Stress resistance of Drosophila transgenic for bovine CuZn superoxide dismutase. Several oxidative and non-oxidative stresses were applied to two transgenic strains of Drosophila melanogaster (designated P(bSOD)5 and P(bSOD)11) that express superoxide dismutase (SOD) at elevated levels, and control strains that express normal SOD levels. Transgenic strain P(bSOD)5 exposed to paraquat (1,1'-dimethyl-4,4'-bipyridinium dichloride), a redox cycling agent that generates superoxide anion when metabolized in vivo, was significantly more resistant to this xenobiotic than control flies. When test flies were subjected to 100% oxygen for 20 min each day, the mean lifespan was 3.62 days for control strain 25, but 4.35 days for both transgenic strains. The mortality curves of strains fed 1% H2O2 were similar, but the median lifespan of 72 h for controls and 64 h for transgenics suggests that the transgenic flies were slightly more sensitive to H2O2. The activity of catalase was the same for all strains. Using starvation resistance as a non-oxidative stress, flies maintained on water without any food had identical survival curves; for all strains, the median lifespan was 72 h. Throughout the lifespan, no statistically significant difference in physical activity was displayed for transgenic versus control flies. Collectively, these data suggest that the increased lifespan previously observed in SOD transgenics is specifically related to resistance to oxidative stresses.


The simultaneous generation of superoxide and nitric oxide can initiate lipid peroxidation in human low density lipoprotein. Oxidation of low density lipoprotein (LDL) has been shown to occur in the artery wall of atherosclerotic lesions in both animal models and human arteries. The oxidant(s) responsible for initiating this process are under intensive investigation and 15-lipoxygenase has been suggested in this context. Another possibility is that nitric oxide and superoxide, generated by cells present in the artery wall, react together to form peroxynitrite which decomposes to form the highly reactive hydroxyl radical. In the present study we have modelled the simultaneous generation of superoxide and nitric oxide by using the sydnonimine, SIN-1 and have investigated its effects on LDL. SIN-1 liberates both superoxide and nitric oxide during autooxidation resulting in the formation of hydroxyl radicals. We have demonstrated that superoxide generated by SIN-1 is not available to take part in a dismutation reaction since it reacts preferentially with nitric oxide. It follows, therefore, that during the autooxidation of SIN-1 little or no superoxide, or perhydroxyl radical will be available to initiate lipid peroxidation. We have shown that SIN-1 is capable of initiating the peroxidation of LDL and also converts the lipoprotein to a more negatively charged form. The SIN-1-dependent peroxidation of LDL is completely inhibited by superoxide dismutase which scavenges superoxide. Neither sodium nitroprusside or S-nitroso-N-acetyl penicillamine, which only produce nitric oxide, are able to modify LDL. These results are consistent with the hypothesis that a product of superoxide and nitric oxide could oxidize lipoproteins in the artery wall and so contribute to the pathogenesis of atherosclerosis in vivo.


Clearance of HBsAg in seven patients with chronic hepatitis B. The natural history of chronic hepatitis B patients who spontaneously cleared serum HBsAg was investigated. A total of 351 patients with chronic hepatitis B were observed in our hospital for at least 3 yr. Seven of these patients became HBsAg negative during the follow-up period. HBsAg disappeared within 6 mo (range = 11 to 169 days, mean = 70 days) after acute elevation of ALT. ALT levels as high as 500 IU were found in three patients, whereas such elevation was not demonstrated in the other four patients. After the disappearance of HBsAg, ALT levels returned to normal in all patients. With one exception, all patients seroconverted to antibody to HBsAg; however, hepatitis B virus DNA remained detectable in serum using the polymerase chain reaction in five patients. The titer of percent inhibition of antibody to HBcAg gradually decreased to less than 70% when a 1:200 dilution of the serum of six patients was used. Four of the patients had active liver disease develop: two had chronic active hepatitis and two had cirrhosis. Three of these four patients subsequently had hepatocellular carcinoma develop. These findings suggest that patients may suffer complications of chronic hepatitis even after normalization of transaminase activities and after the clearance of HBsAg. Thus hepatitis B virus should be considered as a possible factor associated with hepatocellular carcinoma even in the absence of HBsAg, particularly if serum hepatitis B virus DNA persists.


Analysis of the p53 tumor-suppressor gene in hepatocellular carcinomas from Britain. Human hepatocellular carcinomas from patients in Britain, an area of low prevalence of hepatocellular carcinoma and low dietary exposure to aflatoxin B1, were analyzed for mutations in the p53 tumor-suppressor gene. Abnormalities in the p53 gene were detected in 2 of 19 hepatocellular carcinomas by polymerase chain reaction--single-stranded conformation polymorphism. Direct sequencing of the evolutionarily conserved regions of p53 (exons 5, 6, 7 and 8), where mutations have been commonly found in a variety of tumors, confirmed that only two hepatocellular carcinomas had mutations in p53, one a 6-bp deletion of codons 158 and 159 (exon 5) and the other a G to A transition at codon 286 (exon 8). No mutations were found in any hepatocellular carcinoma in exons 6 and 7; in particular all tumors had wild-type sequence at codon 249, which has been reported to be a mutational hot spot in the p53 gene in hepatocellular carcinomas from high incidence areas such as China and southern Africa. Abnormalities in p53 expression were examined by immunohistochemistry and found in 1 of the 19 hepatocellular carcinomas. These findings show that p53 mutations are infrequently involved in the malignant transformation of hepatocytes in an area of low hepatocellular carcinoma prevalence. They support the suggestion of a possible link between dietary exposure to aflatoxin and selective G to T mutations at codon 249 of the p53 gene. Our observations also indicate that hepatitis B virus infection alone, present in six of the hepatocellular carcinomas examined, does not account for the specificity for codon 249 mutations reported from endemic areas.


Risk factors linked to tumor recurrence of human hepatocellular carcinoma after hepatic resection. A total of 238 patients who received curative hepatic resections during the last 10 yr were observed to search for the risk factors linked to early tumor recurrence of human hepatocellular carcinoma after hepatectomy. The results revealed that tumor size, tumor appearance and DNA ploidy were the factors in predicting tumor recurrence after resection for hepatocellular carcinoma. Patients with a tumor size less than or equal to 5 cm or a tumor appearance of the solitary type had better disease-free survival than did those with a tumor size greater than 5 cm or a tumor appearance of multiple/daughter nodule types (p < 0.05). Although patients with pattern III (aneuploid with > or = 2 G0/G1 peaks) hepatoma had fewer statistically significant differences (p = 0.19) than did those with pattern I (diploid) or pattern II (aneuploid with single G0/G1 peak) tumors in predicting tumor recurrence, they did have poorer results in terms of the overall survival rate (p < 0.05). We conclude that patients with hepatocellular carcinoma having the aforementioned risk factors should be observed closely.


Incidence and diagnostic features of macroregenerative nodules vs. small hepatocellular carcinoma in cirrhotic livers. In Japan, the presence of a large regenerative nodule within a cirrhotic liver, referred to as a macroregenerative nodule or adenomatous hyperplasia, is thought to play a role in the pathogenesis of hepatocellular carcinoma. These lesions, however, have received little attention outside of Japan. We examined 110 sequentially explanted cirrhotic livers for the presence of such nodules. By gross examination, 19 livers (17.3%) had 40 nodules (10 livers had more than one nodule) between 0.8 and 3.5 cm in diameter. By histological examination, 28 of these were macroregenerative nodules and 12 were hepatocellular carcinomas. Three of these hepatocellular carcinomas, however, appeared to have arisen in association with a macroregenerative nodule. We found that the architectural features of thickened cell plates, formation of trabeculae and loss of reticulin were usually very helpful in differentiating benign macroregenerative nodules from hepatocellular carcinoma. The incidence of macroregenerative nodules in our series was similar to that seen in the Japanese studies, and although we feel that they may play a role in the pathogenesis of carcinoma, we do not believe their presence is necessary for the development of hepatocellular carcinoma.


Reactivation of cytomegalovirus in patients with cirrhosis: analysis of 122 cases. Human cytomegalovirus causes severe and often fatal infections in immunocompromised patients. After organ transplantation cytomegalovirus in peripheral blood mononuclear cells is thought to be activated by alloreaction and to spread because of immunosuppression, and it may cause endogenous cytomegalovirus diseases. Patients with cirrhosis, one group of candidates for liver transplantation, often show various grades of immunosuppression before transplantation. To evaluate the status of cytomegalovirus infection in cirrhotic patients and its relevance to the degree of immunosuppression, we examined the presence of cytomegalovirus in mononuclear cells by polymerase chain reaction and immunocytochemical analysis. We studied 122 patients with definite cirrhosis and 43 normal volunteers. All cirrhotic patients (100%) and 40 (93%) of 43 normal controls were seropositive for cytomegalovirus. Cytomegalovirus DNA was detected by polymerase chain reaction in 77 (63.1%) of 122 seropositive cirrhotic patients, but in only 1 (2.5%) of 40 seropositive normal controls (p < 0.01). Cytomegalovirus antigen could not be detected in mononuclear cells by immunocytochemical staining with monoclonal antibodies. Cytomegalovirus DNA-positive patients have a greater impairment of liver function than do cytomegalovirus DNA-negative patients; this fact is manifested by delayed indocyanine green retention rates and elevated serum bilirubin levels (p < 0.05). Lymphocyte proliferative response induced by phytohemagglutinin and natural killer cell activity were also significantly lower in cytomegalovirus DNA-positive patients as compared with cytomegalovirus DNA-negative patients (p < 0.01). Our data suggest that the reactivation of cytomegalovirus may have already occurred in patients with cirrhosis before transplantation.


Anti-elastin antibodies in patients with lung cancer. The aim of this study was to investigate anti-elastin antibodies of the IgG and IgM types in sera of patients suffering from lung cancer, using the DOT immunobinding assay. We studied 96 pathological and 40 control sera. Anti-elastin antibodies were found to be present in 45% of patients with small cell lung cancer, 19% of subjects with adenocarcinoma and not-identified lung tumor and 15% of patients with squamous cell lung cancer. They circulated in 5% of control persons only. The highest values of their titers were observed in the advanced stages of disease. In 55% of anti-elastin antibody positive small cell lung cancer patients, antibodies were of the IgM type, suggesting the initial step of the autoimmunization to elastin.


Drug rehabilitation in west Malaysia: an overview of its history and development. West Malaysia has had a long history of drug use, beginning as early as the 1800s. While early use was primarily restricted to Chinese coolies and Indian immigrant laborers, the 1970s saw drug use become the domain of the youth of Malaysia and achieve the proportions of a national crisis. This paper traces the early origins of drug use and misuse in Malaysia, its development and expansion during the 1970s and the current efforts at eradication and rehabilitation. This examination of Malaysian efforts in response to the spread of drug use or misuse may provide researchers and practitioners in other countries some historical and cross cultural perspectives on current international efforts at eradicating similar problems.


Effective outpatient drug treatment organizations: program features and selection effects. This research identifies program features that predict outpatient drug treatment outcomes. Treatment effectiveness is measured at the organizational level of analysis in a nationally representative sample of non-methadone outpatient drug misuse treatment organizations (N = 394). Multivariate analyses are conducted to identify program features at various stages of the client career that are related to client outcomes after controlling for client characteristics, organizational characteristics, and social area characteristics. Results indicate that effective non-methadone outpatient drug misuse treatment is related to a number of program features including adequate staff levels, quality assurance efforts, and client follow-up, as well as selection factors that reflect client problem severity.


Neural and endothelial regulatory peptides, their possible involvement in inflammation. The vasoactive regulatory peptides produced by neural and endothelial tissues contribute to normal vascular function and can modify the inflammatory process. Modern microscopic imaging techniques are revealing the plasticity of this neurovascular peptidergic system, highlighting changes in the synthesis, release, binding and degradation of vasoactive peptides during the inflammatory process. Initial changes may represent normal protective mechanisms against tissue injury, while subsequently a loss of neurovascular regulation may contribute to the pathogenesis of some inflammatory conditions. Understanding the complexity of these regulatory systems provides novel insights into the inflammatory process and suggests new possibilities for therapeutic approaches to human disease. In this review we examine the potential for modern microscopic imaging techniques to help elucidate the complexity of the inflammatory process, as illustrated by the involvement of the vasoactive neural- and endothelial-derived regulatory peptides substance P, calcitonin gene-related peptide, neuropeptide Y and endothelin.


Posttreatment exposure to camptothecin enhances the lethal effects of x-rays on radioresistant human malignant melanoma cells. Little is known about the molecular mechanisms responsible for the survival recovery process(es) (known as potentially lethal damage repair), which occurs in mammalian cells following ionizing radiation. Previously, we presented data indicating a role for the DNA unwinding enzyme, topoisomerase I, in DNA repair. We now demonstrate that camptothecin, a specific inhibitor of topoisomerase I, causes dramatic radiosensitization of an extremely resistant human melanoma (U1-Mel) cell line. Camptothecin radiosensitized U1-Mel cells when it was administered either during or immediately following x-irradiation. U1-Mel cells were optimally radiosensitized with 4 microM camptothecin for a period of 4-6 hrs after x-irradiation. Enhanced cell killing by camptothecin was proportional to the initial extent of damage created by x-irradiation; the higher the dose of ionizing radiation, the greater the radiosensitization. The apparent synergy observed with camptothecin and x-rays was irreversible; camptothecin-treated U1-Mel cells were not able to carry out PLDR in a 48 hr period after the drug was removed. We hypothesize that the administration of camptothecin causes lesion modification through a topoisomerase I-mediated mechanism. These data support a role for topoisomerase I in DNA repair and indicate that camptothecin, or more effective derivatives, may have clinical use.


Attenuation of reflex pressor and ventilatory responses to static contraction by an NK-1 receptor antagonist. The chemical messengers released onto second-order dorsal horn neurons from the spinal terminals of contraction-activated group III and IV muscle afferents have not been identified. One candidate is the tachykinin substance P. Related to substance P are two other tachykinins, neurokinin A (NKA) and neurokinin B (NKB), which, like substance P, have been isolated in the dorsal horn of the spinal cord and have receptors there. Whether NKA or NKB plays a transmitter/modulator role in the spinal processing of the exercise pressor reflex is unknown. Therefore, we tested the following hypotheses. After the intrathecal injection of a highly selective NK-1 (substance P) receptor antagonist onto the lumbosacral spinal cord, the reflex pressor and ventilatory responses to static muscular contraction will be attenuated. Likewise, after the intrathecal injection either of an NK-2 (NKA) receptor antagonist or an NK-3 (NKB) receptor antagonist onto the lumbrosacral spinal cord, the reflex pressor and ventilatory responses to static contraction will be attenuated. We found that, 10 min after the intrathecal injection of 100 micrograms of the NK-1 receptor antagonist, the pressor and ventilatory responses to contraction were significantly (P < 0.05) attenuated. Mean arterial pressure was attenuated by 13 +/- 3 mmHg (48%) and minute volume of ventilation by 120 +/- 38 ml/min (34%). The cardiovascular and ventilatory responses to contraction before either 100 micrograms of the NK-2 receptor antagonist or 100 micrograms of the NK-3 receptor antagonist were not different (P > 0.05) from those after the NK-2 or the NK-3 receptor antagonists.(ABSTRACT TRUNCATED AT 250 WORDS)


Effect of exercise training on intracellular free Ca2+ transients in ventricular myocytes of rats. The purpose of this study was to test the hypothesis that exercise training induces enhanced intracellular free Ca2+ (Cai) availability to the contractile elements of cardiac cells. Cai transients were directly measured in single isolated contracting ventricular myocytes from exercise-trained (EX) and sedentary control (SED) rats. Male Sprague-Dawley rats underwent 16 wk of progressive treadmill exercise (32 m/min, 8% grade, 1.5 h/day) (EX) or were cage confined (SED). EX rats had lower resting heart rate and elevated skeletal muscle oxidative capacity. Cai was measured with the fluorescent Cai indicator fura-2. Simultaneous video monitoring indicated that myocytes suspended in physiological salt solution were quiescent until stimulated electrically at a frequency of 0.2 Hz (12-36 V, 2-ms duration). Stimulated Cai transients, measured from changes in fura-2 fluorescence, were similar in cells from EX and SED groups. Peak shortening, time to peak shortening, velocity of shortening, contraction duration, and time to half-relaxation were also similar in cells from EX and SED rats. Ryanodine (10 microM) was applied to eliminate the contribution of Ca2+ release from sarcoplasmic reticulum to the Cai transient. Verapamil was applied to eliminate the contribution of voltage-gated Ca2+ channels to Cai transients. Cai transients were also similar in cells from EX and SED groups after these pharmacological interventions. These results suggest that treadmill training of rats does not alter Cai availability to the contractile elements in isolated ventricular myocytes.


Amiloride-sensitive Na+ pathways in capillary endothelial cell swelling during hemorrhagic shock. We recently discovered that the endothelium of skeletal muscle capillaries swells in the low-flow ischemia induced by hemorrhagic shock. The present study was undertaken to determine the Na+ transmembrane pathways involved in this swelling, since hypoxic cell swelling is attributed to an influx of Na+ and water. In an initial series of experiments, amiloride (5 mg/kg body wt), which blocks multiple Na+ pathways, was infused intravenously into anesthetized rabbits 30 min prior to shock (40% single-withdrawal hemorrhage). Intravital microscopy of treated capillaries in the rabbit tenuissimus muscle showed that after a 1-h shock period, there was no endothelial cell swelling, as evidenced by no measurable change in the width of red blood cells traversing the capillary. In contrast, the swollen endothelium of untreated capillaries reduced the luminal diameter by 20-25% with a preserved stationary abluminal membrane. The specific effects of amiloride on Na+ transport were investigated with amiloride analogues. Animal pretreatment with 5-(N,N-hexamethylene)amiloride, a selective inhibitor of Na(+)-H+ activity, in a dose of 0.5 mg/kg did not significantly mitigate shock-induced swelling; however, a dose of 1 mg/kg completely prevented it. Phenamil, a selective inhibitor of Na+ channel conductance, even at a potent dosage of 0.5 mg/kg, did not affect swelling. These results suggest a primary role for Na(+)-H+ exchange in endothelial cell swelling during hemorrhagic shock, possibly as a means to regulate cellular pH, which may become acidic during ischemia. Narrowed capillaries with elevated hydraulic resistances could delay and diminish resumption of microcirculatory flow on shock resuscitation.


Effect of bradykinin on airway neural responses in vitro. We investigated the effects of bradykinin (BK) on airway excitatory nonadrenergic noncholinergic (e-NANC) and cholinergic nerves in vitro. Neural responses were elicited by electrical field stimulation in guinea pig airways in vitro before and after the addition of BK (10(-10)-10(-7) M). Captopril (10(-5) M) and phosphoramidon (10(-6) M) were added to prevent degradation of BK, and all neural responses were measured in the presence of indomethacin (10(-5) M) and propranolol (10(-6) M). BK potentiated e-NANC responses in bronchi in a concentration-dependent manner (10(-10)-10(-7) M) without changing concentration-response curves to exogenously applied substance P (10(-10)-10(-5) M). BK significantly potentiated e-NANC neural constrictor responses by 22 +/- 7% at 10(-8) M (mean +/- SE, n = 5, P < 0.05) and 32 +/- 7% at 10(-7) M (n = 8, P < 0.01), compared with changes in time-matched control tissues (7 +/- 2%, n = 8). The potentiation of e-NANC responses by BK was abolished by pretreatment with a specific B2-receptor antagonist, HOE 140 (10(-7) M). Cholinergic constrictor responses elicited to electrical field stimulation were not affected by the addition of BK (up to 10(-7) M). These results suggest that BK potentiates e-NANC bronchoconstrictor responses prejunctionally via a B2-receptor.


Levels of epsilon, an essential replication subunit of Escherichia coli DNA polymerase III, are controlled by heat shock proteins. In Escherichia coli, epsilon, the proofreading subunit of DNA polymerase III, is encoded by dnaQ. A random search for mutants that affect the expression of dnaQ revealed that mutations in the genes encoding the heat shock proteins (HSPs) DnaK, DnaJ, and GrpE result in dramatic decreases in the cellular levels of epsilon. dnaQ is arranged in an overlapping divergent transcriptional unit with rnhA, which encodes RNase H1, and mutations in the same HSPs also reduced the apparent levels of RNase H1. The HSPs had only small effects on transcriptional fusions to these genes; thus, it is likely that they operate primarily at the protein level. Since survival and mutagenesis after DNA damage are affected by epsilon and RNase H1, HSPs may have a broad influence on various aspects of DNA replication and repair.


Stimulation of glucose catabolism in Escherichia coli by a potential futile cycle. Fifteen-fold overexpression of phosphoenolpyruvate synthase (Pps) (EC 2.7.9.2) in Escherichia coli stimulated oxygen consumption in glucose minimal medium. A further increase in Pps overexpression to 30-fold stimulated glucose consumption by approximately 2-fold and resulted in an increased excretion of pyruvate and acetate. Insertion of two codons at the PvuII site in the pps gene abolished the enzymatic activity and eliminated the above-described effects. Both the active and the inactive proteins were detected at the predicted molecular weight by polyacrylamide gel electrophoresis. Therefore, the observed physiological changes were due to the activity of Pps. The higher specific rates of consumption of oxygen and glucose indicate a potential futile cycle between phosphoenolpyruvate (PEP) and pyruvate. A model for the stimulation of glucose uptake is presented; it involves an increased PEP/pyruvate ratio caused by the overexpressed Pps activity, leading to a stimulation of the PEP:sugar phosphotransferase system.


Mechanism of glutamate uptake in Zymomonas mobilis. The energetics of the anaerobic gram-negative bacterium Zymomonas mobilis, a well-known ethanol-producing organism, is based solely on synthesis of 1 mol of ATP per mol of glucose by the Entner-Doudoroff pathway. When grown in the presence of glucose as a carbon and energy source, Z. mobilis had a cytosolic ATP content of 3.5 to 4 mM. Because of effective pH homeostasis, the components of the proton motive force strongly depended on the external pH. At pH 5.5, i.e., around the optimal pH for growth, the proton motive force was about -135 mV and was composed of a pH gradient of 0.6 pH units (internal pH 6.1) and a membrane potential of about -100 mV. Measurement of these parameters was complicated since ionophores and lipophilic probes were ineffective in this organism. So far, only glucose transport by facilitated diffusion is well characterized for Z. mobilis. We investigated a constitutive secondary glutamate uptake system. Glutamate can be used as a nitrogen source for Z. mobilis. Transport of glutamate at pH 5.5 shows a relatively high Vmax of 40 mumol.min-1.g (dry mass) of cells-1 and a low affinity (Km = 1.05 mM). Glutamate is taken up by a symport with two H+ ions, leading to substantial accumulation in the cytosol at low pH values.


Regulation of virulence-associated plasmid genes in enteroinvasive Escherichia coli. The transposon TnphoA was used for construction of gene fusions and for studies of gene regulation in an enteroinvasive strain of Escherichia coli. Several plasmid-encoded virulence genes (e.g., the ipaB and virG operons) of such enteroinvasive strains are subject to coordinated thermoregulation involving both operon-specific (the VirB and VirF activators) and global regulators. The nucleoid-associated E. coli protein H-NS was shown to be a negative regulator as judged by studies using H-NS gene deletion mutations and by increasing the level of H-NS protein in the cells. An increased gene dosage of H-NS led to enhanced repression of the ipa and virG operons, particularly at low (30 degrees C) growth temperature. The cyclic AMP receptor protein complex, which is another global transcriptional regulator in E. coli, was not required for the regulation of ipa and virG expression. The virG operon was expressed in an activator-independent manner in cells lacking H-NS protein. We suggest that the role of the VirF activator is to counteract the silencing effect of H-NS.


Autoregulation of the stability operon of IncFII plasmid NR1. The stb locus of IncFII plasmid NR1, which mediates stable inheritance of the plasmid, is composed of an essential cis-acting DNA site located upstream from two tandem genes that encode essential stability proteins. The two tandem genes, stbA and stbB, are transcribed as an operon from promoter PAB. Using PAB-lacZ gene fusions, it was found that the stb operon is autoregulated. A low-copy-number stb+ plasmid introduced into the same cell with the PAB-lacZ fusion plasmid repressed beta-galactosidase activity about 5-fold, whereas a high-copy-number stb+ plasmid repressed beta-galactosidase about 15-fold. The details of autoregulation were analyzed by varying the concentrations of StbA and StbB to examine their effects on expression from the PAB-lacZ fusion plasmid. StbB protein by itself had autorepressor activity. Although StbA protein by itself had no detectable repressor activity, plasmids that encoded both stbA and stbB repressed more effectively than did those that encoded stbB alone. Plasmids with a mutation in stbA had reduced repressor activity. One mutation in stbB that inactivated the stability function also reduced, but did not eliminate, repressor activity. Repressor activity of the mutant StbB protein was effectively enhanced by stbA. These results indicate that StbB serves two functions, one for stable inheritance and one for autoregulation of the stb operon, both of which may be influenced by StbA protein.


MxiJ, a lipoprotein involved in secretion of Shigella Ipa invasins, is homologous to YscJ, a secretion factor of the Yersinia Yop proteins. Shigella flexneri causes bacillary dysentery by invading epithelial cells of the colonic mucosa. The invasion process requires the synthesis and secretion of the virulence plasmid-encoded Ipa proteins. Using TnphoA mutagenesis, we have identified two virulence plasmid genes, mxiJ and mxiM, that encode proteins exported by the general export pathway. Analysis of the MxiJ and MxiM deduced amino acid sequences suggested that mxiJ and mxiM might encode lipoproteins, which was confirmed by [3H]palmitate labeling of MxiJ:PhoA and MxiM:PhoA fusion proteins. A mxiJ mutant was unable to invade HeLa cells, to induce the formation of plaques on confluent monolayers of HeLa cells, and to provoke keratoconjunctivitis in guinea pigs. In addition, secretion of seven polypeptides, including IpaA, IpaB, and IpaC, was abolished in the mxiJ mutant. Sequence comparisons indicated that MxiJ and MxiH, which is encoded by a gene located upstream from mxiJ, are homologous to the Yersinia enterocolitica YscJ and YscF proteins, respectively.


Purification and characterization of a mutant DnaB protein specifically defective in ATP hydrolysis. The dnaB gene of Escherichia coli encodes an essential DNA replication enzyme. Fueled by the energy derived from the hydrolysis of ATP to ADP+P(i), this enzyme unwinds double-stranded DNA in advance of the DNA polymerase. While doing so, it intermittently stimulates primase to synthesize an RNA primer for an Okazaki fragment. To better understand the structural basis of these and other aspects of DnaB function, we have initiated a study of mutant DnaB proteins. Here, we report the purification and characterization of a mutant DnaB protein (RC231) containing cysteine in place of arginine at residue 231. The mutant protein attains a stable, properly folded structure that allows association of six promoters to form a hexamer, as is also true for wild-type DnaB. Further, the mutant protein interacts with ATP, the nonhydrolyzable ATP analog adenosine-5'-O-(3-thiotriphosphate) (ATP gamma S), ADP, and poly(dT), and it stimulates primase action. It is, however, profoundly deficient in ATP hydrolysis, helicase activity, and replication activity at the chromosomal origin of replication. In addition, while general priming reactions with wild-type DnaB and ATP elicited the synthesis of short primers, reactions with DnaB and ATP gamma S or with RC231 and either ATP or ATP gamma S stimulated the synthesis of significantly longer primers. On the basis of these observations, we suggest that primase interacts directly with DnaB throughout primer synthesis during general priming, until dissociation of DnaB from DNA or ATP hydrolysis by DnaB disrupts the interaction and leads to primer termination.


FtsL, an essential cytoplasmic membrane protein involved in cell division in Escherichia coli. We have identified a gene involved in bacterial cell division, located immediately upstream of the ftsI gene in the min 2 region of the Escherichia coli chromosome. This gene, which we named ftsL, was detected through characterization of TnphoA insertions in a plasmid containing this chromosomal region. TnphoA topological analysis and fractionation of alkaline phosphatase fusion proteins indicated that the ftsL gene product is a 13.6-kDa cytoplasmic membrane protein with a cytoplasmic amino terminus, a single membrane-spanning segment, and a periplasmic carboxy terminus. The ftsL gene is essential for cell growth and division. A null mutation in ftsL resulted in inhibition of cell division, formation of long, nonseptate filaments, ultimate cessation of growth, and lysis. Under certain growth conditions, depletion of FtsL or expression of the largest ftsL-phoA fusion produced a variety of cell morphologies, including Y-shaped bacteria, indicating a possible general weakening of the cell wall. The FtsL protein is estimated to be present at about 20 to 40 copies per cell. The periplasmic domain of the protein displays a sequence with features characteristic of leucine zippers, which are involved in protein dimerization.


Escherichia coli is able to grow with negligible sodium ion extrusion activity at alkaline pH. The Escherichia coli mutant NM81, which is deficient in the nhaA gene for the sodium/proton antiporter, still has a sodium ion extrusion activity because of a second antiporter encoded by nhaB (E. Padan, N. Maisler, D. Taglicht, R. Karpel, and S. Schuldiner, J. Biol. Chem. 264:20297-20302, 1989). By chance, we have found that E. coli pop6810 already contains a mutation affecting the sodium ion circulation, probably in or near nhaB, and that its delta nhaA mutant, designated RS1, has no sodium ion extrusion activity at alkaline pH. The growth of RS1 was inhibited completely by 0.1 M sodium, whereas growth inhibition of NM81 was observed only at sodium concentrations greater than 0.2 M. RS1 grew at a normal rate in an alkaline medium containing a low sodium concentration. Furthermore, RS1 grew with a negligible proton motive force in the alkaline medium containing carbonyl cyanide m-chlorophenylhydrazone. The transport activities for proline and serine were not impaired in RS1, suggesting that these transport systems could be driven by the proton motive force at alkaline pH. These findings led us to conclude that the operation of the sodium/proton antiporter is not essential for growth at alkaline pH but that the antiporter is required for maintaining a low internal sodium concentration when the growth medium contains a high concentration of these ions.


Discovery and characterization of a new transposable element, Tn4811, in Streptomyces lividans 66. Transposition of a new 5.4-kb transposon, Tn4811, of Streptomyces lividans to the melC operon of Streptomyces antibioticus on plasmid pIJ702 was discovered. The nucleotide sequence of this copy of Tn4811, which contained an imperfect (9 of 11 bp) terminal inverted repeat, five putative Streptomyces coding sequences for an oxidoreductase and its transcription regulator, and three transposition-related proteins, was determined. SLP- strains of S. lividans contained one copy (A) of Tn4811, while SLP2+ strains contained an additional copy (B) on the SLP2 plasmid. The nucleotide sequences at three insertion junctions of Tn4811 were determined. Copy B lacked 41 bp from the left end. At the other five junctions the duplication of a putative 3-bp target sequence (TGA) was observed. A sequence of less than 3 kb homologous to Tn4811 was present in S. antibioticus. DNA homologous to Tn4811 was not detected in 14 other Streptomyces species.


Mutation of alanine 623 in the third cytoplasmic loop of the rat thyrotropin (TSH) receptor results in a loss in the phosphoinositide but not cAMP signal induced by TSH and receptor autoantibodies. Thyrotropin (TSH) and IgG preparations from patients with Graves' disease increase inositol phosphate as well as cAMP formation in Cos-7 cells transfected with rat TSH receptor cDNA. Mutation of alanine 623 in the carboxyl end of the third cytoplasmic loop of the TSH receptor, to lysine or glutamic acid, results in the loss of TSH- and Graves' IgG-stimulated inositol phosphate formation but not in stimulated cAMP formation. There is no effect of the mutations on basal or P2-purinergic receptor-mediated inositol phosphate formation. The mutations do not affect transfection efficiency or the synthesis, processing, or membrane integration of the receptor, as evidenced by the unchanged amount and composition of the TSH receptor forms on Western blots of membranes from transfected cells. The mutations increase the affinity of the TSH receptor for [125I]TSH and decrease Bmax; however, cells with an equivalently decreased Bmax as a result of transfection with lower levels of wild type receptor do not lose either TSH-induced inositol phosphate formation or cAMP signaling activity. Thus, in addition to discriminating between ligand-induced phosphatidylinositol bisphosphate and cAMP signals, the mutation appears to cause an altered receptor conformation which affects ligand binding to its large extracellular domain.


The Epstein-Barr virus LMP1 gene product induces A20 zinc finger protein expression by activating nuclear factor kappa B. A20 is an inducible zinc finger protein that confers resistance to tumor necrosis factor alpha cytotoxicity. A survey of various cell lines revealed that A20 was constitutively expressed in Epstein-Barr virus (EBV)-immortalized B-cells. Transfection experiments demonstrated that the EBV latent membrane protein LMP1 induced A20 expression. LMP1 is a transforming protein of EBV that has dramatic effects on cell growth, activation, and survival. An integral membrane phosphoprotein, LMP1 bears no homology to other recognized membrane signaling molecules, and its signal transduction pathway is not known. However, studies using the A20 promoter demonstrated that LMP1 transcriptionally activates the A20 gene through cis-acting kappa B sites. In addition, electrophoretic mobility shift assays confirmed LMP1-inducible binding of an NF-kappa B-like factor to kappa B sequences within the A20 promoter. This is the first report implicating NF-kappa B in signaling by LMP1, a fundamentally important viral transforming protein.


Secretion of thioredoxin by normal and neoplastic cells through a leaderless secretory pathway. Thioredoxin, despite its function as an intracellular disulfide reducing enzyme and its lack of a signal sequence, has been found to play some roles extracellularly. Here we show that thioredoxin is actively secreted by a variety of normal and transformed cells, including fibroblasts, airway epithelial cells, and activated B and T lymphocytes. Neither brefeldin A nor dinitrophenol, two drugs that block transport through the exocytic pathway, inhibit secretion of thioredoxin, indicating that the latter does not follow the classical ER-Golgi route. The secretory mechanism for thioredoxin shares several features with the alternative pathway described for interleukin-1 beta, such as the potentiating effect on secretion of several unrelated drugs and the sensitivity to methylamine. However, unlike interleukin-1 beta, thioredoxin is not detected in membrane-bound compartments of secreting cells. In addition, when COS7 are transfected with plasmids encoding pro-interleukin-1 beta or thioredoxin, only the latter is detectable extracellularly.


An engineered mutant of vaccinia virus DNA topoisomerase I is sensitive to the anti-cancer drug camptothecin. Although highly homologous to the other eukaryotic type I DNA topoisomerases, vaccinia virus DNA topoisomerase I is distinct in its resistance to the anti-cancer drug camptothecin. After comparison of available sequences of sensitive and resistant type I topoisomerases, the aspartic acid at position 221 of vaccinia virus topoisomerase I is mutated to a valine. The resulting mutant protein is partially active. In contrast to the wild type enzyme, the relaxation of supercoiled DNA is inhibited by camptothecin. Its cleavage reaction with DNA is enhanced by camptothecin due to inhibition of religation of DNA. This demonstrates that even though the size of vaccinia virus is only about one-third that of the other camptothecin-sensitive topoisomerases, it has a potential interaction site for camptothecin.


Cytochrome aa3 of Rhodobacter sphaeroides as a model for mitochondrial cytochrome c oxidase. Purification, kinetics, proton pumping, and spectral analysis. Aerobically grown Rhodobacter sphaeroides synthesizes a respiratory chain similar to that of eukaryotes. We describe the purification of the aa3-type cytochrome c oxidase of Rb. sphaeroides as a highly active (Vmax > or = 1800 s-1), three-subunit enzyme from isolated, washed cytoplasmic membranes by hydroxylapatite chromatography and anion exchange fast protein liquid chromatography. The purified oxidase exhibits biphasic kinetics of oxidation of mammalian cytochrome c, similar to mitochondrial oxidases, and pumps protons efficiently (H+/e- = 0.7) following reconstitution into phospholipid vesicles. A membrane-bound cytochrome c is associated with the aa3-type oxidase in situ, but is removed during purification. The EPR spectra of the Rb. sphaeroides enzyme suggest the presence of a strong hydrogen bond to one or both of the histidine ligands of heme a. In other respects, optical, EPR, and resonance Raman analyses of the metal centers and their protein environments demonstrate a close correspondence between the bacterial enzyme and the structurally more complex bovine cytochrome c oxidase. The results establish this bacterial oxidase as an excellent model system for the mammalian enzyme and provide the basis for site-directed mutational analysis of its energy transducing function.


Cytochrome aa3 of Rhodobacter sphaeroides as a model for mitochondrial cytochrome c oxidase. The coxII/coxIII operon codes for structural and assembly proteins homologous to those in yeast. The coxII/coxIII operon of Rhodobacter sphaeroides cytochrome c oxidase has been sequenced and characterized by insertional inactivation/complementation analysis. The organization of the genes in this locus (coxII.orf1.orf3.coxIII) is the same as that of the equivalent operon of Paracoccus denitrificans (ctaC.ctaB.ctaG.ctaE), but unlike that of other bacteria whose cytochrome oxidase genes have been characterized so far. The predicted amino acid sequence homology with eukaryotic oxidases is also higher for Rb. sphaeroides (and P. denitrificans) than for other bacterial versions of the enzyme. The inactivation of coxII results in loss of the characteristic cytochrome oxidase spectrum from membranes of the mutant strain. Full recovery requires introduction into the bacterium of the complete operon containing coxII.orf1.orf3.coxIII; partial complementation yielding a spectrally altered enzyme is achieved with a plasmid containing coxII or coxII.orf1.orf3. These results indicate that the peptides ORF1, ORF3, and COXIII are all required for assembly of native cytochrome c oxidase, suggesting an oxidase-specific assembly or chaperonin function for the ORFs in Rb. sphaeroides similar to that observed for the homologous gene products in yeast, COX10 and COX11.


A truncated recombinant alpha subunit of Gi3 with a reduced affinity for beta gamma dimers and altered guanosine 5'-3-O-(thio)triphosphate binding. The baculovirus-based expression system was adapted to express alpha subunits of the complete (alpha i3) and an amino-terminally truncated (alpha i3') form of Gi3 and of two complete forms of Gs (alpha s-L and alpha s-S). Subunits encoded in full length cDNAs were obtained with yields of 40-60 mg of recombinant protein/liter of cells, of which alpha i3 was between 30 and 50% soluble, but alpha s subunits were only 5-10% soluble. Only the complete alpha i3 was myristoylated. alpha i3 was purified in four steps. The purified protein bound 0.8-0.9 mol of guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) per mol of protein and had one predominant contaminant which was identified as a truncated form that begins with methionine 18 instead of methionine 1. Both the full length alpha i3 and the truncated alpha i3' formed trimers with human erythrocyte beta gamma as seen by their migration in sucrose density gradients and by an increased rate of ADP ribosylation by pertussis toxin, but compared to alpha i3, alpha i3' interacted with beta gamma with a reduced affinity and dissociated upon warming. At 32 degrees C, only full length alpha i3 was ADP-ribosylated; at 4 degrees C, alpha i3 and alpha i3' were both ADP-ribosylated, with the truncated form requiring approximately 200-fold higher concentrations of beta gamma. A genetically engineered alpha i3' (alpha i3[18-354]) was also expressed in Sf9 cells. Yields, assessed as saturable GTP gamma S binding sites, were 3-5 mg per liter. Scatchard analysis showed that truncation of the amino terminus interferes with the ability of Mg2+ to promote high affinity binding of GTP gamma S. We conclude that the G protein alpha subunit amino terminus is not essential for interaction with beta gamma dimers, but rather is important in determining the affinity of the alpha subunit for both the beta gamma dimers and guanine nucleotide.


Purification and characterization of heparin lyases from Flavobacterium heparinum. Heparin lyase I has been purified from Flavobacterium heparinum and has been partially characterized (Yang, V. C., Linhardt, R. J., Berstein, H., Cooney, C. L., and Langer, R. (1985) J. Biol. Chem. 260, 1849-1857). There has been no report of the purification of the other polysaccharide lyases from this organism. Although all three of these heparin/heparan sulfate lyases are widely used, with the exception of heparin lyase I, there is no information on their purity or their physical and kinetic characteristics. The absence of pure heparin lyases and a lack of understanding of the optimal catalytic conditions and substrate specificity has stood in the way of the use of these enzymes as reagents for the specific depolymerization of heparin and heparan sulfate into oligosaccharides for structure and activity studies. This paper describes a single, reproducible scheme to simultaneously purify all three of the heparin lyases from F. heparinum to apparent homogeneity. Heparin lyase I (heparinase, EC 4.2.2.7), heparin lyase II (no EC number), and heparin lyase III (heparitinase, EC 4.2.2.8) have molecular weights (by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and isoelectric points (by isoelectric focusing) of M(r) 42,800, pI 9.1-9.2, M(r) 84,100, pI 8.9-9.1, M(r) 70,800, pI 9.9-10.1, respectively. Their amino acid analyses and peptide maps demonstrate that while these proteins are different gene products they are closely related. The kinetic properties of the heparin lyases have been determined as well as the conditions to optimize their activity and stability. These data should improve the application of these important enzymes in the study of heparin and heparan sulfate.


Ras (CXXX) and Rab (CC/CXC) prenylation signal sequences are unique and functionally distinct. Rab proteins typically lack the consensus carboxyl-terminal CXXX motif that signals isoprenoid modification of Ras and other isoprenylated proteins and, instead, terminate in either CC or CXC sequences (C = cysteine, X = any amino acid). To compare the functional relationship between the Ras CXXX and the Rab CC/CXC motifs, we have generated chimeric Ras proteins terminating in Rab carboxyl-terminal CC or CXC sequences. These mutant Ras proteins were not isoprenylated in vitro or in vivo, demonstrating that the CC and CXC sequences alone are not sufficient to replace a CXXX sequence to signal Ras isoprenoid modification. Surprisingly, chimeric Ras/Rab proteins terminating in significant lengths of carboxyl-terminal sequences from Rab1b (7-139 residues), Rab2 (5-151 residues), or Rab3a (12 residues) were also not isoprenylated. These results demonstrate that the sequence requirements for isoprenoid modification of Rab proteins are more complex than the simple tetrapeptide CXXX sequence for isoprenoid modification of Ras proteins and suggest that the Rab geranylgeranyl transferase(s) requires recognition of protein conformation to signal the addition of geranylgeranyl groups. Finally, competition studies demonstrate that a common geranylgeranyl transferase activity is responsible for the modification of Rab proteins terminating in CC or CXC motifs.


Feedback inhibition of Ca2+ release by Ca2+ is the underlying mechanism of agonist-evoked intracellular Ca2+ oscillations in pancreatic acinar cells. Oscillations of free intracellular Ca2+ concentration ([Ca2+]i) are known to occur in many cell types during physiological cell signaling. To identify the basis for the oscillations, we measured both [Ca2+]i and extracellular Ca2+ concentration ([Ca2+]o) to follow the fate of Ca2+ during stimulation of [Ca2+]i oscillations in pancreatic acinar cells. [Ca2+]i oscillations were initiated by either t-butyloxycarbonyl-Tyr(SO3)-Nle-Gly-Tyr-Nle-Asp-2-phenylethyl ester (CCK-J), which mobilized Ca2+ from the inositol 1,4,5-trisphosphate (IP3)-insensitive pool, or low concentration of cholecystokinin octapeptide (CCK-OP), which mobilized Ca2+ from the IP3-sensitive internal pool. Little Ca2+ efflux occurred during the oscillations triggered by CCK-J or CCK-OP in spite of a large average increase in [Ca2+]i. When internal store Ca2+ pumps were inhibited with thapsigargin (Tg) during [Ca2+]i oscillations, a rapid Ca2+ efflux occurred similar to that measured in intensely stimulated, nonoscillatory cells. Tg also stimulated 45Ca efflux from internal pools of cells stimulated with CCK-J or a low concentration of CCK-OP. Hence, a large fraction of the Ca2+ released during each spike is reincorporated by the internal store Ca2+ pumps. Surprisingly, when the increase in [Ca2+]i during stimulation of oscillations was prevented by loading the cells with 1,2-bis(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid, a persistent activation of Ca2+ release and Ca2+ efflux occurred. This was reflected as a persistent increase in [Ca2+]o in cells suspended at low [Ca2+]o or persistent efflux of 45Ca from internal stores of cells maintained at high [Ca2+]o. Since agonist-stimulated Ca2+ release evidently remains activated when [Ca2+]i is highly buffered, the primary mechanism determining Ca2+ oscillations must include an inhibition of Ca2+ release by [Ca2+]i. Loading the cells with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid had no apparent effect on the levels or kinetics of IP3 formation in agonist-stimulated cells. This suggests that [Ca2+]i regulated the oscillation by inhibition of Ca2+ release independent of its possible effects on cellular levels of IP3.


Binding of netropsin to a DNA triple helix. The interaction of netropsin, a minor groove binding drug, with T-A-T triple helix and A-T double helix was studied using circular dichroism spectroscopy and thermal denaturation. The triple helix was made by an oligonucleotide (dA)12-x-(dT)12-x-(dT)12, where x is a hexaethylene glycol chain bridged between the 3' phosphate of one strand and the 5' phosphate of the following strand. This oligonucleotide is able to fold back on itself to form a very stable triplex. Changing the conditions allows the same oligonucleotide in a duplex form with a (dT)12 dangling arm. Circular dichroism spectroscopy demonstrates that netropsin can bind to the triple helical structure. Spectral analysis shows that the bound drug exhibits a conformation and an environment similar in double-stranded and in triple-stranded structure. However, the binding constant to the triple-stranded structure is found smaller than the binding constant to the double-stranded one. Thermal denaturation experiments demonstrate that netropsin destabilizes the triplex whereas it stabilizes the duplex.


The reduction of cytochrome b558 and the activity of the respiratory burst oxidase from human neutrophils. It is known that in respiratory burst oxidase preparations engaged in O2- production, cytochrome b558, a characteristic oxidase component, is partly reduced. This result has been interpreted in terms of a mechanism in which cytochrome b558 functions as an electron-carrying component of the respiratory burst oxidase, its level of reduction reflecting a steady-state partitioning of the cytochrome between reduced and oxidized forms as it ferries electrons from NADPH to oxygen. Kinetic arguments based on this interpretation have supported the proposal that the cytochrome is reduced at a rate sufficient to account for the rate of O2- production by activated neutrophils. We have confirmed the partial reduction of cytochrome b558 in neutrophil cytoplasts and in oxidase preparations exposed to NADPH, but have found that the reduction of the cytochrome bears no apparent relation to the activity of the oxidase, and can occur when NADPH is added to neutrophil membrane preparations that are unable to manufacture O2-. We therefore conclude that the NADPH-dependent reduction of cytochrome b558 seen in these preparations is unlikely to be a reflection of a catalysis-related steady state and that inferences drawn from such observations regarding the kinetic competence of the cytochrome may need to be reconsidered.


Localization of the single-stranded DNA binding site in the thrombin anion-binding exosite. Single-stranded DNA molecules containing a 15-nucleotide consensus sequence have been reported to inhibit thrombin activity. The mechanism of the inhibition was studied using a consensus 15-mer oligonucleotide and two recombinant mutant thrombins: the anion-binding exosite mutant thrombin R70E, and thrombin K154A, in which the mutation was located in a surface loop outside of the exosite. The consensus 15-mer oligonucleotide inhibited both fibrinogen-clotting and platelet-activation activities of plasma-derived thrombin, recombinant wild type thrombin, and mutant thrombin K154A in a sequence-specific and dose-dependent manner, whereas it did not inhibit either activity of mutant thrombin R70E. The 15-mer oligonucleotide also inhibited thrombomodulin-dependent protein C activation by plasma-derived thrombin. In competition equilibrium binding experiments, binding of 125I-labeled diisopropyl phosphoryl-thrombin to thrombomodulin was completely inhibited by the consensus 15-mer oligonucleotide with a Kd value of 2.68 +/- 0.16 nM. These results suggest that Arg-70 in the anion-binding exosite of thrombin is a key determinant for interaction with specific single-stranded DNA molecules, and that binding of single-stranded DNA molecules to the exosite prevents the interaction of thrombin with fibrinogen, the platelet thrombin receptor, and thrombomodulin.


Thyrotropin-releasing hormone binding to the mouse pituitary receptor does not involve ionic interactions. A model for neutral peptide binding to G protein-coupled receptors. Thyrotropin-releasing hormone, TRH (< Glu-His-Proamide), and [N tau-Me-His]TRH (MeTRH) are present as neutral and positively charged forms at physiologic pH, and it was possible that they bind to the TRH receptor (TRH-R) as charged (protonated) species. Binding affinities of TRH and MeTRH to endogenous rat TRH-Rs and to transfected wild type mouse TRH-Rs decreased below pH 7.1. Half-maximal decreases in binding occurred at the approximate pK alpha values of these ligands. Asp to Ala mutations in extracellular loop 1, TM-4, and TM-5 did not decrease binding affinity, but an Asp to Ala mutation in TM-2 caused the affinity to decrease 8-fold. The pH dependences of binding of MeTRH, however, were similar in wild type and all mutant receptors and were consistent with the protonated form of MeTRH binding less well. Thus, the binding of TRH to its receptor does not involve ionic interactions and may be a prototype for binding of neutral peptide ligands to G protein-coupled receptors.


Expression, purification, and properties of the plasma membrane Ca2+ pump and of its N-terminally truncated 105-kDa fragment. Isoform 4b of the human plasma membrane Ca2+ pump was expressed in COS cells and in the baculovirus system (Sf9 cells). A 105-kDa pump fragment lacking the first two transmembrane domains and the so-called transduction domain was also expressed. The expression level was 2-4 times the background in COS cells and at least 7 times in the baculovirus system. Tests on membranes from both systems showed that the expressed pump was active. The expressed pump and the 105-kDa fragment were isolated from Sf9 cell membranes by calmodulin affinity chromatography. The pump had Ca(2+)-dependent ATPase activity with a calmodulin stimulation factor of 3, formed a La(3+)-stabilized phosphoenzyme, and had a KM (Ca2+) in the presence of calmodulin of about 1 microM. The 105-kDa fragment, assayed by the phosphoenzyme test on COS or Sf9 cell membranes or by ATPase measurements after isolation from Sf9 cells, proved inactive. Laser confocal microscopy on Sf9 cells showed that both the pump and the 105-kDa fragment were apparently associated with the plasma membrane. The expressed pump in COS and Sf9 cells and the endogenous pump in a number of other cell lines had a slower gel mobility (i.e. a higher apparent molecular mass) than the erythrocyte pump.


Noncatalytic cGMP-binding sites of amphibian rod cGMP phosphodiesterase control interaction with its inhibitory gamma-subunits. A putative regulatory mechanism of the rod photoresponse. The cGMP phosphodiesterase (PDE) of retinal rods plays a central role in phototransduction. Illumination leads to its activation by a rod G-protein (Gt, transducin), thus causing a decrease in intracellular cGMP concentration, closure of plasma membrane cationic channels gated by cGMP, and development of the photoresponse. The PDE holoenzyme is an alpha beta gamma 2 tetramer. The alpha- and beta-subunits each contain one catalytic and one, or possibly two, noncatalytic cGMP-binding sites. Two identical gamma-subunits serve as protein inhibitors of the enzyme. Their inhibition is removed when they bind to Gt-GTP during PDE activation. Here we report that the noncatalytic cGMP-binding sites regulate the binding of PDE alpha beta with PDE gamma and as a result determine the mechanism of PDE activation by Gt. If the noncatalytic sites are empty, Gt-GTP physically removes PDE gamma from PDE alpha beta upon activation. Alternatively, if the noncatalytic sites are occupied by cGMP, Gt-GTP releases PDE gamma inhibitory action but remains bound in a complex with the PDE heterotetramer. The kinetic parameters of activated PDE in these two cases are indistinguishable. This mechanism appears to have two implications for the physiology of photoreceptor cells. First, the tight binding of PDE gamma with PDE alpha beta when the noncatalytic sites are occupied by cGMP may be responsible for the low level of basal PDE activity observed in dark-adapted cells. Second, occupancy of the noncatalytic sites ultimately controls the rate of PDE inactivation (cf. Arshavsky, V. Yu., and Bownds, M. D. (1992) Nature 357, 416-417), for the GTPase activity that terminates PDE activity is slower when these sites are occupied and Gt stays in a complex with PDE holoenzyme. In contrast GTPase acceleration is maximal when the noncatalytic sites are empty and Gt-PDE gamma dissociates from PDE alpha beta. Because cGMP levels are known to decrease upon illumination over a concentration range corresponding to the binding constants of the noncatalytic sites, the binding might be involved in determining the lifetime of activated PDE, after a single flash and/or during dark adaptation.


Positive regulation of mu-calpain action by polyphosphoinositides. Whether calcium is the only major intracellular activator of calpain has not yet been established. Here we demonstrate that polyphosphoinositides may play critical roles in the activation process of mu-calpain. Experiments with purified enzyme, substrate (fodrin), and phospholipids show that only polyphosphoinositides but not other lipids significantly promote calpain action in the physiological intracellular calcium range of 10(-7) to 10(-6) M. The effect of polyphosphoinositide is exerted through both a reduction in the calcium concentration required for calpain autolysis and an increase in the Vmax of the proteolytic reaction. Neomycin, a polyphosphoinositide-binding antibiotic, inhibits both polyphosphoinositide-assisted proteolysis in test tubes and calcium-induced calpain activation coupled with substrate proteolysis in intact cells. This implies that the presence of polyphosphoinositides may actually be a prerequisite for calpain activation inside cells.


An exonuclease requiring an intact helical stem for specificity produces the 3' end of Acanthamoeba castellanii 5 S RNA. A nuclear extract from Acanthamoeba castellanii which contains all of the components necessary for specific transcription of a 5 S RNA gene was separated into fractions required for specific transcription initiation and an additional fraction needed in the reconstituted system to produce the 3' end characteristic of mature 5 S RNA. The latter fraction contained a novel processing activity characterized by an exonuclease specific for highly structured RNAs, including 5 S RNA. An intact helical stem formed between the 5' and 3' ends of the 5 S RNA precursor determines the 3' nucleotide. In addition, the presence of ATP is required for specific processing. However, the possibility has not been ruled out that ATP inhibits a nonspecific ribonuclease in the extract since processing proceeds into the helical stem in its absence.


Purification and properties of pancreatic glycine N-methyltransferase. Glycine N-methyltransferase (GNMT) regulates the ratio of S-adenosylmethionine to S-adenosylhomocysteine. It is very abundant in liver cytosol and earlier studies have shown it to be present in high concentrations in the pancreas. We have previously reported that liver GNMT is allosterically inhibited by 5-methyltetrahydrofolate pentaglutamate (5-CH3-H4PteGlu5), and proposed that this represents a metabolic control mechanism which links the de novo synthesis of methyl groups to the methylating ability of the liver. We now report that pancreatic GNMT also contains bound folate in vivo. Purified pancreatic GNMT is inhibited by reduced folate polyglutamates in vitro. The KI for the synthetic (R,S)5-CH3-H4PteGlu5 is 2.4 x 10(-7) M. The natural (S) form of 5-CH3-H4PteGlu5 is tightly bound and has a Kd of 1.3 x 10(-7) M. One mole is bound per enzyme tetramer. These studies suggest that GNMT is important in the regulation of methyl group metabolism in the pancreas as well as in the liver.


Characterization of a minimal promoter element required for transcription of the mouse type II beta regulatory subunit (RII beta) of cAMP-dependent protein kinase. The 5'-flanking DNA of the mouse RII beta subunit of the cAMP-dependent protein kinase gene was characterized by transient transfection of RII beta-CAT constructs into mouse neuroblastoma cells (NB2a) and Chinese hamster ovary (CHO) cells and by gel mobility shift and footprinting assays. The minimal promoter of the RII beta gene was composed of two adjacent functional elements. A 3'-element which supported enhanced CAT activity was located between base pairs (bp) -267/-168 from the translation initiation start site. CAT plasmids containing these RII beta sequences showed 12- and 16-fold increased CAT activity in the NB2a and CHO cells, respectively, compared to the basic CAT vector. Plasmids containing 20 additional bp 5' to the -267/-168 fragment showed 2-fold more CAT activity than the shorter fragment in NB2a cells, while CAT activity in CHO cells was nearly the same for both constructs. CAT plasmids containing only this 20-bp fragment showed 9- and 13-fold increased CAT activity in NB2a and CHO cells, respectively. The core promoter of the RII beta gene lacked classical TATA and CAT sequences, but contained 3 copies of the Sp1 core consensus sequence. Gel mobility shift assays using 32P-labeled 5'-flanking DNA containing bp -291/-49 and nuclear extracts from NB2a and CHO cells displayed several retarded bands in the gels suggesting complex formation with nuclear DNA-binding factors. Unlabeled DNA containing bp -291/-49 blocked the appearance of all retarded bands. Competition using an oligonucleotide corresponding to the Sp1 DNA-binding site effectively blocked the appearance of the two more slowly migrating bands but did not affect the major rapidly migrating bands. DNase I footprinting analysis using purified Sp1 protein confirmed that Sp1 could bind to the Sp1 sites. Methylation interference and mutational analysis showed that one of the faster migrating bands was the result of factor binding to the DNA sequence adjacent to the Sp1 sites. Additional tissue-specific nuclear-binding factor sequences were detected upstream of the core promoter. Our data suggest that the core promoter of the RII beta gene can initiate transcription from the DNA around the Sp1 sites but that there are tissue-specific nuclear factor-binding sites located distal to the Sp1 sites.


Homology in the structure and the prosthetic groups between two different terminal ubiquinol oxidases, cytochrome a1 and cytochrome o, of Acetobacter aceti. Acetobacter aceti produces two different terminal oxidases dependent on the culture conditions, shaking and static cultures. Cells grown on shaking culture contain cytochrome a1, while cytochrome o is present in cells grown on static culture. Cytochrome a1 and cytochrome o of A. aceti were compared especially with respect to the protein structure and the prosthetic groups. Cytochrome a1 exhibited lower CN sensitivity and higher affinity for O2 than cytochrome o. Both terminal oxidases consisted of four nonidentical polypeptides of which the molecular sizes were identical between both enzymes. Cytochrome a1 cross-reacted with an antibody raised against cytochrome o at the same level as cytochrome o did, and an antibody elicited against cytochrome a1 cross-reacted with both cytochrome o and cytochrome a1 at the same intensity, which indicates that both oxidases are indistinguishable immunochemically. Furthermore, almost the same peptide mapping pattern with chymotrypsin was observed in subunit I and in subunit II between both terminal oxidases, and the amino-terminal sequences in the subunit II of both oxidases were identical at least in their 10 amino acids. As for the prosthetic groups, both oxidases were shown to contain two heme-irons and one copper atom. Further, high performance liquid chromatography analysis of the heme moieties extracted from both the purified enzymes indicated that cytochrome a1 contains hemes b and a at a ratio of 1 to 1, whereas cytochrome o contains the same amounts of hemes b and o. Thus, data indicate that cytochrome a1 and cytochrome o of A. aceti are cytochrome ba and cytochrome bo ubiquinol oxidases, respectively, and that both oxidases have a closely similar protein structure and prosthetic groups, in which only heme a in the heme/copper binuclear center of cytochrome a1 is replaced by heme o in that of cytochrome o.


A third form of the G protein beta subunit. 2. Purification and biochemical properties. Visual excitation in cones is thought to involve a cone-specific G protein (cone transducin) that transduces the light signal detected by the cone visual pigment into an increase in the enzymatic activity of a cGMP phosphodiesterase. In the preceding paper, we have shown that the G beta 3 isoform of G proteins is specifically localized in bovine cone photoreceptors and proposed that it might be a component of cone transducin. We reported here the purification from bovine retinal extract of a cone-specific T beta 3 gamma complex (where T is transducin), which is composed of a G beta 3 subunit and an immunochemically distinct G gamma subunit. Our purification of this complex is based on a two-stage procedure; the first stage consists of a series of column chromatographies that yield a mixture of purified T beta gamma substantially enriched in T beta 3 gamma, and the second stage involves the removal of all of the rod-specific T beta 1 gamma from the mixture using an affinity column of immobilized monoclonal antibodies directed against the rod T gamma subunit of transducin. Using this procedure, we were able to obtain sufficient amounts of T beta 1 gamma and T beta 3 gamma to begin a comparative study of their properties. We showed that T beta 3 gamma is distinguishable from T beta 1 gamma by isoelectric focusing under nondenaturing conditions. The G beta 3 polypeptide of T beta 3 gamma also migrates slightly slower than the G beta 1 polypeptide of T beta 1 gamma on denaturing polyacrylamide gels. Analysis of the interactions of T beta 3 gamma with other retinal proteins indicated that it has a lower affinity for the T alpha subunit of rod transducin but appears to complex with a phosducin-like protein. The differences in the intrinsic biochemical properties of T beta 3 gamma as compared to T beta 1 gamma may partially account for the lower light sensitivity of cones.


Signal transduction within the nucleus by mitogen-activated protein kinase. The nucleus is an important target of signal transduction by growth factor receptors that stimulate mitogen-activated protein (MAP) kinases. We tested the hypothesis that MAP kinases have a signaling role within the nucleus by examining the effect of the expression of a human MAP kinase isoform (p41mapk) in tissue culture cells. The expressed p41mapk was found to be localized in both the cytoplasmic and nuclear compartments of the cells. Significantly, the expression of p41mapk caused an increase in the phosphorylation of a nuclear substrate: Ser62 of c-Myc. Phosphorylation at Ser62 stimulated the activity of the NH2-terminal transactivation domain of c-Myc. Thus, p41mapk causes the phosphorylation and regulation of a physiologically significant nuclear target of signal transduction. These data establish that at least one MAP kinase isoform has a nuclear role during signal transduction.


Involvement of cysteine residues in catalysis and inhibition of human aldose reductase. Site-directed mutagenesis of Cys-80, -298, and -303. In order to study the potential role of cysteinyl residues in catalysis and inhibition of human aldose reductase, mutants containing cysteine to serine substitution at positions 80 (ALR2:C80S), 298 (ALR2:C298S), and 303 (ALR2:C303S) were constructed. Mutation of Cys298 resulted in the most profound changes, as ALR2:C298S displayed 4- to 5-fold elevation in K'm(NADPH), K'm(DL-glyceraldehyde), and kcat(DL-glyceraldehyde) relative to wild type aldose reductase as well as a 10-fold higher Ki for the aldose reductase inhibitor sorbinil. Wild type and mutant reductases were equally sensitive to tolrestat, a structurally different reductase inhibitor. Carboxymethylation of the wild type enzyme or the C80S and C303S mutants led to a modest decrease in kcat as well as an increase in K'm(DL-glyceraldehyde) and Ki(sorbinil). These parameters were not significantly changed when ALR2:C298S was subjected to carboxymethylation. Lithium sulfate caused activation of ALR2:WT, C80S, and C303S but did not significantly affect the activity of ALR2:C298S. The differential sensitivity of wild type and mutant reductases to inhibition by sorbinil and tolrestat, before and after carboxymethylation, indicates that these inhibitors bind at different sites. These results suggest that Cys-298 is present near the active site and constitutes a regulatory group which controls the catalytic activity and inhibitor sensitivity of the enzyme.


Molecular diversity in the adenylylcyclase family. Evidence for eight forms of the enzyme and cloning of type VI. The conservation of amino acid sequence among types I-IV adenylylcyclase has made it possible to apply the polymerase chain reaction to examine the extent of the molecular diversity within this family of enzymes. cDNA templates from rat heart, liver, kidney, guinea pig brain and testes, and mouse skeletal muscle were amplified with primers specific to adenylylcyclase sequences. Evidence was obtained for a total of eight distinct gene products divisible into five subfamilies. Five of the products correspond to regions from cloned forms of adenylylcyclase, while three are previously unidentified. As many as seven different adenylylcyclases are expressed in rat heart, liver, and kidney based on this analysis. Two newly identified polymerase chain reaction (PCR) products were utilized to screen a rat cDNA library from H35 Reuber hepatoma cells. A 6080-nucleotide cDNA contains an open reading frame encoding the 1166-amino acid type VI protein which has a predicted topography similar to that of other adenylylcyclases. The type VI message is abundantly expressed in rat heart, kidney, and brain. Human embryonal kidney cells stably expressing the cDNA showed an enhanced response to isoproterenol that could be inhibited by carbachol in intact cells. Increases in intracellular Ca2+ contribute to the inhibitory effect of carbachol.


Membrane association of the myristoylated alanine-rich C kinase substrate (MARCKS) protein appears to involve myristate-dependent binding in the absence of a myristoyl protein receptor. The myristoylated alanine-rich C kinase substrate, or MARCKS protein, has been implicated in several cellular processes, yet its physiological function remains unknown. We have studied the molecular basis of its membrane association in a cell-free system in order to help elucidate its regulation and function. First, we showed that the MARCKS protein incorporated [3H]myristate when its mRNA was translated in vitro in reticulocyte lysates. The myristoylated protein bound rapidly to freshly fractionated cell membranes, while a nonmyristoylated mutant associated to a much lesser extent (< 15% of wild type). To determine whether this binding was due to a specific cytoplasmic-face protein "receptor," as is seen with pp60v-src, we pretreated the membranes in several ways. Prior treatment of membranes with heat (100 degrees C for 3 min) or trypsin did not affect subsequent MARCKS binding. Binding was markedly decreased in 50 mM EDTA, 0.5 M NaCl, or 1.0% Triton X-100; it was restored to normal after removal of the NaCl and EDTA but was still decreased after removal of the Triton X-100. These findings argued against the existence of a protein receptor for the MARCKS protein on cellular membranes. Finally, MARCKS protein phosphorylated in vitro with protein kinase C bound to the cell membranes to the same extent as the nonphosphorylated protein; this binding was also unaffected by an excess of a synthetic peptide corresponding to the phosphorylation site domain of the protein. We conclude that, at least in this in vitro system, the membrane association of the MARCKS protein is primarily dependent on the amino-terminal myristate moiety and does not appear to involve a specific cytoplasmic-face protein receptor.


Transcriptional repression of interleukin-6 gene by adenoviral E1A proteins. Transcription of interleukin-6 (IL-6) gene in human HepG2 and HeLa cells was induced by treatment with interleukin-1 (IL-1), tumor necrosis factor-alpha (TNF-alpha), phorbol 12-myristate 13-acetate, or dibutyryl cyclic AMP. These agents enhanced the expression of chloramphenicol acetyltransferase (CAT) activity in cells transfected with chimeric CAT genes driven by the transcriptional regulatory regions of human IL-6 gene. Both induced and basal levels of CAT expression were severely repressed upon co-transfection of expression vectors encoding the adenoviral E1A289R or E1A243R protein. The conserved region 1 of E1A proteins was required for this activity. IL-6-CAT expression could also be induced by co-transfecting expression vectors containing cDNAs of the catalytic subunit of protein kinase A or c-jun. E1A repressed transcriptional induction by these agents as well. Similar inhibition was observed when a CAT gene driven by the NF kappa B element of the IL-6 gene was used as a reporter plasmid. In a cell line stably transfected with the E1A gene, IL-1 or TNF-alpha failed to induce IL-6 mRNA. Electrophoretic mobility shift assays were carried out with nuclear extracts of these cells using, as probes, the NF kappa B element or the multiple regulatory element of the IL-6 gene. With either probe, additional faster migrating DNA-protein complexes were formed in the extracts of E1A-expressing cells as compared with the extracts of the corresponding control cells. Experiments with NF kappa B antibody revealed differences between the different DNA-protein complexes formed in the extract of E1A-expressing cells. These observations suggest that E1A represses IL-6 gene transcription by interfering with the formation of appropriate DNA-protein complexes.


Morphological assessment of hepatoma cells (HepG2) microencapsulated in a HEMA-MMA copolymer with and without Matrigel. Hepatoma cells (HepG2), an anchorage-dependent cell line, were microencapsulated in a HEMA-MMA polyacrylate membrane to which the cells do not adhere. This environment was altered by the coencapsulation of Matrigel, a reconstituted extracellular matrix derived from the Engelbreth-Holm-Swarm (EHS) mouse tumor basement membrane, to provide sites for cell attachment. The effect on the cells of these two capsule microenvironments during a 2-week in vitro culture period was assessed by examining the spatial arrangement, morphology, and viability of the cells using light microscopy and scanning electron microscopy (SEM). In preparation for microscopy, dissolution of the polymer was prevented by the use of frozen sections embedded in a water-soluble compound. Similarly, freeze cleavage of conductively stained capsules permitted SEM observation of the capsule interior along with ultrastructural detail of the cells. In the absence of Matrigel, cells in HEMA-MMA capsules were found to form aggregates in intracapsular pockets with central necrosis occurring at day 7 in large aggregates. The coencapsulation of HepG2 cells with Matrigel, resulted in an initially uniform distribution of essentially individual cells with aggregates appearing later within the Matrigel. Many cells within these capsules had remained viable when examined up to day 14 with only limited cellular necrosis, implying a favorable environment for microencapsulated HepG2 cells.


Toxicity test of biodegradable polymers by implantation in rabbit cornea. To evaluate whether or not the corneal micropocket implantation is effective for determining the toxicity of polymeric materials, currently used biodegradable polymers such as poly(lactic acid) (PLA), poly(glycolic acid) (PGA), LA-GA copolymers, and three kinds of poly(2-cyano-acrylate)s (PCA) were implanted in a rabbit corneal pouch and the tissue responses were observed macroscopically and microscopically. It was found that PLA induced no vascularization, whereas a residual solvent and ethylene oxide gas remaining in the PLA matrix invoked vascularization. Vascularization clearly took place when PGA was implanted in the cornea, which became opaque, probably because of cellular infiltration. In the case of PCA implantation, severe inflammation as well as vascular invasion occurred in the initial stage. It is likely that these tissue reactions were caused by the leachables from the implanted materials, the extent being dependent on the leaching rate and the toxicity. It was concluded that the corneal micropocket assay is a good means to detect trace amounts of leachables from implanted materials without sacrificing the animals with the implanted materials.


Biodegradable rods versus Kirschner wire fixation of wrist fractures. A randomised trial. In a prospective trial, biodegradable polyglycolic acid rods were compared with Kirschner wires for fixation of wrist fractures (Frykman types I, II, V and VI). Fifteen patients were randomly assigned to each treatment group. There was no significant difference between the groups with regard to age, sex ratio and fracture type. Kapandji's pinning technique was used in all cases. There were no significant differences in the results obtained in both groups at final follow-up. At three months and six months the functional results of the Kirschner-wire group were, however, significantly better (p < 0.05), due to numerous transient complications from foreign-body reactions to the polyglycolic acid rods. The use of polyglycolic acid rods is therefore not recommended for the fixation of distal radial fractures.


Functional dissection of the phosphorylated termini of fission yeast DNA topoisomerase II. Fission Yeast DNA topoisomerase II (165 kD) consists of an enzymatically active 125-kD core, approximately 10-kD NH2-terminal and 30-kD COOH-terminal domains. The question addressed in the present study is what is the role of the topo II termini. Although deletion of either the NH2 or the COOH terminus is viable, deletion of both termini is lethal; the termini share an essential role for viability. We show here that topo II phosphorylation sites are localized in the terminal domains, but dephosphorylated topo II is still active. The topo II terminal sequences are required for nuclear localization; topo II double terminal deletion mutants are deficient for nuclear targeting, whereas wild-type and single deletion mutant topo IIs are transported into the nucleus with different efficiencies. Functional subdomains in the NH2 terminus are further dissected; we identified a 15 amino acid nuclear localization sequence (NLS) which is essential for viability and nuclear localization when the COOH terminus is deleted. This NLS could be substituted with SV-40 large T-antigen NLS. Two other functional subdomains were found; a non-essential acidic stretch which is phosphorylated and apparently enhances the nuclear localization and an essential hydrophilic stretch of unknown function. Motifs similar to these three NH2-terminal subdomains are also found in the COOH terminus. Our results support the possibility that phosphorylation of topo II does not play an essential role in fission yeast.


Antibodies against 70-kD heat shock cognate protein inhibit mediated nuclear import of karyophilic proteins. Previously, we found that anti-DDDED antibodies strongly inhibited in vivo nuclear transport of nuclear proteins and that these antibodies recognized a protein of 69 kD (p69) from rat liver nuclear envelopes that showed specific binding activities to the nuclear location sequences (NLSs) of nucleoplasmin and SV-40 large T-antigen. Here we identified this protein as the 70-kD heat shock cognate protein (hsc70) based on its mass, isoelectric point, cellular localization, and partial amino acid sequences. Competition studies indicated that the recombinant hsc70 expressed in Escherichia coli binds to transport competent SV-40 T-antigen NLS more strongly than to the point mutated transport incompetent mutant NLS. To investigate the possible involvement of hsc70 in nuclear transport, we examined the effect of anti-hsc70 rabbit antibodies on the nuclear accumulation of karyophilic proteins. When injected into the cytoplasm of tissue culture cells, anti-hsc70 strongly inhibited the nuclear import of nucleoplasmin, SV-40 T-antigen NLS bearing BSA and histone H1. In contrast, anti-hsc70 IgG did not prevent the diffusion of lysozyme or 17.4-kD FITC-dextran into the nuclei. After injection of these antibodies, cells continued RNA synthesis and were viable. These results indicate that hsc70 interacts with NLS-containing proteins in the cytoplasm before their nuclear import.


Overexpression of human alpha-galactosidase A results in its intracellular aggregation, crystallization in lysosomes, and selective secretion. Human lysosomal alpha-galactosidase A (alpha-Gal A) was stably overexpressed in CHO cells and its biosynthesis and targeting were investigated. Clone AGA5.3-1000Mx, which was the highest enzyme overexpressor, produced intracellular alpha-Gal A levels of 20,900 U/mg (approximately 100 micrograms of enzyme/10(7) cells) and secreted approximately 13,000 U (or 75 micrograms/10(7) cells) per day. Ultrastructural examination of these cells revealed numerous 0.25-1.5 microns crystalline structures in dilated trans-Golgi network (TGN) and in lysosomes which stained with immunogold particles using affinity-purified anti-human alpha-Gal A antibodies. Pulse-chase studies revealed that approximately 65% of the total enzyme synthesized was secreted, while endogenous CHO lysosomal enzymes were not, indicating that the alpha-Gal A secretion was specific. The recombinant intracellular and secreted enzyme forms were normally processed and phosphorylated; the secreted enzyme had mannose-6-phosphate moieties and bound the immobilized 215-kD mannose-6-phosphate receptor (M6PR). Thus, the overexpressed enzyme's selective secretion did not result from oversaturation of the M6PR-mediated pathway or abnormal binding to the M6PR. Of note, the secreted alpha-Gal A was sulfated and the percent of enzyme sulfation decreased with increasing amplification, presumably due to the inaccessibility of the enzyme's tyrosine residues for the sulfotransferase in the TGN. Overexpression of human lysosomal alpha-N-acetylgalactosaminidase and acid sphingomyelinase in CHO cell lines also resulted in their respective selective secretion. In vitro studies revealed that purified secreted alpha-Gal A was precipitated as a function of enzyme concentration and pH, with 30% of the soluble enzyme being precipitated when 10 mg/ml of enzyme was incubated at pH 5.0. Thus, it is hypothesized that these overexpressed lysosomal enzymes are normally modified until they reach the TGN where the more acidic environment of this compartment causes the formation of soluble and particulate enzyme aggregates. A significant proportion of these enzyme aggregates are unable to bind the M6PR and are selectively secreted via the constitutive secretory pathway, while endogenous lysosomal enzymes bind the M6PRs and are transported to lysosomes.


Regulation of Cu,Zn-superoxide dismutase in bovine pulmonary artery endothelial cells. To evaluate the regulation of endothelial cell Cu,Zn-SOD, we have exposed bovine pulmonary artery endothelial cells in culture to hyperoxia and hypoxia, second messengers or related agonists, hormones, free radical generating systems, endotoxin, and cytokines and have measured Cu,Zn-SOD protein of these cells by an ELISA developed in our laboratory. Control preconfluent and confluent cells in room air contained 196 +/- 18 ng Cu,Zn-SOD/10(6) cells. A23187 (0.33 microM), forskolin (10 microM), isobutylmethylxanthine (0.1 mM), dexamethasone (1 microM), triiodothyronine (1 microM) and retinoic acid (1 microM) failed to alter this level of Cu,Zn-SOD. Exposure to anoxia and hyperoxia both elevated the level approximately 1.5-2.0-fold over 20% oxygen-exposed controls at 48-72 hr. Similarly, exposures to glucose oxidase (0.0075 units/ml), menadione (12.5 microM), xanthine-xanthine oxidase (10 microM, 0.03 units/ml) and H2O2 (0.0005%) increased the level up to two-threefold over controls at 24-48 hr. Lipopolysaccharide, TGF beta 1, TNF alpha, and Il-1 also increased levels of cellular Cu,Zn-SOD, but only in proliferating cells. Il-2, Il-4, interferon-gamma, and GM-CSF had no effect on Cu,Zn-SOD. All treatments that elevated SOD resulted in inhibition of cellular growth, but decreased growth of cells at confluence alone was not associated with increased Cu,Zn-SOD. We propose from these studies that Cu,Zn-SOD of endothelial cells is not under conventional second messenger or hormonal regulation, but that up-regulation of the enzyme is associated with (and perhaps stimulated by) free-radical or oxidant production that also may be influenced by availability of certain cytokines under replicating conditions.


Establishment of a Chinese hamster ovary cell line that expresses grp78 antisense transcripts and suppresses A23187 induction of both GRP78 and GRP94. GRP78, a 78,000 dalton protein residing in the endoplasmic reticulum, is postulated to play important roles in protein folding and cell survival during calcium and other physiological stress. Here we describe the construction of an eukaryotic expression vector for the constitutive expression of grp78 antisense RNA and the creation of a CHO cell line, 78WO, which expresses high levels of the grp78 antisense RNA through amplification of the stably transfected antisense vector. We observed that whereas 78WO maintains a basal level of GRP78 similar to that of control cells, GRP78 is no longer inducible by A23187. The 78WO cells have undergone a compensatory increase in grp78 transcription such that the effects of antisense are cancelled out at the protein level under nonstressed conditions. In these same cells, GRP94, a 94,00 dalton ER protein, is also rendered noninducible by A23187. This provides the first evidence that the regulation of two ER proteins might be coupled such that the failure to induce GRP78 results in the down-regulation of GRP94. The 78WO cell line grows with a doubling time of about 26 hr and exhibits decreased tolerance to A23187, suggesting the GRPs contribute to cell viability under calcium stress. The establishment of this cell line, which can be stably maintained, will provide a useful tool for testing whether the induction of the GRPs is important for protein folding or transport and whether their enhanced synthesis is the cause or consequence of a variety of physiological adaptations.


Rapid method for automated on-line extraction and fractionation of plasma leukotrienes and 12-hydroxy-5,8,10,14-eicosatetraenoic acids by reversed-phase high-performance liquid chromatography. A method is described for automated on-line extraction and fractionation of plasma leukotrienes (LTs) and (5Z,8Z,10E,14Z)-(12S)-hydroxy-5,8,10,14-eicosate traenoic acid [12(S)-HETE] by reversed-phase high-performance liquid chromatography (RP-HPLC). This method was utilized to assess the accuracy of leukotriene B4 (LTB4) and leukotriene C4 (LTC4) determinations obtained by direct immunoassay of guinea pig plasma. Plasma LTB4 levels were significantly higher (p less than 0.01) and plasma LTC4 levels were unchanged when immunoassays were performed post versus pre RP-HPLC fractionation. Rapid separation, high recovery and baseline separation of LTB4, LTC4 and 12(S)-HETE, and minimal hardware requirements clearly demonstrate the general utility of this method.


Preparative isolation of angiotensin-converting enzyme from human lung. Angiotensin-converting enzyme from human lung was purified to apparent homogeneity using a five-step purification procedure consisting of ammonium sulfate precipitation, ion-exchange chromatography on DEAE Sephadex A-50, gel permeation on Sephadex G-200, chromatofocusing on a polybuffer exchange (PBE 94) column and high-performance liquid chromatographic gel permeation on a Bio-Sil TSK-250 column. This procedure gave an approximately 700-fold purification with a 20% yield compared to a 550-fold purification and a 1% yield with an affinity chromatography-based procedure. The 20-fold greater yield of the five-step procedure offers a major advantage for preparative use in the structural characterization of angiotensin-converting enzyme.


Complementary use of counter-current chromatography and preparative reversed-phase high-performance liquid chromatography in the separation of a synthetic mixture of brominated tetrachlorofluoresceins. A synthetically prepared mixture of brominated 4,5,6,7-tetrachlorofloresceins was separated by a combination of preparative reversed-phase high-performance liquid chromatography and high-speed counter-current chromatography. Two new lower-brominated subsidiary colors of D&C Red Nos. 27 and 28 (phloxine B), 4',5'-dibromo-4, 5,6,7-tetrachlorofluorescein and 2',4',5'-tribromo-4,5,6, 7-tetrachlorofluorescein, were isolated and characterized by 1H NMR and chemical ionization mass spectrometry.


The effect of 17 alpha-hydroxyprogesterone caproate/oestradiol valerate on the development and outcome of early pregnancies following in vitro fertilization and embryo transfer: a prospective and randomized controlled trial. A prospective and randomized study was performed to investigate the effect of supportive hormones on the development and outcome of early pregnancies following in vitro fertilization and embryo transfer. Immediately after pregnancies were confirmed endocrinologically on day 15 after oocyte collection, 17 alpha-hydroxyprogesterone caproate and oestradiol valerate (PC/EV) was administered to half of the patients in a randomly controlled trial. One group received supportive treatment until the twelfth week of gestation, whereas the other group received no treatment at all. Both groups were followed up by measurements of serum levels of human chorionic gonadotrophin (HCG), oestradiol and progesterone every three days until day 35, when a first ultrasound examination was carried out to confirm fetal vitality. Subsequently, pregnancies were monitored sonographically at regular intervals. In cases of miscarriage, dilatation and curettage was performed and histology analysed. A total of 120 pregnant patients entered the study: 55 of them received hormone treatment and 65 patients were controls. Within the treatment group 48 (89%) clinically ongoing pregnancies were observed, five (9%) miscarriages occurred after the seventh week of gestation and one preclinical pregnancy was noted. In the control group, 38 (59%) ongoing pregnancies were observed, nine (14%) miscarriages occurred and 17 (27%) preclinical pregnancies were recorded. Two ectopic pregnancies, one in each group, were excluded from evaluation. A significantly higher percentage of pregnancies were intact at 7 weeks of gestation after treatment with PC/EV (p less than or equal to 0.01).


The use of gonadotrophin releasing hormone agonists for in vitro fertilization and other assisted procreation techniques: experience from three centres. Gonadotrophin releasing hormone (GnRH) agonists have had a profound impact on infertility treatment by in vitro fertilization and related procreation techniques. Its combination with human menopausal gonadotrophin (HMG) for superovulation has increased the success rate in patients with premature luteinizing hormone (LH) surges, a poor response, endometriosis or polycystic ovarian disease. Although most in vitro fertilization (IVF) centres routinely use GnRH agonists for reasons of a better planning of activities, its superiority as a first-line treatment over the more conventional regimes of clomiphene citrate (CC)/HMG or HMG alone is still debated. An increase of the number and maturity of recovered oocytes was reported by most groups, but an inverse relationship between the number of oocytes and embryo quality and pregnancy rate after cryopreservation has been frequently mentioned. The possible complications of the combined use of a GnRH agonist with HMG include the formation of ovarian 'follicle' cysts and ovarian hyperstimulation syndrome; luteal phase defects are also common. Routine luteal phase support with progesterone and oestradiol valerate or human chorionic gonadotrophin (HCG) is required. GnRH agonists have proved to be safe drugs, side-effects are minor and sporadic and the congenital malformation rate in GnRHa/HMG treatment is comparable to the spontaneous rates reported in national IVF registers.


A 4-year controlled clinical study into the use of a ceramic hydroxylapatite implant material for the treatment of periodontal bone defects. 10 patients with chronic adult periodontitis who had greater than 1 tooth with infra-bony pockets were treated at the test defects by periodontal flap procedures with implantation of hydroxylapatite particles; the control defects were treated by the same surgical procedures but without the implant. A total of 58 test defects and 59 control defects were treated. Each defect had measurements carried out at given sites on the involved tooth surfaces, the sites being considered for subsequent tabulation purposes under the category of shallow (less than 3 mm) moderate (3-6 mm) and deep (greater than 6 mm) initial pocket depths. There were 146 and 152 shallow sites, 216 and 241 moderate sites and 140 and 133 deep sites, at test and control sites, respectively. Measurements of recession, probing pocket depths and probing attachment levels were made at 6 months and 1, 2, 3 and 4 years. At all sites over the period of the study, for the moderate and deep initial pockets there was a significant reduction in probing depths and an increase in the probing attachment levels. At the 4th year of assessment for the initially deep pockets, the reduction in probing depths was significantly greater for the sites treated with the implant material. In view of the difficult clinical problem posed by the treatment of teeth with deeper periodontal bone defects, further research using either this type of implant material or similar material should be considered.


Comparative clinical and microbiological effects of topical subgingival application of metronidazole 25% dental gel and scaling in the treatment of adult periodontitis. The aim of the study was to compare the clinical and microbiological effects of topical application of a metronidazole gel and a single session of subgingival scaling in the treatment of adult periodontitis. An open, randomized controlled clinical study design was employed. Each of 24 subjects received the 2 treatments simultaneously each in 2 randomly selected quadrants of the dentition. The metronidazole, 25% gel was applied subgingivally on days 0 and 7. Scaling was carried out in one quadrant on day 0 and in one quadrant on day 7. Clinical and microbiological examinations were carried out before treatment and on days 21, 49, 91, 133, and 175 of the experimental period. The microbiological analyses included determination of total anaerobically cultivable bacteria, and relative proportions of anaerobes, aerobes, black-pigmented anaerobic Gram negative rods, Actinobacillus actinomycetemcomitans, streptococci, and spirochetes. Both treatments were effective in reducing probing pocket depth and bleeding on probing. Metronidazole tended to be a little better than scaling during the study period and the clinical effects of both treatments persisted during the whole 6 months observation period. Local metronidazole treatment induced a significant and long-lasting shift in the subgingival flora towards a composition more compatible with health and comparable to that obtained by mechanical debridement. Proportions of black pigmented anaerobes including Prevotella intermedia, and the number of spirochetes were significantly reduced after both treatments with a concomitant increase in the proportions of streptococci. While scaling resulted in a statistically significant increase in the proportion of A. actinomycetemcomitans, this was avoided after metronidazole treatment.(ABSTRACT TRUNCATED AT 250 WORDS)