tested. The serotype of these penicillin-resistant strains was also determined. The strains that belonged to the predominant serotype 9 were resistant only to penicillin. All six erythromycin- and clindamycin-resistant strains belonged to serotype 6 and three of them were also resistant to chloramphenicol and tetracycline (plus penicillin).


Computed tomography and magnetic resonance imaging of the nasopharynx. Various radiological techniques are used for the evaluation of nasopharyngeal lesions--conventional lateral and base projections, computed tomography (CT), magnetic resonance imaging (MRI), and angiography. Owing to its multiplanar imaging capability, MRI is the optimal modality for investigation of the anatomy and demonstration of the pathology. Gadolinium-DTPA enhancement helps to delineate the lesions and their extension, especially into the intracranial cavity; however, since fat and bone marrow enhance prominently, MR fat suppression technique should be utilized in certain cases. A detailed description is given of the clinical and radiological (CT, MRI) features of the different benign (adenoid tissue hypertrophy, Thornwaldt cyst, juvenile angiofibroma) and malignant diseases, 80% of which are squamous cell carcinomas.


Emergence of quinolone-imipenem cross-resistance in Pseudomonas aeruginosa after fluoroquinolone therapy. Emergence of resistance to fluoroquinolones was observed in two clinical isolates of Pseudomonas aeruginosa after ciprofloxacin or norfloxacin monotherapy. In the first case, the resistant variants exhibited quinolone-imipenem cross-resistance (MIC of norfloxacin and ciprofloxacin: 16 mg/L; MIC of imipenem: 8 mg/L), although the patient had never received imipenem treatment, while the strain from the second case remained imipenem-susceptible (MIC of norfloxacin or ciprofloxacin: 8 mg/L; MIC of imipenem: 2 mg/I). The frequency of in-vitro emergence of variants resistant to imipenem and fluoroquinolones was studied for the two strains, with imipenem or fluoroquinolones as selecting agents. Ciprofloxacin and three other quinolones (norfloxacin, temafloxacin and tosufloxacin) selected imipenem-resistant variants in a similar way to imipenem for the first strain, but not for the other. In contrast, imipenem did not select quinolone-resistant variants from either strain. For both strains, killing curves demonstrated that a bactericidal effect could be obtained with a drug combination (2 x MIC of ciprofloxacin and 2 x MIC of imipenem) without any selection of resistant mutants after 24 h, thereby suggesting the possible use of this combined regimen for treating severe P. aeruginosa infection.


Optimal stimulation of paralyzed muscle after human spinal cord injury. Muscle properties change profoundly as a result of disuse after spinal cord injury. To study the extent to which these changes can be reversed by electrical stimulation, tibialis anterior muscles in complete spinal cord-injured subjects were stimulated for progressively longer times (15 min, 45 min, 2 h, and 8 h/day) in 6-wk intervals. An index of muscle endurance to repetitive stimulation doubled (from 0.4 to 0.8), contraction and half-relaxation times increased markedly (from 70 to approximately 100 ms), but little or no change was measured in twitch or tetanic tension with increasing amounts of stimulation. The changes observed with 2 h/day of stimulation brought the physiological values close to those for normal (control) subjects. A decrease in the stimulation period produced a reversal of the changes. No effects were observed in the contralateral (unstimulated) muscle at any time, nor was there evidence of decreased numbers of motor units in these subjects secondary to spinal cord injury. Motor unit properties changed in parallel with those of the whole muscle. The occasional spasms occurring in these subjects are not sufficient to maintain normal muscle properties, but these properties can largely be restored by 1-2 h/day of electrical stimulation.


Nasal effects of bradykinin and capsaicin: influence on plasma protein leakage and role of sensory neurons. Nasal insufflation with bradykinin induces nasal discomfort, rhinorrhea, and nasal blockage, all features of rhinitis. We recently showed these effects to be mediated by the B2-receptor subtype, which has been identified at neural and vascular sites. To investigate the relative contribution of capsaicin-sensitive sensory neural stimulation to the action(s) of bradykinin, two randomized double-blind placebo-controlled studies have been undertaken comparing the nasal effects of single-dose administrations of bradykinin (1.88 x 10(-3) M) and capsaicin (3.28 x 10(-5) M). In comparison with placebo, both bradykinin and capsaicin induced nasal pain/discomfort (P less than 0.01) and rhinorrhea (P less than 0.02). Bradykinin significantly increased nasal airways resistance (P less than 0.005) and plasma protein exudation (P less than 0.02). No such changes were identified after nasal challenge with capsaicin. These findings suggest that bradykinin-induced nasal discomfort and rhinorrhea are neurally mediated, whereas the effects on nasal airways resistance and plasma protein exudation are due to a direct vascular action. In addition, these findings question the role of capsaicin-sensitive sensory neurons in nasal vasculature responses, because no vascular effects of capsaicin could be identified in the human nasal mucosa.


Effects of endotoxic shock on diaphragmatic function in mechanically ventilated rats. Diaphragmatic function was investigated in mechanically ventilated rats during endotoxic shock (group E, n = 18) and after saline solution injection (group C, n = 8). Endotoxic shock was produced by a 1-min injection of Escherichia coli endotoxin (10 mg/kg iv) suspended in saline. Diaphragmatic strength was assessed before (T0) and 15 (T15) and 60 (T60) min after injection by measuring transdiaphragmatic pressure (Pdi) generated during bilateral phrenic stimulation at 0.5, 10, 20, 30, 50, and 100 Hz. Diaphragmatic neuromuscular transmission was assessed by measuring the integrated electrical activity of the diaphragm. Diaphragmatic endurance was assessed 75 min after injection from the rate of Pdi decline after a 30-s continuous 10-Hz phrenic stimulation. In 16 additional animals, diaphragmatic glycogen content was determined 60 min after inoculation with endotoxin (n = 8) or 0.9% sodium chloride solution (n = 8). Diaphragmatic resting membrane potential (Em) was measured in 16 additional animals 60 min after endotoxin (n = 8) or saline injection (n = 8). Mean blood pressure decreased from 74 +/- 3 to 53 +/- 6 mmHg at T60 in group E, whereas it was maintained in group C. At T60 Pdi was decreased in group E for frequencies of 50 and 100 Hz and was associated with a decreased diaphragmatic electromyographic activity of 25.3 +/- 2.5 and 26.5 +/- 5.2% for 50- and 100-Hz stimulations, respectively, in comparison with T0 values.(ABSTRACT TRUNCATED AT 250 WORDS)


Conducting airway gas exchange: diffusion-related differences in inert gas elimination. We studied CO2 and inert gas elimination in the isolated in situ trachea as a model of conducting airway gas exchange. Six inert gases with various solubilities and molecular weights (MW) were infused into the left atria of six pentobarbital-anesthetized dogs (group 1). The unidirectionally ventilated trachea behaved as a high ventilation-perfusion unit (ratio = 60) with no appreciable dead space. Excretion of higher-MW gases appeared to be depressed, suggesting a MW dependence to inert gas exchange. This was further explored in another six dogs (group 2) with three gases of nearly equal solubility but widely divergent MWs (acetylene, 26; Freon-22, 86.5; isoflurane, 184.5). Isoflurane and Freon-22 excretions were depressed 47 and 30%, respectively, relative to acetylene. In a theoretical model of airway gas exchange, neither a tissue nor a gas phase diffusion resistance predicted our results better than the standard equation for steady-state alveolar inert gas elimination. However, addition of a simple ln (MW) term reduced the remaining residual sum of squares by 40% in group 1 and by 83% in group 2. Despite this significant MW influence on tracheal gas exchange, we calculate that the quantitative gas exchange capacity of the conducting airways in total can account for less than or equal to 16% of any MW-dependent differences observed in pulmonary inert gas elimination.


Cloning, sequencing, and expression of the Zymomonas mobilis fructokinase gene and structural comparison of the enzyme with other hexose kinases. The frk gene encoding the enzyme fructokinase (fructose 6-phosphotransferase [EC 2.7.1.4]) from Zymomonas mobilis has been isolated on a partial TaqI digest fragment of the genome and sequenced. An open reading frame of 906 bp corresponding to 302 amino acids was identified on a 3-kbp TaqI fragment. The deduced amino acid sequence corresponds to the first 20 amino acids (including an N-terminal methionine) determined by amino acid sequencing of the purified protein. The 118 bp preceding the methionine codon on this fragment does not appear to contain a promoter sequence. There was weak expression of the active enzyme in the recombinant Escherichia coli clone under control of the lac promoter on the pUC plasmid. Comparison of the amino acid sequence with that of the glucokinase enzyme (EC 2.7.1.2) from Z. mobilis reveals relatively little homology, despite the fact that fructokinase also binds glucose and has kinetic and structural properties similar to those of glucokinase. Also, there is little homology with hexose kinases that have been sequenced from other organisms. Northern (RNA) blot analysis showed that the frk transcript is 1.2 kb long. Fructokinase activity is elevated up to twofold when Z. mobilis was grown on fructose instead of glucose, and there was a parallel increase in frk mRNA levels. Differential mRNA stability was not a factor, since the half-lives of the frk transcript were 6.2 min for glucose-grown cells and 6.6 min for fructose-grown cells.


Isolation and characterization of rhizobitoxine mutants of Bradyrhizobium japonicum. To explore the role of rhizobitoxine in Bradyrhizobium-legume symbiosis, 11 rhizobitoxine mutants of B. japonicum USDA61 were isolated on the basis of their inability to synthesize the toxin in culture. Each mutant is prototrophic and symbiotically effective on soybean, cowpea, siratro, and Glycine soja. The rhizobitoxine mutants differ in their chlorosis phenotypes and rhizobitoxine production in planta. As expected, one group of mutant fail to make toxin in planta, resulting in the absence of chlorosis. Another group of mutants causes severe chlorosis on all cultivars of soybean tested. Surprisingly, this group of mutants makes more rhizobitoxine in soybean nodules than the wild-type strain does. This phenotype is only observed on soybean and not on other hosts such as cowpea, siratro, or G. soja. The remaining mutants all produce rhizobitoxine in planta but vary in the amount of toxin they produce and the severity of chlorosis they induce in soybean plants. Biochemical analysis of mutants demonstrates that one mutant is unable to synthesize serinol, a molecule hypothesized to be an intermediate in rhizobitoxine biosynthesis. By using these mutants, it was found that rhizobitoxine plays no apparent role in the nodulation of rj1 soybeans. Recently, it was found that inhibition of ethylene biosynthesis allows Rhizobium meliloti to overcome nitrate inhibition of nodule formation on alfalfa. Because rhizobitoxine also inhibits ethylene biosynthesis, we tested the ability of mutants which accumulate high levels of toxin in planta to overcome nitrate inhibition of nodule formation on soybean plants and found that the nodule formation induced by the wild type and that induced by mutant strains were equally suppressed in the presence of nitrate.


Permeability and charge-dependent adsorption properties of the S-layer lattice from Bacillus coagulans E38-66. We investigated the permeability properties of the oblique S-layer lattice from Bacillus coagulans E38-66 after depositing cell wall fragments on a microfiltration membrane, cross-linking the S-layer protein with glutaraldehyde, and degrading the peptidoglycan with lysozyme. Comparative permeability studies on such multilayered S-layer membranes and suspended S-layer vesicles from thermophilic members of the family Bacillaceae with use of the space technique (M. Sára and U. B. Sleytr, J. Bacteriol. 169:4092-4098, 1987) revealed identical molecular exclusion limits (M. Sára and U. B. Sleytr, J. Membr. Sci. 33:27-49, 1987). Examination of the S-layer lattice from B. coagulans E38-66 with the S-layer membrane technique revealed unhindered passage for molecules up to the size of myoglobin (M(r) 17,000). The molecular dimensions of this protein (2.8 by 3.2 by 4.5 nm) correspond approximately to the size of the ovoid-shaped pore previously shown by high-resolution electron microscopy of negatively stained S-layer self-assembly products (D. Pum, M. Sára, and U. B. Sleytr, J. Bacteriol. 171:5296-5303, 1989). Chemical modification of the S-layer protein and comparative labeling, adsorption, and permeability studies clearly demonstrated that (i) in the native state, free amino and carboxyl groups are present on the outer S-layer face and in the interior of the pores and (ii) electrostatic interactions between these groups prevent unspecific adsorption of the S-layer in vivo.


Identification of pilR, which encodes a transcriptional activator of the Pseudomonas aeruginosa pilin gene. Two regulatory mutants of Pseudomonas aeruginosa, R1 and RA, that affect transcription of the pilin gene were isolated. This was done by introducing a plasmid carrying a fusion of the pilin gene's promoter with the lacZ gene into a bank of P. aeruginosa DNA mutagenized with the transposon Tn5G. The block in pilin expression in these mutants was shown to be at the level of transcription, since these mutants did not synthesize either pilin mRNA or pilin antigen. A restriction fragment derived from the R1 mutant that contains the entire transposon plus flanking chromosomal DNA was cloned and used as a probe to screen a cosmid library of P. aeruginosa DNA. Cosmids that could complement the pilin expression defect in both R1 and RA were isolated. The gene inactivated in R1 was sequenced. This gene, designated pilR, encodes an approximately 50-kDa polypeptide which exhibits significant similarity to the NtrC family of response regulators of the two-component regulatory system. PilR contains the amino-terminal aspartic acid residues which are conserved among the response regulators, suggesting that pilin gene transcription is regulated via a phosphotransfer mechanism in which PilR is phosphorylated by an as yet unidentified protein kinase.


osmY, a new hyperosmotically inducible gene, encodes a periplasmic protein in Escherichia coli. A new osmotically inducible gene in Escherichia coli, osmY, was induced 8- to 10-fold by hyperosmotic stress and 2- to 3-fold by growth in complex medium. The osmY gene product is a periplasmic protein which migrates with an apparent molecular mass of 22 kDa on sodium dodecyl sulfate-polyacrylamide gels. A genetic fusion to osmY was mapped to 99.3 min on the E. coli chromosome. The gene was cloned and sequenced, and an open reading frame was identified. The open reading frame encoded a precursor protein with a calculated molecular weight of 21,090 and a mature protein of 18,150 following signal peptide cleavage. Sequencing of the periplasmic OsmY protein confirmed the open reading frame and defined the signal peptide cleavage site as Ala-Glu. A mutation caused by the osmY::TnphoA genetic fusion resulted in slightly increased sensitivity to hyperosmotic stress.


Cloning, sequencing, and transcriptional regulation of viuA, the gene encoding the ferric vibriobactin receptor of Vibrio cholerae. A 74-kDa iron-regulated outer membrane protein of Vibrio cholerae acts as the receptor for the V. cholerae iron-siderophore complex, ferric vibriobactin. MBG14, a mutant of V. cholerae 0395 containing a TnphoA insertion in a gene designated viuA, lacks this 74-kDa outer membrane protein and is unable to bind or utilize exogenous ferric vibriobactin. Introduction of a plasmid containing the complete viuA coding sequence and 513 bp of upstream DNA into MBG14 restored ferric vibriobactin utilization to the mutant. The DNA insert in this plasmid was sequenced, revealing a single open reading frame of 2,061 bp, encoding a deduced protein of 687 amino acids with a predicted molecular mass of 76,417 Da and a predicted initial signal sequence of 37 amino acids. ViuA showed only weak homology to two iron-regulated outer membrane proteins in Escherichia coli, IutA and FecA. Construction of viuA::TnphoA gene fusions allowed study of the regulation of viuA expression by iron. This regulation in E. coli was dependent on the fur gene. Northern (RNA) blot analysis of RNA from wild-type V. cholerae grown in high- and low-iron media revealed a monocistronic viuA message that was negatively regulated by iron at the transcriptional level. Primer extension analysis identified a single transcriptional start site, located 243 bp above the translational start site. The promoter region of viuA contained two interrupted dyad symmetric nucleotide sequences, overlapping the -10 and -35 boxes, each similar to the E. coli Fur binding consensus sequence. Another iron-regulated gene in V. cholerae that is negatively regulated by fur, irgA, requires a positive transcriptional activator (irgB) for expression. However, a strain of V. cholerae mutant in irgB was unaffected in viuA expression. These studies suggest that there is conserved, global coordinate iron regulation in V. cholerae by fur; additional regulatory factors, superimposed upon the fur system, may provide more precise control of individual iron-regulated genes.


Dissection of the Salmonella typhimurium genome by use of a Tn5 derivative carrying rare restriction sites. A polylinker with rare restriction sites was introduced into a mini-Tn5 derivative. These sites include M.XbaI-DpnI (TCTAGATCTAGA), which is rare in most bacterial genomes, SwaI (ATTTAAAT) and PacI (TTAATTAA), which are rare in G+C-rich genomes, NotI (GCGGCCGC) and SfiI (GGCCN5GGCC), which are rare in A+T-rich genomes, and BlnI (CCTAGG), SpeI (ACTAGT), and XbaI (TCTAGA), which are rare in the genomes of many gram-negative bacteria. This Tn5(pfm) (pulsed-field mapping) transposon carries resistance to chloramphenicol and kanamycin to allow selection in a wide variety of background genomes. This Tn5(pfm) was integrated randomly into the Salmonella typhimurium and Serratia marcescens genomes. Integration of the new rare SwaI, PacI, BlnI, SpeI, and XbaI sites was assayed by restriction digestion and pulsed-field gel electrophoresis. Tn5(pfm) constructs could be valuable tools for pulsed-field mapping of gram-negative bacterial genomes by assisting in the production of physical maps and restriction fragment catalogs. For the first applications of a Tn5(pfm), we bisected five of the six largest BlnI fragments in the S. typhimurium genome, bisected the linearized 90-kb pSLT plasmid, and used Tn5(pfm) and Tn10 to trisect the largest BlnI fragment.


Serial cerebrospinal fluid corticotropin-releasing hormone concentrations in healthy and depressed humans. CRH, a hypothalamic peptide that is the most potent ACTH secretagogue known, also appears to be produced in the cerebral cortex and spinal cord. Depressed patients have blunted responses to exogenous CRH and normal to high concentrations of CRH immunoreactivity in single morning samples of lumbar cerebrospinal fluid (CSF). Although these data suggest that depression may be associated with hypersecretion of CRH, it has also been postulated that central nervous system insufficiency of CRH might have a pathophysiological role in certain depressive syndromes. We continuously sampled lumbar CSF via indwelling subarachnoid catheters from 1100-1700 h and measured CRH at 10-min intervals in depressed patients and normal subjects. A standardized mixed liquid meal was administered at 1300 h. CSF CRH was strikingly reduced in depressed patients compared to normal subjects [4.2 +/- 1.1 pmol/L vs. 13 +/- 2.1 pmol/L (mean +/- SEM), respectively, P less than 0.01 by Wilcoxon test]. CSF CRH concentrations rose progressively during the experiment in both groups, suggesting a diurnal rhythm and, possibly, response to a test meal. CRH had a very brief half-life in CSF (less than 10 min), suggesting that the spinal cord is the origin of CRH in lumbar CSF. The rapid transients in CSF CRH concentration demonstrate that single samples provide very limited information. There were no intraindividual correlations between CSF CRH concentrations and those of either plasma ACTH or cortisol, both of which rose in response to eating. The present data show that impaired central nervous system secretion of CRH can exist during states of severe depression.


Interleukin-1 (IL-1)-induced IL-6- and IL-6-receptor-mediated release of human chorionic gonadotropin by choriocarcinoma cell lines (Jar and HCCM-5) activates adenosine 3',5'-monophosphate-independent signal transduction pathway. The status of preservation of the ability to secrete cytokines, such as interleukin-1 (IL-1), tumor necrosis factor-alpha (TNF alpha), and IL-6, and the cytokine-mediated regulatory cascade was investigated in four choriocarcinoma cell lines. Each cell line constitutively produced IL-6, but not IL-1 alpha, IL-1 beta, or TNF alpha. Jar and HCCM-5 cells responded to IL-6, releasing hCG by direct activation of IL-6 receptors (IL-6-R) with IL-6. Both cell lines also responded to IL-1, but failed to responded to TNF alpha. When stimulated with recombinant IL-1 alpha, both cell lines released IL-6 and activated the IL-6-R system to release hCG, whereas stimulation with TNF alpha failed to release hCG. The experiments showed that both the Jar and HCCM-5 cell lines possessed a partially intact cytokine-mediated cascade, suggesting that IL-1-induced IL-6 release and IL-6-R activation operate in an autocrine manner. In contrast, NUC-1 and SCH cells failed to respond to IL-6, IL-1, or TNF alpha. Although 8-bromo-cAMP, which is a cAMP analog, stimulates hCG release by Jar cells, it failed to stimulate IL-6 release. Moreover, cAMP-mediated hCG release was not blocked by PM1, an anti-IL-6-R antibody. This suggests that elevation of the cytoplasmic cAMP level might activate a pathway different from the IL-6- and IL-6-R-dependent pathway. Moreover, IL-1- and IL-6-mediated hCG release was not blocked by H8, a cAMP-dependent kinase inhibitor, which further suggests that the IL-1- and IL-6-mediated pathway functions independently of the cAMP-dependent pathway in releasing hCG in Jar cells.


Effects of low doses of transdermal 17 beta-estradiol on carbohydrate metabolism in postmenopausal women. The impact of a 3-month continuous administration of transdermal estradiol (E2-TTS 50; 50 micrograms/day) or oral conjugated estrogen (CE; 0.625 mg/day) on glucose and lipid metabolism was investigated in two groups (n = 15/group) of postmenopausal women. Fasting levels of glucose, insulin, and C-peptide; C-peptide/insulin ratio (index of hepatic insulin clearance); and their responses to a 75-g oral glucose tolerance test (OGTT) were evaluated before and after 3 months of continuous estrogen administration. E2-TTS 50 modified carbohydrate metabolism, decreasing fasting insulin levels (P less than 0.01) and increasing the pancreatic islet response to glucose challenges, as indicated by an increased integrated value of the C-peptide curve associated with OGTT (P less than 0.05). Despite greater C-peptide secretion, integrated peripheral insulin after OGTT was decreased (P less than 0.05). The resulting increase in the integrated curve of the molar C-peptide/insulin ratio (P less than 0.01) indicated elevated hepatic insulin clearance after E2-TTS 50 administration. CE treatment did not modify carbohydrate metabolism, except for reducing fasting glucose levels (P less than 0.01). Neither therapy modified lipid metabolism, but a slight increase in circulating triglycerides (P less than 0.01) was observed during CE administration. Our data show that the addition of low doses of natural estrogens does not negatively influence glucose and lipid metabolism in postmenopausal women. By contrast, reversal of postmenopausal hypoestrogenism to early follicular phase estrogenic values with E2-TTS 50 administration seems to exert a beneficial effect on glucose metabolism by increasing hepatic insulin clearance.


Human chorionic gonadotropin (hCG) interacts directly with recombinant human TSH receptors. We have previously shown that highly purified urinary hCG has the potential to both stimulate the intracellular accumulation of cyclic AMP and induce growth of immortalized rat thyroid cells. We have now compared the ability of recombinant human TSH and purified urinary hCG preparations to stimulate Chinese hamster ovary (CHO) cells which have been transfected with the human TSH receptor. Only transfected CHO cells expressing recombinant TSH receptors, but not control CHO cells, were stimulated by hCG to release cyclic AMP in a dose-related manner and the effect of 100 IU of HCG was equivalent to approximately 9.2 uU of rec-hTSH. These data demonstrate that hCG interacts directly with the human TSH receptor.


Physical and physiological specification of magnetic pulse stimuli that produce cortical damage in rats. The effects of transcranial magnetic pulse stimuli on the brain tissue of rats were examined. In Experiment I, 52 male albino rats received pulsed magnetic stimulation of the head. Stimulus intensity, number of stimulations, stimulated sites, and interval between last stimulation and sacrifice for neuropathological examination were varied. High stimulus intensity (2.8 T) and 100 or more stimulations produced clearly defined microvacuolar changes in the neuropil portion of cortical layers 2-6 (especially layers 3 and 4) in 12 of 24 animals. Fewer stimulations and lower intensities produced no such effects in 28 rats given that stimulation. Midline stimulation and stimulation over the left hemisphere produced similar results. No other brain, ocular, or spinal structures manifested such changes. Lesions were present in animals that had intervals up to 30 days between the last stimulation and perfusion. In Experiment II with 18 animals, compound motor action potentials (CMAPs) evoked by magnetic stimulation of the cortical motor area and recorded from the right lower extremity were examined. The electromyographic threshold was 0.83 T. Further increases in stimulus intensity produced increases in CMAP amplitude, up to approximately 1.9 T. It was noted that the lesion-producing intensity (2.8 T) was 0.9 T greater than the intensity needed for near-asymptotic reactions and was 3.4 times the CMAP threshold value.


Acoustic chiasm V: inhibition and excitation in the ipsilateral and contralateral projections of LSO. When this series of experiments was begun in 1984, the activity of each lateral superior olive (LSO) in the mammalian hindbrain was known to encode the hemifield of acoustic space containing a sound source. However, the almost random bilaterality of its ascending projections seemed to jumble that identification before reaching the midbrain. At the same time, electrophysiological studies of LSO and its efferent target in the inferior colliculus, along with the strictly contralateral deficits in sound localization resulting from unilateral lesions above the level of the superior olives, indicated that hemifield allegiance was largely maintained (though reversed) at the midbrain. Here we present seven lines of biochemical evidence, some combined with prior ablations, supporting the notion that the anatomical segregation of the ipsilateral and contralateral fibers ascending from the LSO is accompanied by a corresponding segregation of their neurotransmitters: most of the ascending ipsilateral projection is probably glycinergic and, hence, inhibitory in effect, while most of the contralateral projection is probably glutamatergic/aspartergic and, hence, excitatory in effect. Taken together, the inhibitory ipsilateral projections and the excitatory contralateral projections serve to amplify functional contralaterality at the higher levels of the auditory system.


Characterization of an immortalized human cell line derived from neonatal foreskin diploid fibroblasts. A new human skin cell line, designated as CCFS-1/KMC, immortalized from human neonatal foreskin diploid fibroblast cells, has been subcultured successfully in vitro for more than 500 passages. This anchorage-dependent cell line possesses many common features of transformation such as morphological and cytoskeletal changes, hypotriploidy, infinite lifespan, increasing plating efficiency and saturation density, and decreasing serum requirement and population doubling time. Human papillomavirus (HPV) type 18 DNA was detected in the cell line before and after immortalization by the polymerase chain reaction (PCR) method. Tumorigenicity, however, was not demonstrated in vivo. The isoenzyme activity of the cell line shows activation of a placental form of alkaline phosphatase and a changing lactate dehydrogenase isoenzyme pattern that is different from transformation by carcinogens. Class I HLA and class II HLA antigens are constitutively expressed on this skin cell line. Here we report that these immortalized human fibroblasts derived from neonatal HPV-18-DNA-contained diploid fibroblasts possess double minute chromosomes (DMs), a karyotypic aberration usually found in cancer cells.


Comparative study of Ki-67 immunostaining and nuclear DNA content in histiofibrous tumors. A comparative assessment was made of dermatofibroma, dermatofibrosarcoma protuberans, and malignant fibrous histiocytoma. The study was comprised of an immunohistochemical investigation using the Ki-67 monoclonal antibody and a quantitative analysis of cell nuclear DNA using flow cytometry. It was found that the index of Ki-67 monoclonal antibody labeling, the histogram pattern, and DNA index correlated with clinical malignancy, suggesting the usefulness of these indices for differential diagnosis and evaluation of prognosis.