Differentiation of renal Na(+)-K(+)-ATPase in control and streptozotocin-induced diabetic mice by G-protein acting toxins and phorbol esters. The specific activity of Na(+)-K(+)-ATPase in the renal medulla and cortex of 50-day-old streptozotocin (STZ)-induced diabetic mice was increased 58% and 50%, respectively, as compared to controls. Km values of Na+ and K+ for this enzyme were unaltered, while that of ATP was decreased in diabetic mice. The Na(+)-K(+)-ATPase in control medulla and cortex was activated by both cholera and pertussis toxins, while this effect was abolished in diabetics. Since dibutyryl cAMP stimulates cortical Na(+)-K(+)-ATPase activity in control mice, the activation effect of cholera toxin on this enzyme might be due to its interaction with a Gs-protein and the persistent stimulation of adenylate cyclase activity, while the effect of pertussis toxin might be due to its masking of the inhibitory action of a Gi-protein on adenylate cyclase activity. However, the protein kinase C (PKC)-associated Na(+)-K(+)-ATPase might also be quiescent in diabetes, because the stimulating effect of phorbol 12,13-dibutyrate (PDBu) and phorbol 12-myristate 13-acetate (PMA) on this enzyme was abolished in diabetic cortex. In addition, nicardipine and ouabain were found to have differential effects on this enzyme derived from control and diabetic mice.


Thrombin is the major serum factor stimulating phosphoinositide turnover, but not DNA synthesis in human neuroblastoma SH-EP cells. Fetal calf serum stimulates both phosphoinositide turnover and DNA synthesis in SH-EP cells. The phosphoinositide turnover-stimulating activity of serum is largely (70%) reduced in the presence of hirudin, a blocker of thrombin activity. Yet, hirudin does not alter the ability of serum to stimulate DNA synthesis. Purified alpha-thrombin is a potent (EC50, 35 pM) stimulator of phosphoinositide turnover in SH-EP cells, but induces DNA synthesis only at much higher concentrations (10 nM-1 microM). Thus, serum thrombin accounts for most of the ability of serum to stimulate phosphoinositide hydrolysis, but not for the effect of serum on cell division, since the concentration of thrombin in serum is not sufficient to induce DNA synthesis. These data suggest that hydrolysis of inositol lipids may not be the main signalling event mediating the mitogenic effects of alpha-thrombin.


Effects of subunit types of the recombinant GABAA receptor on the response to a neurosteroid. When vertebrate brain poly(A)+ RNA is expressed in Xenopus oocytes the response of the GABA receptors formed is found to be inhibited allosterically by a neurosteroid, pregnenolone sulphate (PS). This negative modulation was reproduced after expressing RNAs encoding bovine GABAA receptor subunits in the combinations alpha i + beta 1, or alpha i + beta 1 + gamma 2 (where i = 1, 2 or 3). The characteristics of this inhibition vary significantly with the type of the alpha subunit (alpha 1, alpha 2, or alpha 3) used. When the bovine gamma 2L alternate form of the gamma 2 subunit was replaced by the human gamma 2S subunit, the behaviour was unchanged: the human gamma 2S subunit used is a newly-cloned form, which encodes a polypeptide with two amino acid differences from the human gamma 2 subunit previously described. The results of co-application of PS and 3 alpha-hydroxy-5 alpha-pregnan-ol-20-one, a neurosteroid which is a positive modulator of the GABAA receptor, indicate that these act at different sites on the receptor. PS also increases the desensitisation of the receptor by GABA. This effect, also, is alpha-subunit-type dependent and occurs by an acceleration of the fast phase of desensitisation.


Endothelin-elicited stimulation of phospholipase C is mediated by guanine nucleotide binding protein(s). In permeabilized C6 glioma cells and NIH 3T3 cells, the peptide endothelin 1 (ET-1) in combination with GTP gamma S stimulates the formation of inositol phosphates. In the presence of 10 microM GTP gamma S, ET-1 induces the formation of inositol phosphates with an EC50 value of 2.5 nM for C6 glioma cells and 1.6 nM for NIH 3T3 cells. The analogous peptide endothelin 3 (ET-3) is less potent than ET-1 in such action. In NIH 3T3 cells, ET-1+GTP gamma S-induced formation of inositol phosphates could be detected after 1 min of stimulation, and it increased for up to 30 min. ET-1-induced effects were partially reduced by pretreatment of the cells with pertussis toxin (1 microgram/ml) in C6 glioma cells, but were unaffected in NIH 3T3 cells. In binding studies in whole C6 cells and NIH 3T3 cells, specific binding for [125I]ET-1 was detected. Cross-linking of [125I]ET-1 in whole C6 cells revealed the presence of two binding proteins for ET-1 of 74 kDa and 55 kDa. ET-1 at 100 nM inhibited the labeling of both proteins by [125I]ET-1. However, ET-3 inhibited the labeling of the 55 kDa protein only. The results provide direct evidence for endothelin receptor coupling to phospholipase C through guanine nucleotide binding (G) proteins. In addition, in C6 cells, endothelin-mediated phospholipase C activation is partially inhibited by pertussis toxin pretreatment. The endothelin receptor involved in phospholipase C stimulation in C6 cells seems to correspond to a 74 kDa protein which binds ET-1 but not ET-3.


The selective endothelin ETA receptor antagonist BQ123 antagonizes endothelin-1-mediated mitogenesis. The mitogenic effects of endothelin isopeptides and the selective ETA receptor antagonist BQ123 were evaluated in rat aortic vascular smooth muscle cells. Endothelin-1 (ET-1) and endothelin-3 (ET-3) produced concentration-dependent increases in [3H]thymidine incorporation (EC50 = 0.1 and 25 nM, respectively). The ETB-selective agonist sarafotoxin 6c did not produce significant effects on [3H]thymidine incorporation. BQ123 produced selective and concentration-dependent inhibition of ET-1-mediated [3H]thymidine incorporation. These data demonstrate that ET-1-mediated mitogenesis in vascular smooth muscle is mediated by ETA receptors.


A newly found angiotensin II receptor subtype mediates cyclic GMP formation in differentiated Neuro-2A cells. In search of the functional role of the newly found angiotensin II (Ang II) binding site which is expressed in differentiated Neuro-2A cells, we found that Ang II causes a marked stimulation of cGMP formation dose-dependently. The stimulation was blocked by the nonselective Ang II receptor antagonist [Sar1,Ile8]Ang II but not by the AT1 antagonist DuP 753 or the AT2 antagonist PD 123319. These results suggest that Ang II increased cGMP level via a new Ang II receptor subtype in differentiated Neuro-2A cells.


Trans-ACPD inhibits cAMP formation via a pertussis toxin-sensitive G-protein. In primary cultured striatal neurons we found that (+-)-trans-1-amino-cyclopentyl-1,3-dicarboxylate (trans-ACPD) could inhibit forskolin-induced cAMP formation in a dose-dependent manner (EC50 156 +/- 38 microM, n = 5, maximal inhibition 37.8 +/- 1.2, n = 37). The trans-ACPD-induced inhibition was totally abolished in neurons preincubated with Bordetella pertussis toxin (1 microgram/ml), demonstrating the involvement of a G-protein. This is the first report in intact neurons of a glutamate metabotropic receptor negatively coupled to cAMP formation.


Evidence for adenosine A2b receptors in the rat pineal gland. We describe the effects of 5'-N-ethylcarboxamidoadenosine (NECA), a mixed A2a/A2b adenosine receptor agonist and 2-[p-(carboxyethyl)-phenylethylamino]-5'-N-ethylcarboxamidoaden osin e (CGS 21680), a selective A2a agonist, on cyclic AMP and N-acetylserotonin synthesis in rat pineal gland. NECA, 1 and 10 microM, increased cyclic AMP by 5- and 25-fold and N-acetylserotonin by 40- and 60-fold respectively, whereas CGS 21680 at the same concentrations was ineffective. These results argue for the presence of adenosine A2b receptors in rat pinealocytes.


Salivary antibody testing in a school outbreak of hepatitis A. During a community-wide outbreak of hepatitis A in Gloucester, UK there was a high attack rate in children attending two city primary schools and a pre-school centre sharing the same site. In September 1990, saliva specimens were collected from 478 (85%) of the 562 children. The prevalence of antibody to hepatitis A virus (anti-HAV), as determined by saliva testing, was 29.6%; highest prevalences were seen in 5-6-year-olds and in children from that area of the city at the centre of the community-wide outbreak. The proportion of immune children with a history of clinical hepatitis varied with age from 1 in 42.7 of under-5-year-olds to 1 in 4.7 of 8-10-year-olds. Six children who received prophylaxis with human normal immune globulin (HNIG) because they were household contacts of cases subsequently became infected. Since there was evidence of transmission outside the school environment it is unlikely that a policy of universal prophylaxis within the schools would have stopped the outbreak. Mass prophylaxis in school outbreaks is only likely to be effective if most transmission is occurring at school and if the target population can be clearly defined. Salivary antibody testing is a simple, practical and acceptable procedure in young children. Salivary antibody surveys in conjunction with vaccination against hepatitis A should provide a cost-effective method for control of future outbreaks.


Glucose deprivation elicits neurofibrillary tangle-like antigenic changes in hippocampal neurons: prevention by NGF and bFGF. A decrement in glucose utilization in brain was previously demonstrated in Alzheimer's disease (AD) and this abnormality has been proposed to play a role in the process of neuronal degeneration. We now report that glucose deprivation in cultured hippocampal neurons can result in antigenic alterations similar to those seen in AD neurofibrillary tangles (NFT) and, ultimately, cell death. Hypoglycemia caused an increase in neuronal immunoreactivity toward tau and ubiquitin antibodies. The antigenic alterations resulted from hypoglycemia-induced elevations in intracellular calcium levels as measured using the calcium indicator dye fura-2. The increased intraneuronal calcium levels, increased tau and ubiquitin immunoreactivities, and neuronal damage resulted from influx through the plasma membrane since they were not observed when cells were incubated in calcium-deficient medium. Neuronal damage and NFT-like antigenic changes were completely prevented by nerve growth factor (NGF) and basic fibroblast growth factor (bFGF), but not by epidermal growth factor (EGF). NGF and bFGF, but not EGF, prevented the aberrant rise in intracellular calcium levels that normally resulted from glucose deprivation. These data are consistent with the possibility that reduced glucose availability to neurons may contribute to the neuronal degeneration that occurs in AD. They also suggest that growth factors may normally protect neurons against hypoglycemic damage.


Three-dimensional structure of (1-36)bacterioopsin in methanol-chloroform mixture and SDS micelles determined by 2D 1H-NMR spectroscopy. Spatial structures of proteolytic segment A (sA) of bacterioopsin of H. halobium (residues 1-36) solubilized in a mixture of methanol-chloroform (1:1), 0.1 M LiClO4 organic mixture, or in perdeuterated sodium dodecyl sulfate (SDS) micelles, were determined by 2D 1H-NMR techniques. 324 and 400 NOESY cross-peak volumes were measured in NOESY spectra of sA in organic mixture and SDS micelles, respectively. The sA spatial structures were determined by local structure analysis, distance geometry calculation with program DIANA and systematic search for energetically allowed side chain rotamers consistent with NOESY cross-peak volumes. The structures of sA are similar in both milieus and have the right-handed alpha-helical region from Pro8 to Met32 with root mean square deviation (RMSD) of 0.25 A between backbone heavy atoms and fit well with Pro8 to Met32 alpha-helical region in electron cryo-microscopy model of bacteriorhodopsin. The N-terminal region Ala2-Gly6 of sA in organic mixture has a fixed structure of two consecutive gamma-turns as 2 * 2(7)-helix (RMSD of 0.25 A) stabilized by the Thr5 NH...O = C Gln3 and Ile4 NH...O = C Ala2 hydrogen bonds while this region in SDS micelles has disordered structure with RMSD of 1.44 A for backbone heavy atoms. The C-terminal region Gly33-Asp36 of sA is disordered in both milieus. Torsion angles chi 1 of sA were unequivocally determined for 13 (SDS) and 11 (organic mixture) of alpha-helical residues and are identical in both milieus.


Implication of brain cdc2 and MAP2 kinases in the phosphorylation of tau protein in Alzheimer's disease. Brain tau protein is phosphorylated in vitro by cdc2 and MAP2 kinases, obtained through immunoaffinity purification from rat brain extracts. The phosphorylation sites are located on the tau molecule both upstream and downstream of the tubulin-binding motifs. A synthetic peptide comprising residues 194-213 of the tau sequence, which contains the epitope recognized by the monoclonal antibody tau-1, is also efficiently phosphorylated in vitro by cdc2 and MAP2 kinases. Phosphorylation of this peptide markedly reduces its interaction with the antibody tau-1, as it has been described for tau protein in Alzheimer's disease. Both cdc2 and MAP2 kinases are present in brain extracts obtained from Alzheimer's disease patients. Interestingly, the level of cdc2 kinase may be increased in patient brains as compared with non-demented controls. These results suggest a role for cdc2 and MAP2 kinases in phosphorylating tau protein at the tau-1 epitope in Alzheimer's disease.


[Secretion of endogenous regulators of Na K ATPase by isolated rat duodenum in osmotic stimulation of its mucous membrane]. The experiments in vitro have shown that perfusion of the isolated duodenum cavity in rats by hypotonic (20 mosmol/l H2O) or hypertonic (500 mosmol/l H2O) solutions of NaCl and mannitol during 10 min stimulates secretion of endogenic factors (activators or inhibitors of enterocytes of Na+, K(+)-ATPase) into the serous incubation solution. Contact of the duodenum mucose surface with isotonic (300 mosmol/l H2O) solutions does not stimulate the secretion of this enzyme regulators. Endogenic substance are thermostable: they retain activity relative to Na+, K+ ATPase of enterocytes after heating (100 degrees C, 10 min duration) of serous incubation solutions.


[Characteristics of potential-dependent calcium channel current of the secretory cell membrane]. The parameters of inward current of potential-dependent calcium channels in cells of the upper and lower lobes of the salivary glands in Chironomus larvae have been studied. The current activation thresholds in cells of both types are near -60 mV, but in cells of the upper lobe the current reaches its maximum values at -20 mV, while in cells of the lower lobe at -10 mV. The current density in cells of the upper lobe is approximately 1.3 times higher and the inactivation time constant is 1.62 less than in cells of the lower lobe. Calcium channels in cells of the upper and lower lobes of glands also differ in metabolic dependence: introduction of cAMP, ATP and Mg2+ into cells of the lower gland lobe causes a considerable increase in the current amplitude, while the same action in cells of the upper lobe--its decrease. The current amplitude in the cells of the lower lobe decreases under the influence of nitrendipine and La, Co, Ni, Cd, Zn cations as well as a decrease of the temperature or pH in the external solution.


Glucose transporter number, function, and subcellular distribution in rat skeletal muscle after exercise training. Endurance exercise training can result in increased rates of insulin-stimulated glucose uptake in skeletal muscle; however, this effect may be lost rapidly once training ceases. To examine a mechanism for these changes, the skeletal-muscle glucose transport system of female rats exercise-trained in wheelcages for 6 wk were studied against a group of untrained female rats. The trained rats were studied immediately following and 2 and 5 days after removal from wheelcages; both trained and untrained rats were studied 30 min after insulin (90 nmol/rat, intraperitoneal) or saline injection. The total number of skeletal-muscle plasma-membrane glucose transporters (R0), total muscle-homogenate and plasma-membrane GLUT4 protein, and rates of plasma-membrane vesicle D-facilitated glucose transport were higher in the exercise-trained rats immediately after exercise training and did not decrease significantly during the 5 days after cessation of training. On the other hand, exercise training did not alter microsomal-membrane total glucose-transporter number or GLUT4 protein, nor did training alter GLUT1 protein in total muscle homogenates nor either membrane fraction. The carrier-turnover number, an estimate of average functional activity of glucose transporters in the plasma membrane, was elevated slightly, but not significantly, in the trained muscle. In both the trained and untrained muscle, insulin administration resulted in translocation of glucose transporters from the microsomal-membrane fraction to the plasma membrane and an increase in the carrier-turnover number.(ABSTRACT TRUNCATED AT 250 WORDS)


Generation of reactive oxygen metabolites by the haemocytes of the mussel Mytilus edulis. Generation of superoxide anion by stimulated haemocytes of Mytilus edulis was demonstrated using dihydrorhodamine 123 and quantified using reduction of nitroblue tetrazolium (NBT). In the presence of zymosan or phorbol myristate acetate, there was an increased reduction of NBT to formazan. The addition of superoxide dismutase (SOD) and iodoacetamide to the incubation medium resulted in a significant reduction in deposition of reduced formazan. Incubation of haemocytes with the SOD inhibitor diethyldithiocarbamate (DDC) gave rise to a small but significant increase in NBT reduction. The production of hydrogen peroxide by haemocytes was quantified using horseradish peroxidase-dependent oxidation of phenol red. The presence of SOD in the incubation medium together with zymosan resulted in a significant increase in H2O2 production. Haemocytes incubated with DDC prior to the assay or with sodium nitroprusside during the assay showed a decrease in H2O2 production with increasing concentration of the inhibitor.


Elevated plasma concentrations of beta-cell tropin (ACTH22-39) in diet-treated type II diabetic patients. beta-Cell tropin, the pituitary peptide ACTH22-39, is a potent insulin secretagogue and stimulates lipogenesis in adipose tissue in rodents. Plasma beta-cell tropin was measured fasting and after glucose infusion (5 mg.kg glucose ideal body weight-1.min0-1 for 90 min) in 10 mild diet-treated non-insulin-dependent (type II) diabetic subjects and 10 control subjects (body mass index) (BMI): 26.4 +/- 3.2 and 24.1 +/- 2.0 kg/m-2, NS, fasting plasma glucose 7.8 +/- 2.7 mM and 4.7 +/- 0.3 mM, respectively). The diabetic subjects had raised fasting plasma beta-cell tropin compared with the normal subjects (geometric mean (1 SD range): 0.49 (0.25-0.96) nM and 0.17 (0.10-0.28) nM, respectively, P = 0.007). In response to the glucose infusion, plasma glucose rose higher in the diabetic subjects (mean +/- 1 SD: 13.7 +/- 3.1 and 9.6 +/- 0.9 mM, P = 0.007) and plasma insulin was impaired in the diabetic compared with the nondiabetic subjects (geometric mean (1 SD range): 14 (8-26) and 34 (18-63), P less than 0.01). beta-Cell tropin concentrations in the diabetic subjects rose to 1.31 (0.74-2.30) nM (P = 0.007), whereas beta-cell tropin did not change in the normal subjects at 0.19 (0.11-0.91) nM. There was no overlap between glucose-stimulated plasma beta-cell tropin in the two groups (P = 0.0002). Pituitary-adrenal function, as assessed by plasma cortisol, did not differ between the two groups when fasting and did not change after the glucose infusion.(ABSTRACT TRUNCATED AT 250 WORDS)


cDNA cloning of the constant region genes of the guinea pig alpha/beta T-cell receptor. A lambda gt11 cDNA library was prepared from activated guinea pig T-lymphocyte blasts. cDNA clones coding for the alpha-chain and beta-chain of the constant region of the guinea pig T-cell receptor were isolated by means of crosshybridization using the corresponding mouse cDNA genes. The guinea pig and the corresponding mouse cDNA genes hybridized in Northern blots with mouse mRNA of the same size. Guinea pig T-cell receptor mRNA showed the same size like its mouse counterpart. In contrast to the alpha-chain clone, genomic Southern blot analysis showed that the identified beta-chain gene fragment undergoes genomic rearrangement during T-cell differentiation.


High expression of stathmin in multipotential teratocarcinoma and normal embryonic cells versus their early differentiated derivatives. Stathmin is a ubiquitous cytoplasmic protein, phosphorylated in response to agents regulating the proliferation, the differentiation and the specialized functions of cells, in a way possibly integrating the actions of diverse concomitant regulatory signals. Its expression is also regulated in relation with cell proliferation and differentiation and reaches a peak at the neonatal stage. To assess the possible role of stathmin at earlier stages of development, we examined its expression and regulation in embryonal carcinoma (EC) and derived cell lines as well as in the early mouse embryo. Interestingly, stathmin is highly abundant in the undifferentiated, multipotential cells of the F9, 1003 and 1009 EC cell lines. Its high expression markedly decreased, both at the protein and mRNA levels, when F9 cells were induced to differentiate into endodermal-like cells with retinoic acid and dibutyryl-cAMP. Stathmin was also much less abundant in differentiated cell lines such as the trophectodermal line TDM-1, as well as in several F9- and 1003-derived cell lines committed to differentiate towards the mesodermal and neuroectodermal lineages but still proliferating. Therefore, the observed decrease of stathmin expression is not related to the reduced proliferation rate but rather to the differentiation of the multipotential EC cells. The immunocytochemical pattern of stathmin expression during early mouse development indicated that stathmin is also highly abundant in the multipotential cells of the inner cell mass of the blastula, whereas it is much lower in the differentiated trophectodermal cells. These results confirm the physiological relevance of the observations with EC cells, and suggest that stathmin, in addition to its high expression at later stages of development and in the adult nervous system, may be considered as a new marker of the multipotential cells of the early mouse embryo.


Stimulation of rat pancreatic tumoral AR4-2J cell proliferation by pituitary adenylate cyclase-activating peptide. In the present work the effects of the novel neuropeptide Pituitary Adenylate Cyclase Activating Peptide (PACAP) on both AR4-2J cell growth and the modulation of ornithine decarboxylase activity were investigated. Both PACAP38 and the amidated form PACAP27 caused a concentration-dependent stimulation of AR4-2J cell growth; the maximal increase was seen at 1 nmol/L (30% above control, P less than 0.01) with a half-maximal effect at 0.01 nmol/L. Ornithine decarboxylase activity was also increased by PACAP in a dose-dependent manner, reaching half-maximal stimulation at 0.5 nmol/L. The addition of 1 nmol/L of somatostatin analog SMS 201-995 totally suppressed PACAP-stimulated AR4-2J cell growth. Vasoactive intestinal polypeptide (3 mumol/L) and 8-bromo-cyclic adenosine monophosphate (1 mmol/L) had no effect on cell proliferation. Treatment of cells by pertussis toxin (25 ng.mL-1.day-1) suppressed PACAP-stimulated AR4-2J cell growth but enhanced PACAP-induced stimulation of adenylate cyclase activity. It was concluded that PACAP stimulates AR4-2J cell proliferation by a mechanism that seems independent of cyclic adenosine monophosphate production. The mitogenic effect of PACAP depends on a pertussis toxin-sensitive G protein and is associated with an increase of ornithine decarboxylase activity.


Polymerase chain reaction to detect human cytomegalovirus in livers of infants with neonatal hepatitis. Neonatal hepatitis is closely related to human cytomegalovirus infection in Taiwan, a conclusion based on serological and urine culture studies. To obtain more direct evidence further relating cytomegalovirus to the pathogenesis of neonatal hepatitis, the cytomegalovirus genome was studied in the liver tissues of 50 infants with neonatal hepatitis using the polymerase chain reaction (PCR). Liver tissues from 26 infants with biliary atresia and another 30 infants and children with diagnoses other than neonatal hepatitis, cholestasis, or hepatitis were also studied for comparison. Sequences from the immediate early gene 1 and 2 regions were used as primers. The liver tissues from 23 (46%) of the 50 infants with neonatal hepatitis were positive for cytomegalovirus genome, whereas those of 2 of the 26 infants with biliary atresia and none of the liver tissues from 30 infants and children without neonatal hepatitis were positive for cytomegalovirus genome, by PCR. The results of PCR correlated well with that of serology and urine culture. This study provides further evidence of cytomegalovirus in the pathogenesis of neonatal hepatitis.


Ischemia/reperfusion-induced feline intestinal dysfunction: importance of granulocyte recruitment. Although previous studies have reported that neutrophils play an important role in mediating the microvascular injury observed after reperfusion of ischemic intestine, the contribution of these phagocytic cells to the mucosal dysfunction remains unclear. Three series of experiments consisting of an untreated group, a short-term monoclonal antibody (MAb) IB4 treatment group (MAb IB4 given on the day of the experiment), and a long-term MAb IB4 treatment group (3-day pretreatment with MAb IB4) were performed using autoperfused segments of cat ileum exposed to 3 hours of ischemia followed by 1 hour of reperfusion. Mucosal myeloperoxidase activity, an index of mucosal granulocyte levels, increased from 12 to 25 U/g wet wt in the untreated group. In the short-term MAb IB4 experiments, baseline values were very similar to those of the untreated group but no increase in myeloperoxidase activity was observed after ischemia/reperfusion. Long-term pretreatment with MAb IB4 reduced baseline values of myeloperoxidase activity to approximately 1 U/g wet wt; the values remained at this level throughout the experiment. The permeability of the mucosal barrier was quantitated by measuring blood-to-lumen clearance to 51Cr-ethyl-enediaminetetraacetic acid (EDTA). The water absorptive capacity of the intestine was also measured. In the untreated group, mucosal permeability to 51Cr-EDTA increased sixfold and water absorption was abolished after reperfusion. Both short-term and long-term administration of MAb IB4 prevented the net fluid loss into the lumen, but only long-term administration of MAb IB4 blunted the increased mucosal permeability induced by ischemia/reperfusion. These data suggest that interstitial granulocytes contribute significantly to the mucosal dysfunction associated with reperfusion of the ischemic intestine.


Studies of pHi in rabbit esophageal basal and squamous epithelial cells in culture. Rabbit esophageal epithelium grown in primary culture enabled the study of intracellular pH (pHi) regulation at two distinct stages in the life cycle of the epithelial cell: basal and mature squamous. pHi was measured in single basal and mature squamous cells after loading with the fluorescent probe 2',7'-bis(carboxyethyl)-5(and -6)carboxyfluorescein at 25 degrees C in a nominally bicarbonate-free HEPES buffer. The results revealed that the resting pHi was higher and the intrinsic buffer capacity lower (at pH values less than or equal to 7.6) in basal compared with mature squamous cells. In addition, both types recovered from an acid load (NH4Cl prepulse) by an Na(+)-dependent, amiloride-inhibitable process consistent with an Na+/H+ antiporter. However, hydrogen ion extrusion rates by the Na+/H+ antiporter, even after taking into account buffer capacity and acid loading, were two to four times as fast for basal as for mature squamous cells. Further, mature squamous cell but not basal cell recovery from an acid load deteriorated with time (3 weeks) in culture. These results establish the use of primary cultures for studying pHi regulation at different stages in the epithelial cell life cycle and document that basal and mature squamous cells show Na+/H+ antiport activity for extrusion of an acid load but that this activity diminishes in effectiveness as the cell matures.


A study of autoimmune gastritis in the postpartum period and at a 5-year follow-up. The presence of autoimmune gastritis was investigated in 54 women with postpartum thyroiditis. Parietal cell antibodies (PCA) specific against H+, K(+)-adenosine triphosphatase (EC 3.6.1.36) were found in 18 women during pregnancy; in 10 of them, a 2-9-fold increase in the PCA level was observed in the postpartum period. At a 5-year follow-up, the initially PCA-positive women still had elevated antibody levels. Hypergastrinemia and low pepsinogen levels were noted in 4 women. In 2 of these women low serum vitamin B12 levels had developed. In 6 of 9 PCA-positive women examined by gastroscopy, biopsy specimens from the gastric body mucosa contained mononuclear cells, mainly T lymphocytes (CD3+) and macrophages (Leu-M3+) combined with an aberrant epithelial expression of HLA-DR. In four patients with chronic gastritis, all parietal cells, as defined by a specific monoclonal antibody, were found to have immunoglobulin G (IgG) deposits by a double-immunostaining method. Three of them had microscopic evidence of atrophy, whereas in 1 patient the body mucosa was intact. In 1 further patient with intact glands at histological examination, the basolateral membrane of some oxyntic glands was coated with IgG. The selective in situ deposition of antibodies associated with histologically intact parietal cells may support the concept that specific autoantibodies participate in the early pathogenesis of parietal cell destruction.


[Risk of osteoporosis in long-term heparin therapy of thromboembolic diseases in pregnancy: attempted prevention with ossein-hydroxyapatite]. Generalized idiopathic osteoporosis and transient osteoporosis of the hip are both rare complications of pregnancy. More frequently, long-term heparin administration to treat deep thrombosis in the legs or pelvis may lead to substantial decreases in bone mass and consequently increased risk of osteoporosis. Therapeutic studies with the aim to counteract the osteoporosis inducing effect of heparins, have not been published to date. In the special situation of pregnancy, most medications used for osteoporosis are contraindicated. In our open randomised study, 9 women on heparin-treatment received daily 6.46 g of the bone preparation OHC (ossein-hydroxyapatite-compound) over a period of 6 months and were compared to 11 women without bone protective treatment. In the OHC-group, good compliance was observed with no side effects and reduced back pain. Bone mass did not change significantly, whilst it dropped significantly statistically in the controls.


[Cervix cancer in HPV16-associated Bowenoid papulosis]. In a 38-year-old female patient with bowenoid papulosis of the anogenital region and an extensive carcinoma in situ of the cervix uteri, HPV16 DNA was found in the biopsy specimens and HPV16/18 DNA was detected in the cervical swab. The known coincidence of both these diseases indicates the necessity for careful regular gynaecological check-ups of patients with bowenoid papulosis.


The animal and human plasma membrane (Ca(2+)+Mg2+)-ATPases--approaches to molecular arrangements of functional parts and oxidative changes. The molecular structures of animal and human plasma membrane (Ca(2+)+Mg2+)-ATPases are not completely understood in part due to the fact that no suitable single crystal is available. The elucidation of the two-dimensional structure is in progress. The amino acid sequences of human erythrocyte and rat plasma membrane Ca2+ pump isoforms as well as of the pig smooth muscle plasma membrane Ca2+ pump are already known. This article reviews the present state of the knowledge in (Ca(2+)+Mg2+)-ATPase research of animal and human plasma membranes performed in the past few years, concerning in particular arrangements of proteolytically cleaved fragments, and relations between the erythrocyte (Ca(2+)+Mg2+)-ATPase in situ and the purified red cell enzyme, oxidative changes. Results of different experimental approaches concerning the structure of (Ca(2+)+Mg2+)-ATPases rather than the applications of the methods used are emphasized.


Ammonia and proton gated channel populations in trigeminal ganglion neurons. The existence of three distinct types of proton-gated sodium currents classified in accordance with their kinetics of desensitization as fast, medium and slow, has been confirmed in the present study on isolated rat trigeminal ganglion neurons. The emphasis was put on the investigation of a peculiar medium type of a chemically gated channel population, using the concentration jump method. The features of the medium-type mechanism allow to distinguish it from the other already known types: it was insensitive to the Ca antagonist verapamil (in contrast to proton-gated sodium current found in dorsal root ganglion neurons), displayed a strong dependence of the kinetics of desensitization on the membrane potential, and (besides the apparent proton-gating) was activated and desensitized by the application of ammonia containing solution at normal pH values. The effect of ammonia itself appeared to be a good tool for the separation of fast and slow proton-gated responses. The results obtained allow to postulate a nonspecific proton-activation of medium-type receptor-channel complexes and their specificity to ammonia (Kd = 10(-4) mol/l) as an agonist.


'uidA-antibiotic-resistance cassettes for insertion mutagenesis, gene fusions and genetic constructions. We have constructed a series of promoterprobe cassettes that provides powerful tools for insertion mutagenesis, transcription fusions and genetic constructions. These cassettes contain the Tn9 chloramphenicol (CmR) and the Tn903 kanamycin (KmR) resistance genes which are expressed in a large variety of microorganisms; these antibiotic-resistance markers were associated with the uidA promoterless gene. This beta-glucuronidase-encoding gene of Escherichia coli K-12 has been successfully used as reporter gene for various organisms including prokaryotes and eukaryotes. The resulting 'uidA-KmR and 'uidA-CmR cassettes (truncated at the ') can be excized with most of the commonly used restriction enzymes. Furthermore, they are borne by ApR or CmR plasmids which facilitate their utilization. These promoter-probe cassettes allow transcriptional signal localization and regulation studies.


Regulation of peripheral-type benzodiazepine receptors following repeated benzodiazepine administration. The effects of chronic benzodiazepine (BZD) treatment on rat peripheral-type benzodiazepine receptors (PBR) were studied. Five days treatment with the PBR ligands RO 5-4864 or diazepam (DZ) up-regulates kidney PBR. In contrast clonazepam, a specific central-type BZD receptor ligand, did not alter 3H-RO 5-4864 binding. Fourteen days administration of DZ produced an up-regulation of kidney and heart PBR and a down-regulation of testicular PBR. The results suggest that chronic BZD exposure differentially regulates PBR in peripheral organs.


Electrophysiological evidence for the presence of ionotropic and metabotropic excitatory amino acid receptors on dopaminergic neurons of the rat mesencephalon: an in vitro study. The actions of the ionotropic and metabotropic excitatory amino acid agonists AMPA, quisqualate, kainate, NMDA and trans-ACDP were studied by means of intracellular electrophysiological recordings from dopaminergic neurons of rat mesencephalon in brain slices. It was observed that all these agents evoked an inward current in cells which were voltage-clamped near the resting potential (-50, -60 mV). The membrane responses produced by AMPA, kainate and quisqualate were associated with an increase of the apparent input conductance while the responses induced by NMDA and trans-ACDP were associated with a decrease in the apparent input conductance. Therefore, stimulation of ionotropic and metabotropic amino acid receptors activates inward currents in the dopaminergic cells by different mechanisms.


Delta opioid receptors mediate seizures produced by intrahippocampal injection of ala-deltorphin in rats. The effects of the selective delta opioid receptor agonist, ala-deltorphin, microinfused into the dorsal hippocampus or into the locus coeruleus, on the electrocorticographic (ECoG) activity continuously quantified in its spectrum were studied in rats. The microinjection into the dorsal hippocampus of ala-deltorphin (0.03, 0.3 and 1.0 nmol) produced dose-dependent motor and ECoG epileptogenic disorders. The microinfusion of similar doses of ala-deltorphin into the locus coeruleus did not evoke motor and ECoG changes. Intrahippocampal injection of naltrindole (10 and 20 pmol) antagonized the epileptogenic effects induced by ala-deltorphin; also naloxone (20 and 50 pmol) was able to prevent the effects induced by ala-deltorphin, although with a minor potency. In conclusion, the present experiments show that ala-deltorphin is a useful tool to characterize central opioid effects mediated by delta receptors in the brain and suggest that the stimulation of this subtype of receptors in the hippocampus is responsible for epileptogenic effects of opiates.


Physiological significance of long-term potentiation. Long-term potentiation (LTP) is a form of synaptic plasticity that follows repetitive, high frequency stimulation of excitatory presynaptic fibers. It produces a long-lasting enhancement of synaptic strength that is usually revealed as an increased size of excitatory postsynaptic potentials. It is most prominent in the hippocampus, although it may be evoked in other brain regions. Use-dependent modifications in synaptic function are probably the basis of learning and memory, thus the study of LTP may represent a useful model for investigating the physiological mechanisms underlying the process of information storage.


Responses of nursing home residents to intrainstitutional relocation. Relocation effects may be positive when residents are prepared for the move. The degree to which they exercise control over their environment and participate in the decision-making process influences the outcome of relocation. Planned interventions may offset adverse effects of stress. Prerelocation involvement of the residents we observed in visiting the new facility, and in selecting their bedrooms and roommates, reduced their anxiety to an acceptable, even positive level. These choices had a direct impact on their quality of life. The greater the number of choices the residents had, the more predictable the new environment became. Any stress generated became a positive, rather than a negative, force.


LAN-1 human neuroblastoma cells are provided of endothelin-1 receptors linked to [Ca++]i elevation. Endothelin-1 (ET-1) produced a dose-dependent increase of intracellular Ca++ concentrations [Ca++]i characterized by an early peak phase and a delayed plateau in LAN-1 human neuroblastoma cells. The ET-1 receptor showed a rapid desensitization since a second pulse application of ET-1 did not elicit a further [Ca++]i increase. Furthermore thapsigargin, an endoplasmic reticulum Ca(++)-ATPase inhibitor, completely abolished the ET-1 induced intracellular Ca++ elevation.


Organization of a medications group for older patients with mental illness. A medications group for older inpatients with mental illness improved their understanding of and compliance with medication regimens.


Geriatric trauma: a case study.

Geriatric trauma: a case study. Major advances in health care have made it possible for a greater number of people to live to advanced age and thereby be exposed to the same risk of injury as the younger segment of the population. Elderly trauma victims, however, possess unique needs that must be addressed. Comorbidity, the aging process, and its relationship to the response to injury, as well as ethical and moral dilemmas such as "do not resuscitate" decisions and cost of care, need to be investigated. Although advanced age clearly predisposes one to an increase in mortality, mortality has also been correlated with complications that develop during the hospital stay. Trauma, especially involving the elderly, is a rich area for nursing research, and a refined knowledge base contributes to improving the quality of trauma care.


Small cell carcinoma with neurosecretory granules arising in an ovarian dermoid cyst. A rare small cell carcinoma with neurosecretory granules arising in an ovarian dermoid cyst with 7 years survival after conservative surgery and adjuvant chemotherapy with cisplatin, adriamycin, and cyclophosphamide is described. Light microscopy showed the typical uniformly small cells with hyperchromic nuclei and scanty basophilic cytoplasm within the mature cystic teratoma. Although none of the immunohistochemical stains were reactive, electron microscopy demonstrated the membrane-bound neurosecretory granules in some tumor cells. The related literature is reviewed and the issues concerning treatment options in this unusual malignancy are discussed.


The perfectionistic patient.

The perfectionistic patient. Family members, residents, and care givers often react negatively when perfection is demanded by the geriatric patient. This type of reaction presents a challenge to the nurse providing care. When a geriatric patient is perfectionistic, it is important to step back, to observe and assess, and to plan appropriate strategies that will facilitate the patient's adjustment. When the behavior is related to situational stressors, it may be appropriate to respond to the stressors and ignore the behavior. When the behavior results from a feeling of loss of control and powerlessness, intervention should be directed in that direction. In any case, actions of the nurse should respond to the patient's need for self-esteem and improved ability to cope with the environment. Responding to the perfectionistic geriatric patient presents a particular challenge to the nurse in long-term care, and the nurse's strategic intervention can significantly increase the patient's ability to adjust to the ongoing changes that accompany the aging process.


Ovarian serous borderline epithelial tumors with multiple retroperitoneal nodal involvement: metastasis or malignant transformation of epithelial glandular inclusions? One of four patients who underwent lymph node excision at exploration for ovarian serous borderline epithelial tumor (OSBT) at Baystate Medical Center was found to have FIGO Stage III C lesion associated with extensive ovarian external (surface) papillary growth, peritoneal implants in the omentum and cul-de-sac, and involvement of multiple pelvic and periaortic lymph nodes by the tumor. Histologically, the lymph nodes showed an admixture of endosalpingeal glandular inclusions with neoplastic tissue identical to the ovarian tumor. The exact histogenesis and the prognostic significance of the nodal involvement by OSBT are still not fully understood. Although there is a small number of reported cases of lymph node involvement associated with OSBT, they are described as examples of nodal metastases or independent primary foci of malignant transformation. This paper presents an interesting association of OSBT with extensive pelvic and periaortic nodal involvement and reviews the relevant literature.


Involvement of opioid receptors in Met-enkephalin modulation of blast-transformation of mouse splenocytes. The influence of Met-enkephalin on mitogenic stimulation of mouse splenocytes was investigated. Met-enkephalin (ME) was shown to suppress proliferation induced by Concanavalin A and activate proliferation induced by Staphylococcus enterotoxin A. Both effects were revealed at low (down to 10(-14) M) concentration of pentapeptide. Naloxone reversed ME influence on cell activation. The number of receptors for naloxone was shown to increase up to 2.5-fold during mitogenic activation. The difference in expression of various types of opioid receptors at mitogenic stimulation was demonstrated by ligand displacement experiments.


Granulocyte-macrophage colony stimulating factor potentiates human polymorphonuclear leukocyte aggregation responses to formyl-methionyl-leucyl-phenylalanine. Polymorphonuclear leukocytes (PMN) are known to be activated by several lymphokines and can be induced to release lysosomal enzymes, prostaglandins (PG), thromboxanes (TX) and lipoxygenase products that may be involved in PMN aggregation responses during inflammatory reactions. Granulocyte-macrophage colony stimulating factor (GM-CSF), a glycoprotein cytokine released by immunocompetent cells, has been found to prime neutrophil responses, such as increased cell aggregation after exposure to various biological stimulants. In this study, we examined the effects of the cytokine GM-CSF on human neutrophilic aggregation stimulated by N-formyl-methionyl-leucyl-phenylalanine (FMLP) and its influence on the production of various arachidonic acid metabolites. Neutrophil aggregation of purified PMNs was measured by the percent change in light transmission in a standard aggregometer, and the arachidonic acid products leukotriene B4 (LTB4) and thromboxane A2 (TXA2) were quantified by radioimmunoassay. We found that GM-CSF and other cytokines, used alone, did not cause any significant increase in aggregation of the PMN. However, prior exposure of PMN to GM-CSF markedly increased the aggregation induced by FMLP as opposed to that detected with PMN stimulated with only FMLP. This priming effect was not observed with PMN preincubated with interleukin-1 (IL-1), tumor necrosis factor (TNF) or interleukin-6 (IL-6). In addition, GM-CSF and IL-6 both failed to stimulate the production of LTB4 and TXA2, products which are known to induce PMN aggregation. These findings provide new evidence suggesting that GM-CSF facilitates the action of FMLP on the adhesion dependent cellular functions of the inflammatory response, serving as an important co-factor in neutrophil aggregation.


Dress for success. A nurse's knowledge of simple clothing adaptations and dressing aids may make the difference between rehabilitation success and failure. Typical chronic diseases that affect older people often result in the loss of independence in daily activities, such as dressing. Losses of functional independence in such basic areas are factors in the institutionalization of elderly persons. The nurse's challenge? Help your clients remain independent. Simple clothing adaptations and dressing aids will help elderly people preserve self-esteem, social contacts, and control of their personal environment.


Pharmacological investigation of the role of leukotrienes in the pathogenesis of experimental NSAID gastropathy. The role of leukotrienes in the pathogenesis of acute gastric ulceration induced by nonsteroidal antiinflammatory drugs was investigated using a rat model. One part of the study involved oral pretreatment with a leukotriene synthesis inhibitor 1 h prior to administration of indomethacin (20 mg/kg per os). Three hours after indomethacin, the extent of macroscopically visible gastric damage was determined, and gastric LTB4 synthesis was determined. The compounds tested were PF-5901, A-64077, nordihydroguaiaretic acid, and L-698,037. Each compound produced dose-related inhibition of gastric LTB4 synthesis and a parallel reduction in the severity of indomethacin-induced damage. The antioxidant properties of these compounds was assessed using an in vitro assay. There was no correlation between the antioxidant properties of the compounds and their ability to reduce the severity of indomethacin-induced gastric damage. In the second part of the study, the effects of intravenous, administration of LTD4 and LTB4 receptor antagonists on indomethacin-induced gastric epithelial damage (measured by permeability to [51Cr]EDTA) were assessed. The two LTD4 receptor antagonists (MK-571 and ICI-204,219) significantly reduced the permeability changes induced by indomethacin, while the two LTB4 antagonists (SC-41930 and LY-255,283) were without significant effect. Despite the reduction of gastric epithelial injury, blockade of LTD4 receptors did not markedly affect the extent of macroscopically visible injury. These data are consistent with the hypothesis that leukotrienes contribute to the epithelial injury and macroscopically visible damage induced by NSAIDs. However, it remains unclear to what extent leukotrienes are involved in the initiation of the injury, as opposed to its amplification.


Oxygen radical production and trapping in subjects with previous Yersinia infection. Previous studies indicate that oxygen radical production by 1-min activation of N-formyl-methionyl-leucyl-phenylalanine-induced chemiluminescence (CL) by whole blood is increased in subjects with previous Yersinia arthritis (YA). This finding is confirmed in the present study and extended further by demonstrating that subjects with previous YA show increased oxygen radical generation and enhanced release of myeloperoxidase from neutrophils and increased CL activity by serum, all factors that can contribute to an increase in the initial activation of CL. Initial activation was not increased in subjects who had had Yersinia enteritis without arthritis; however, intracellular oxygen radical production by purified neutrophils was significantly increased, suggesting that the cells had been primed in vivo. The water-soluble antioxidants of plasma samples tested by a peroxyl radical-trapping assay were much the same in subjects with previous YA, in subjects with previous Yersinia enteritis without arthritis, and in healthy subjects. The results suggest that aberrations in neutrophil oxygen radical production play a role in the pathogenesis of YA.


Priming of human neutrophil functions by tumor necrosis factor: enhancement of superoxide anion generation, degranulation, and chemotaxis to chemoattractants C5a and F-Met-Leu-Phe. Recombinant human tumor necrosis factor-alpha (rTNF) stimulated increased generation of superoxide anion (O2-) by human neutrophils in a concentration-dependent fashion. Preincubation of human neutrophils with rTNF (2.2-2200 units/ml) for 10 min enhanced the subsequent generation of O2- in response to C5a and f-Met-Leu-Phe (FMLP). Recombinant TNF did not enhance O2- generation by neutrophils stimulated with phorbol myristate acetate (PMA). Recombinant TNF alone failed to induce release of myeloperoxidase (MPO) and lysozyme by neutrophils. However, it did enhance the release of MPO and lysozyme by neutrophils stimulated with C5a and FMLP, but not with PMA. Although rTNF alone (0.001-50,000 units/ml) was not chemotactic for neutrophils, preincubation of neutrophils with rTNF (0.001-0.1 units/ml) enhanced the chemotactic activity of suboptimal concentrations of C5a (0.1 nM) and FMLP (5 nM). Neutrophils treated with high concentrations of rTNF (100-10,000 units/ml) showed inhibition of random movement and of chemotaxis induced by C5a or FMLP. We conclude from these studies that rTNF primes neutrophils for enhanced responses to subsequent stimuli and thus may augment the inflammatory response by increased oxidant production and lysosomal enzyme release and promote down-regulation of chemotactic movement.


L-arginine-dependent killing of intracellular Ehrlichia risticii by macrophages treated with gamma interferon. Thioglycolate-induced murine peritoneal macrophages infected with Ehrlichia risticii and treated in vitro with gamma interferon (IFN-gamma) developed antiehrlichial activity that eliminated the intracellular bacteria. This antiehrlichial activity was suppressed by NG-monomethyl-L-arginine, a competitive inhibitor of nitric oxide synthesis from L-arginine, but not by L-tryptophan. Increased levels of nitrite, an oxidative product of nitric oxide, were measured in cultures of infected macrophages treated with IFN-gamma. Sodium nitroprusside, which spontaneously releases nitric oxide, also showed the antiehrlichial activity. The antiehrlichial activity by reactive nitrogen intermediates was not mediated by elevation of the cellular concentration of cyclic GMP since the addition of 8-bromo-cyclic GMP itself had no influence on ehrlichial infection of macrophages. Addition of the intracellular iron chelator deferoxamine also inhibited E. risticii infection in vitro. These results suggest that intracellular E. risticii survival is iron dependent and that production of reactive nitrogen intermediates triggers iron loss from critical target enzymes of E. risticii, leading to lethal metabolic inhibition. However, addition of excess FeSO4, ferric citrate, or iron-saturated transferrin did not counteract the antiehrlichial effect induced by IFN-gamma.


Humoral response to Porphyromonas (Bacteroides) gingivalis in rats: time course and T-cell dependence. In this study, we describe the time course and T-cell dependence of the serum antibody response to the periodontopathogen Porphyromonas (Bacteroides) gingivalis in an experimental rat model. Normal Fischer rats were challenged by a local injection of P. gingivalis (2 x 10(8) bacteria) into gingival tissue or by the administration of a similar number of bacteria by the intravenous (i.v.) route on days 0, 2, and 4. Serum antibody activity was detected within 1 week and peaked at 8 weeks after gingival challenge. A similar but lower response was seen for rats challenged by the i.v. route. The response in both groups of rats was mainly of the immunoglobulin G (IgG) isotype; some IgM but no IgA antibody activity was detected. Analysis of the IgG subclass revealed mainly IgG2c in animals challenged locally in the gingiva with P. gingivalis, whereas IgG2b predominated in rats challenged by the i.v. route. The importance of T cells in the response was established by demonstrating the absence of serum IgG antibodies in nude rats after a local challenge of gingival tissue with P. gingivalis. Nude rats given purified splenic T cells from normal rats immunized systemically with P. gingivalis prior to a local gingival challenge showed a rapid appearance of serum antibody activity that peaked between 4 and 6 weeks. This initial peak occurred 2 to 4 weeks earlier than that seen in normal animals. Fluorescence-activated cell sorter analysis of splenic lymphoid cells from these nude rats revealed a helper T-cell population. The levels of serum IgG antibodies in nude rats given nonimmune T cells rose slowly, and the antibodies were mainly of the IgG2a and IgG2b subclasses. Nude rats given immune T cells showed a rapid increase primarily in IgG2b antibody levels following a local gingival challenge. These findings suggest that the immune helper T-cells contributed to the rapid development of the response to P. gingivalis. Furthermore, it is likely that the IgG subclass response to P. gingivalis in these nude rats was related to the splenic origin of the T cells used for adoptive transfer.


An unusual pagC::TnphoA mutation leads to an invasion- and virulence-defective phenotype in Salmonellae. Two phenotypes believed to contribute to the pathogenesis of Salmonella infections are macrophage survival and invasion of epithelial cells. It was recently observed that the Salmonella macrophage survival factor PagC has significant amino acid similarity to the Yersinia invasion factor Ail. This observation raised the possibilities that macrophage survival is in part determined by the pathway of entry and that PagC confers an entry mechanism that does not trigger the microbicidal activities of the macrophage. Thus, we sought to investigate the role of PagC in invasion by examining (i) the invasion phenotype of pagC mutants and (ii) the invasion phenotype of Escherichia coli carrying pagC. A previously identified invasion-defective TnphoA insertion mutant of Salmonella enteritidis was found to have TnphoA inserted into the signal sequence-encoding region of pagC; the pagC allele from this mutant, SM5T, was designated pagC64. In contrast, Salmonella typhimurium carrying the pagC1 allele (a TnphoA insertion mutation, downstream of the region encoding the signal sequence) was not defective for invasion. Further analysis of these two pagC alleles suggested that the invasion-defective phenotype associated with pagC64 is not due to the loss of PagC function but rather is due to the synthesis of a hybrid PagC-alkaline phosphatase protein that is aberrantly localized, most likely to the inner membrane, and thus may prevent proper localization or function of a factor(s) required for efficient invasion. The observation that pagC did not confer an invasive phenotype to E. coli further suggests that PagC is not an invasion factor. A cloned pagC gene complemented the macrophage survival defect of S. typhimurium pagC1 mutants, but the cloned ail gene did not. Together these results suggest that the structural similarity between PagC and Ail may not extend to a similarity in function. Interestingly, S. enteritidis carrying the pagC64 allele that results in both an invasion defect and a macrophage survival defect was less virulent for mice infected intragastrically or intraperitoneally than was S. enteritidis carrying the pagC1 allele that results only in a macrophage survival defect.


Genetic analysis of Pseudomonas aeruginosa adherence: distinct genetic loci control attachment to epithelial cells and mucins. Infection of mucosal tissues by the opportunistic pathogen Pseudomonas aeruginosa is initiated by attachment of the bacterium to host tissues. To gain a better understanding of this interaction, we used two methods to isolate mutants of P. aeruginosa with altered adherence to cultured A549 cells and to mucins. First, from a population of nonpiliated mutants of P. aeruginosa mutagenized with transposon Tn5G, we have isolated variants that are defective in binding to both A549 cells and respiratory mucins. Using a cloned transposon plus flanking DNA from one such mutant as a DNA probe, we have isolated plasmids from a cosmid bank, which, upon reintroduction to the original mutants, restored adhesion to both A549 cells and mucin. The second strategy to identify genes involved in adhesion used mutagenesis of P. aeruginosa N1G, an rpoN mutant which is unable to bind to either A549 cells or mucin, with transposon Tn5 containing an outward-directed promoter. From this bank of mutagenized P. aeruginosa N1G, two classes of adhesion variants were isolated; one class attached to A549 cells and to mucin, and the other class restored binding of the rpoN mutant to mucin but not to A549 cells. These findings suggest that P. aeruginosa can express at least two adhesins distinct from pili, one recognizing receptors shared by epithelial cells and mucins and the other recognizing mucins alone.


Jumping translocations originate clonal rearrangements in SV40-transformed human fibroblasts. A comparative study of chromosomal rearrangements occurring in 4 independent clones obtained from SV40-transformed cornea and skin human fibroblasts was performed. Rearrangements principally affect some constitutive heterochromatin and, to a lesser degree, telomeric regions. This results in multiple exchanges between a limited number of chromosome structures, i.e., in jumping translocations. Such rearrangements occur even at early passages and some of them give rise to clonal rearrangements that accumulate at late passages. This process is responsible for progressive modification of the karyotypes, principally characterized by deletion of a number of chromosome segments. Thus, clonal rearrangements are selected among many others not occurring at random. The selective pressure retaining clonal rearrangement seems to be similar for the 4 independent clones, since selection of the derivative chromosomes leads to the same imbalances, whatever the origin of the clone. This sequence of events recalls that of human solid tumors, since jumping rearrangements are generally observed in pre-malignant conditions or in low-grade malignancies, whereas clonal rearrangements leading to typical imbalances are detected in more advanced malignant tumors.


Detection of distinct Epstein-Barr virus genotypes in NPC biopsies from southern Chinese and Caucasians. Using the polymerase chain reaction (PCR) to analyze paraffin sections from 12 Caucasian patients, we detected only the prototype F Epstein-Barr virus (EBV) in 10 specimens from patients with nasopharyngeal carcinoma (NPC). This is in contrast to the higher frequency of association of "f" variants in NPC biopsies from Southern Chinese. The results of EBV genotyping support evidence that the EBV strains associated with NPC in the Southern Chinese population differ from those found in Caucasians. DNA sequencing confirmed that a simple point mutation is responsible for the restriction-fragment-length polymorphism which distinguishes the prototype F virus from the "f" variant.


The role of mucin in colon-cancer metastasis. Mucinous colorectal cancer often presents at an advanced stage. We have previously observed that mucin production by human colon-cancer cells correlates with their ability to colonize the liver in experimental animal models. The present study was undertaken in order to further elucidate the mechanisms by which production of mucin by colon-cancer cells affects metastasis. Cell lines showing high mucin production (HMP) (HM 7, HM 3 and LS LiM 6) demonstrated increased adherence to basement membrane proteins and invaded a reconstituted basement membrane to a greater extent than their counter-part cell lines showing low mucin production (LMP) (LS174T and LM 12). Adherence of the LMP parental cell line LS174T to various matrix proteins was potentiated by the addition of purified human colon-cancer mucin in a dose-dependent fashion. HMP cell lines secreted more proteolytically active type-IV collagenase than LMP lines, and collagenase activity was further stimulated by purified mucin in a dose-dependent manner. Specific inhibition of mucin O-glycosylation by benzyl-alpha-N-acetylgalactosamine significantly affected each of the metastasis-related events, with the greatest effect on the HMP cell lines. The present data further indicate that mucin may play an important role in the metastatic process.


Expression of genes related to the human erbB, erbA, pdgf and pdgf-r in tumors of different etiology in Xiphophorus. The melanoma determining Tu locus of the teleost Xiphophorus contains an accessory gene, x-erbB*a, which is closely related to the EGF receptor gene family, and is probably oncogenic. x-erbB*a exists in allelic forms that are specific for distinct Tu-loci, and shows high homology to a non-allelic non-oncogenic counterpart x-erbB*i which is transcribed into mRNA of 4.6 kb in non-tumorous and tumorous tissues of fish harboring and lacking Tu. Expression of a 4.0-kb mRNA in tumors (melanoma and fibrosarcoma) of different etiology is strictly correlated with the inheritance of X. maculatus x-erbB*a alleles; transcripts of 8.0 kb were detected in melanoma and carcinoma of fish harboring a certain x-erbB*a of X. variatus. The expression of the putative x-erbB*a transcripts parallels the stage of malignancy of the tumor. The expression of the xiphophorine EGF receptor gene (x-erbB) was detected in almost all tumors, is strongly enhanced in carcinoma, and is positively correlated with the degree of malignancy of melanoma and fibrosarcoma. Some tumors show expression of erbA-related genes. The PDGF receptor mRNA is expressed in all tumors analyzed and shows enhanced expression in malignant tumors of neurogenic, epithelial and mesenchymal origin. Expression of x-pdgf was observed in several cases of melanoma, but more frequently in carcinoma and fibrosarcoma. We conclude that x-erbB*a might be involved in initiation of tumors of different cellular origin and etiology in fish harboring Tu, as well as in the determination of the malignancy of the tumor. Furthermore, we assume that x-erbB*i, x-erbB, x-pdgf and x-pdgf-r play a role in secondary events in tumorigenesis by, e.g., conferring a selective growth advantage to the tumor cells.


Single-steep transformation of human breast epithelial cells by SV40 large T oncogene. Normal human mammary epithelial cell (HMEC) cultures originating from 2 mammoplasty reduction surgical samples were transfected with replication-defective SV 40 DNA. Two independent cell lines designated as S2T2 and S1T3, selected for their increased proliferation potential and lifespan, were propagated for greater than 22 months in culture. They maintained a near-diploid karyotype with few chromosomal markers such as trisomy 1q (S1T3) and trisomy 8q (S2T2), which are most common in breast cancer in vivo. Immortalized S1T3 cells were not tumorigenic, whereas S2T2 cells produced slowly growing tumors in nude mice. One tumor was propagated in vitro and the transformed NS2T2 cell line subsequently raised 100% large tumors in the nude mouse. Rearrangement of the SV40 genome was observed in NS2T2 cells, which was not associated with increased expression of large T antigen. S1T3, S2T2 and transformed NS2T2 cell lines expressed cytokeratins CK18, CK19, the mammary-specific antigen DF3, and functional EGF receptors. Single-step immortalization and malignant transformation of human breast epithelial cells can thus occur upon transfection with SV40 large T oncogene. The chromosomal abnormalities observed in these cell lines suggest that they could offer a model for the study of breast-tumor progression in vitro.


Rhabdoid tumor of the skin.

Rhabdoid tumor of the skin. A tumor in the skin of a 42-year-old man was analyzed by light and electron microscopic study and immunohistochemistry. The tumor cells were large and contained eosinophilic, periodic acid-Schiff (PAS)-positive inclusions in the cytoplasm. Immunohistochemically, the neoplasm was positive for intermediate filaments, cytokeratin, vimentin, desmin, and alpha-1-antichimotrypsin, and negative for S-100 and leukocyte common antigen (LCA). Ultrastructurally, the cytoplasm and cellular processes of the cells were inundated with intermediate filaments, some of which were tightly bundled. Junctional complexes and secretory granules were absent. These features suggest a rhabdoid tumor: a malignant tumor of uncertain origin.


Cardiac effects of caloric restriction-mechanisms and potential hazards. The heart is not spared from the catabolic effects of undernutrition, but is subject to the same degree of weight loss as skeletal muscle. Pumping performance, however, is not reduced in proportion to myocardial wasting and thus functional protection prevents failure of the hypotrophic heart. In animal experiments, no major qualitative changes in myocardial composition apart from reduced myosin ATPase activity have been found. This reduction in ATPase activity might be associated with down-regulation of thyroid hormones and the development of insulin resistance and serve as an energy saving adaptation. Increased cardiac sensitivity to adrenergic stimulation may also constitute a means to increase heart performance in situations with augmented circulatory demands. On the other hand, increased sensitivity and maximum response to adrenergic stimulation might render the heart more susceptible to arrhythmia, and thus explain sudden unexpected death following rapid weight loss.


Effectiveness of phenylpropanolamine in the management of moderate obesity. Phenylpropanolamine (PPA), an over-the-counter drug, is used for weight reduction but its effectiveness is controversial. One hundred and one ambulatory subjects (85 female; 16 male), 21-61 years old, 15-45% overweight but otherwise healthy, were studied. The study was divided into two phases: (i) a double blind (DB), placebo-controlled (P) phase in which all subjects took placebo for two weeks and subsequently took PPA, 75 mg sustained release or placebo for six weeks and (ii) an extended double blind phase in which subjects chose to continue up to 20 weeks. All subjects were instructed on a 5023 kJ (1200 kcal) diet. Both groups lost weight at weeks 4, 6 and 8, but the weight loss was greater for the PPA treated (2.59 kg) than for the placebo treated (1.07 kg) subjects (P = 0.01). Dropout was 29.4% for PPA and 44% for placebo treated subjects. In the 36 subjects who chose to continue in the extended double blind study, the difference persisted (PPA 5.1; placebo 0.4 kg) (P = 0.02). No difference between the groups was observed in blood pressure, pulse rate or subjective side effects. In spite of greater weight loss on PPA, patients did not report a greater anorexic effect. We conclude that PPA enhances weight loss in subjects treated with a hypocaloric diet and is free of untoward side effects.


Body fat distribution in pre- and post-menopausal women: metabolic and anthropometric variables and their inter-relationships. The aim of the study was to compare body fat distribution and metabolic variables in pre- and post-menopausal women. Body fat distribution was measured using abdominal circumference and computerized tomography. No significant differences were found between the two groups as regards body weight, body mass index, waist-hip ratio and total abdominal adipose tissue areas. Subcutaneous abdominal adipose tissue areas were significantly higher in premenopausal women whereas visceral abdominal adipose tissue areas and the subcutaneous to visceral abdominal adipose tissue area ratios were significantly higher in post-menopausal subjects. After adjusting for body mass index, no significant differences emerged between the two groups as regards total abdominal adipose tissue areas, waist circumference, hip circumference and waist-hip circumference ratio; subcutaneous abdominal adipose tissue areas were significantly lower and both visceral abdominal adipose tissue areas and visceral to subcutaneous abdominal adipose tissue area ratios significantly higher in post-menopausal women (P less than 0.01). Basal glucose, sum of blood glucose values during oral glucose tolerance test and blood cholesterol values were significantly higher in the post-menopausal group (P less than 0.05), while no significant difference was observed in sum of blood insulin values during oral glucose tolerance test. Basal plasma insulin values, systolic blood pressure and diastolic blood pressure were higher in post-menopausal women, though the differences were not significant. Only blood cholesterol was significantly higher in post-menopausal women after adjusting for visceral abdominal adipose tissue areas. Positive correlations emerged between age and waist-hip ratio (P less than 0.05), visceral abdominal adipose tissue areas and the visceral to subcutaneous abdominal adipose tissue area ratio (P less than 0.001). A negative correlation was established between age and subcutaneous abdominal adipose tissue areas (P less than 0.01). There was a significant correlation between visceral abdominal adipose tissue areas and metabolic and haemodynamic variables in both pre- and post-menopausal women. In pre-menopausal women, body mass index correlated with basal glucose, basal insulin, sum of glucose during oral glucose tolerance test and systolic and diastolic blood pressure (P less than 0.05). When stepwise multiple regression analysis was used to evaluate the joint effect of anthropometric variables on metabolic variables, visceral abdominal adipose tissue area proved to be the most powerful variable for predicting metabolic disorders. Our data suggest that visceral abdominal adipose tissue areas and visceral to subcutaneous abdominal adipose tissue area ratios increase with age. Obesity correlates directly with the amount of subcutaneous fat, but not with the distribution pattern.(ABSTRACT TRUNCATED AT 400 WORDS)


Evaluation of an intensive weight control program using a priori criteria to determine outcome. This study evaluated the effectiveness of a comprehensive obesity treatment program which incorporated open-ended treatment duration, pre-treatment assessments, protein-sparing modified fasts (PSMFs), and the use of six a priori outcome categories to analyse outcomes. Subjects were the first 291 obese individuals (mean weight = 235 lb, 66% overweight) to participate in the program's intake procedures. Data obtained after 55 weeks of treatment (on average) showed that the program seemed quite effective for 65% of those who participated for at least 12 weeks (mean = 62 and 30 lb lost in the two successful groups). Analyses supported the continued use of pre-treatment assessments, extended treatment times, and a priori categorizations of outcomes. In addition, correlational analyses showed that binge eating, high levels of psychological distress, and low income levels were associated with poorer outcomes.


Long-term exercise training and retirement in genetically obese rats: effects on food intake, feeding efficiency and carcass composition. Short-term physical exercise (EX) can reduce body weight and fat gain in obese humans and animals. However, the beneficial effects of physical exercise are not long-lasting. In this study, the effects of long-term physical exercise and retirement from exercise (R) on body weight, body composition and fat distribution were examined in genetically obese (OB) and lean (LE) female rats. Fifty OB and 45 LE rats, four weeks old, were divided into EX (swimming, 2h/day, 5 days/week) or sedentary (SD) groups. At the end of the 28th week of treatment, EX groups were further divided into continued EX or R groups for another 11-12 weeks. It was found that at the end of the 28th week EX had reduced the rate of weight gain in OB and LE rats. Percentage body fat was only reduced in OBEX rats and this was achieved by a significant reduction of subcutaneous fat mass. At the end of the 40th week, EX had further reduced the weight gain, fat mass and body fat percentage in OBEX rats while only body fat percentage was reduced in the LEEX group. Retirement from exercise reversed these phenomena. Thus there were no differences between OBSD and OBEX-R rats in body weight, fat mass and percentage body fat. However, the OBEX-R group had a significantly higher amount of internal fat than the other two OB groups. Therefore, exercise, even long-term to cover the entire fat cell proliferation period, still only exerted temporary beneficial effects in OB rats. After retirement, the beneficial effects all disappeared rapidly.(ABSTRACT TRUNCATED AT 250 WORDS)


The acute effects of corticotropin-releasing factor on energy expenditure in lean and obese women. Corticotropin-releasing factor (CRF) has been implicated in the development of obesity in genetically obese rodents. We have investigated the effect of 100 micrograms of intravenous CRF on energy expenditure in women, comparing the response in obese and lean volunteers. In response to CRF, energy expenditure as measured by indirect calorimetry increased rapidly with a peak response in both groups reached by two minutes with a ten minute post-CRF response averaging 9.0% in the lean and 11.0% in the obese. Subsequently, energy expenditure remained elevated for a longer duration in the lean compared to the obese. Overall, the total 30 min cumulative metabolic rise was similar in the lean and obese. The increments in energy expenditure were associated with elevation of plasma noradrenaline levels, suggesting the possible involvement of the sympathetic nervous system. The adrenocorticotrophic (ACTH) and cortisol responses to CRF were similar in obese and lean. Intravenous administration of CRF therefore acutely increases energy expenditure in both lean and obese healthy subjects.


Phase II feasibility study of high dose epirubicin plus etoposide and cisplatin (HDEEC) regimen in small cell lung cancer. Seventeen patients with small cell lung cancer entered a phase II trial testing the feasibility of adding high dose epirubicin (100-120 mg/m2, day 1) in combination with etoposide (60-80 mg/m2, days 1-5) and cisplatin (70 mg/m2, day 1) courses repeated every three weeks. Complete responders received thoracic (40 Gy) and prophylactic cranial (30 Gy) irradiation. Sixteen patients were evaluable for response and toxicity. Myelosuppression was the dose-limiting side effect. Neutropenic fever was observed in eight patients (53%) and stomatitis in six (40%). No patient had a greater than 14% decline in the cardiac ejection fraction. Strict adherence to the dose-schedule designed was impossible as doses were trimmed and delayed in 30% of instances. The overall objective response rate was 81% (95% confidence limits, 62% to 100%), in limited disease there were complete remissions in 57%. With a 16 months median follow-up, overall median survival was 13 months. This study was unable to prove the feasibility of epirubicin escalation when added to etoposide-cisplatin combination, hampering the dose-intensification Norton-Simon model test.


Evaluation of CHIP (iproplatin) in recurrent pediatric malignant solid tumors. A phase II study (Pediatric Oncology Group). CHIP (325 mg/M2), a second generation cisplatin derivative, was administered intravenously every 3 weeks to 85 pediatric patients with recurrent sarcomas (19), osteosarcomas (20), neuroblastoma (23), germ cell tumors (10), and other malignant tumors (7). Thirty-eight of them had been previously exposed to cisplatin. Partial remissions were only observed in 3 of 23 (13% SE = 7%) patients having neuroblastoma. Severe thrombocytopenia (65%) and neutropenia (35%) were the dose limiting factors.


Role of amino acid residue 187 of poliovirus polypeptide 2C in determining the guanidine trait. Although mutations in codons 164 and 179 of poliovirus polypeptide 2C can establish guanidine resistance, the majority of guanidine-resistant and guanidine-dependent viruses contain an M----L change at amino residue 187 along with other critical mutations. The change at residue 187 probably contributes to the guanidine phenotype along with a variety of other modifications in polypeptide 2C, possibly altering conformation of the polypeptide during the initiation step of viral RNA synthesis.


Quantitative immunoelectron microscopic localization of Na, K-ATPase alpha-subunit in the epithelial cells of rat vestibular apparatus. Na, K-ATPase was quantitatively localized in the epithelial cells of rat vestibular apparatus such as macula utriculi, macula sacculi and crista ampullaris. Immunogold localization method was carried out at the saturation level of antibody using an affinity purified antibody against the alpha-subunit of rat kidney Na, K-ATPase. Numerous gold particles were found on the basolateral membrane of the dark cells, a small number of gold particles were found on the basolateral membrane of the transitional epithelium cells and hair cells, but the luminal surface membranes of the hair cells, transitional epithelium cells, planum semilunatum cells and dark cells were rarely labeled by gold particles. Significance of the abundant localization of Na, K-ATPase on the basolateral surface of the dark cells in the production of endolymph was discussed.


Germ cell tumors.

Germ cell tumors. The development of modern surgical staging and effective chemotherapy regimens has markedly improved outcome of treatment of ovarian germ cell tumors. Almost all patients with completely resected tumors will survive their disease. Those with tumors other than dysgerminoma should all receive adjuvant chemotherapy. Patients with stage IA dysgerminoma can safely be observed, whereas those of higher stage should probably receive chemotherapy, although radiotherapy may be an option in selected patients, particularly older ones or those with other serious illness. Patients with advanced disease should all be treated with chemotherapy. Most dysgerminoma patients will be cured, as will many with other cell types; however, there is room for improvement in the latter group, and continued investigation is appropriate.


Resolution and characterization of two forms of phosphoinositide-specific phospholipase C from bovine rod outer segments. Two forms (I and II) of phospholipase C, specific for phosphatidyl inositol 4,5-bisphosphate, were resolved from bovine retinal rod outer segment (ROS) cytosol by DEAE-Sepharose column chromatography. The two isozymes showed reproducible differences in their catalytic properties in spite of similar substrate specificity and hydrolyzed specifically inositol 4,5-bisphosphate in a Ca(2+)-dependent fashion. In the presence of deoxycholate (DOC), pH optima were at 6.5 and 7.0 for phospholipase C I and II, respectively. Maximal phosphatidylinositol 4,5-bisphosphate hydrolysis rates were obtained at 10(-4) and 10(-5)M Ca2+ for phospholipase C I and II, respectively. Treatment with cAMP-dependent protein kinase did not alter either isozyme activity. Further purification steps were prevented by the extreme lability of the isozymes.


Ribosome-bound elongation factor 2 escapes phosphorylation by Ca2+/calmodulin-dependent protein kinase III. The activity of eukaryotic elongation factor 2 is regulated by phosphorylation catalysed by a highly specific Ca2+/calmodulin-dependent protein kinase. Phosphorylated EF2 binds to ribosomes with decreased affinity. The present evidence indicates that EF2 prebound to ribosomes is protected from phosphorylation, just as earlier evidence indicated that ribosome-bound EF2 is protected from ADP-ribosylation catalysed by diphtheria toxin. Ribosome-inactivating proteins ricin and gelonin, by interfering with the EF2-ribosome interaction, allow full phosphorylation of EF2.


Glucocorticoids modulate the induction of BLTE/granzyme A activity in the murine T cell hybridoma PC60. The cytolytic granule-associated serine esterase granzyme A cleaves the synthetic substrate benzyloxycarbonyl-L-lysinate-thiobenzylester (BLT) and has been described as a marker for cytotoxic T lymphocyte (CTL) activation. We recently showed that BLT-esterase activity (BLTE activity) can be induced in the murine CTL-hybridoma PC60 by exogenous interleukin-1 (IL-1) and/or a rise of the intracellular cAMP level, although cAMP does not act as a second messenger for IL-1 in this system. The present study demonstrates that glucocorticoids (GC) such as dexamethasone and hydrocortisone efficiently inhibit the induction of BLTE activity by IL-1 and/or cAMP and downregulate the basal BLTE levels in PC60 cells; these results could be reproduced in part with progesterone and were steroid class-specific, since estrogen did not affect the induction of BLTE activity. The GC-induced effects on the production of BLTE activity required the activation of specific GC receptors, since induction of the activity could be restored upon addition of the contragestative drug RU 38486; they further could not be related to any alteration of the cellular metabolism of arachidonic acid and did not appear to be mediated by secreted macromolecules such as lipocortins.


Hydroxyl radical formation by UV-irradiated epidermal cells. To elucidate the mechanism of sunlight-induced skin damage, guinea pigs were exposed to UV light (280-320 nm, UV B, 4 J/cm2) and a homogenate of the epidermis was examined by means of the thiobarbituric acid (TBA) test. Three hours after the exposure, TBA-malondialdehyde adducts had increased while glutathione reductase activity had decreased, indicating lipid peroxidation. To detect the initial species, spin trapping with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) was applied to a suspension of illuminated epidermal cells (0.5 J/cm2). An ESR signal obtained only with irradiation comprised a 1:2:2:1 quartet [a(N)= a(beta H) = 1.49 mT] attributable to a spin adduct of hydroxyl radicals. These results suggest that sunlight exposure of skin may lead to hydroxyl radical generation and simultaneous lipid peroxidation.


EMD 52692 (bimakalim), a new potassium channel opener, attenuates luminol-enhanced chemiluminescence and superoxide anion radical formation by zymosan-activated polymorphonuclear leukocytes. We investigated the relationship of potassium channel activation on modulation of oxidative respiratory bursts in canine neutrophils. Generation of superoxide anion radicals in opsonized zymosan-activated cells was determined using the technique of ferricytochrome c reduction. Preincubation of cells with the selective potassium channel opener, EMD 52692 (1-100 microM), attenuated superoxide anion radical production. Furthermore, EMD 52692 also produced a concentration-dependent inhibition of luminol-enhanced chemiluminescence by activated neutrophils. Glyburide, a selective antagonist of ATP-sensitive potassium channels, prevented the modulatory effect of EMD 52692 on both superoxide anion generation and luminol-enhanced chemiluminescence. The results suggest that ATP-sensitive potassium channels may play a significant role in regulating oxygen-derived free radical production in neutrophil-induced tissue injury.


Transmembrane signal transduction and osmoregulation in Escherichia coli: functional importance of the transmembrane regions of membrane-located protein kinase, EnvZ. EnvZ is a membrane-located protein kinase which modulates expression of the ompF and ompC genes through phosphotransfer signal transduction in Escherichia coli. Previously, we developed an in vitro method for analyzing the intact form of EnvZ in isolated cytoplasmic membranes, and demonstrated that this particular form of EnvZ exhibits the ability not only of OmpR phosphorylation but also OmpR dephosphorylation. Taking advantage of this in vitro system, in this study, to assess the structural and functional importance of the membrane-spanning (transmembrane) regions of EnvZ, a set of mutant envZ genes, each of which specifies a mutant EnvZ protein with a single amino acid replacement within or very near the transmembrane regions, were isolated and characterized in terms of their in vivo osmoregulatory phenotypes and in vitro EnvZ-OmpR phosphotransfer activities. On the basis of the results, it was suggested that the transmembrane regions of EnvZ play roles in transmembrane signaling and consequent modulation of the kinase/phosphatase activity exhibited by the cytoplasmic domain in response to an osmotic stimulus.


Polymerized albumin receptor on rat liver cells. The polymerized albumin hypothesis was proposed for the mechanism of a hepatitis B virus (HBV) infection of human liver parenchymal cells on the basis that a receptor for polymerized albumin treated with glutaraldehyde was detected on isolated human liver parenchymal cells. However, some controversy exists regarding this hypothesis, because a receptor for formaldehyde-treated bovine serum albumin (f-BSA) has been found on liver non-parenchymal cells. Therefore, we characterized the uptake of polymerized rat serum albumin (p-RSA) and f-BSA by rat liver in vivo, and their bindings to liver cells in vitro. Most p-RSA and f-BSA was taken up by the liver after intravenous administration, and the uptake of p-RSA was inhibited by a 1,000-fold excess of f-BSA. In addition, more than 80% of p-RSA taken up by the liver was found in the non-parenchymal cells, and the remainder was found in the parenchymal cells. P-RSA as well as f-BSA could bind to isolated rat liver parenchymal and non-parenchymal cells. Furthermore, p-RSA and f-BSA could bind to isolated rat liver cell plasma membranes, and these bindings were completely inhibited by 1,000-fold excess of either f-BSA or p-RSA. These results indicate that there is a receptor, which can recognize both p-RSA and f-BSA, on not only rat liver non-parenchymal cells but also the parenchymal cells. It is also indicated that the receptor on the parenchymal cells as well as the non-parenchymal cells is involved in the in vivo uptake of p-RSA.(ABSTRACT TRUNCATED AT 250 WORDS)


Human plasma gelsolin reversibly binds Mg-ATP in Ca(2+)-sensitive manner. Gelsolin is a Ca(2+)-regulated actin-modulating protein found in a variety of cellular cytoplasm and also in blood plasma. Affinity separation of human plasma gelsolin was successfully accomplished by eluting the protein with a low concentration of nucleoside polyphosphate from immobilized Cibacron Blue F3GA (1, 2). This finding was followed by the demonstration that the protein had one class of ATP binding site with Kd = 2.8 x 10(-7) M, which saturated at an ATP/gelsolin ratio of 0.6 in the absence of Ca2+ (3). To obtain further information on the nucleotide binding properties of gelsolin, binding studies were done in the presence of EGTA with GTP, ADP, and GDP by equilibrium dialysis. Incubation of plasma gelsolin with GTP resulted in binding of 0.6 mol of GTP per mol of protein with a dissociation constant of 1.8 x 10(-6) M, indicating that ATP binds to gelsolin with higher affinity than GTP. Neither ADP nor GDP at up to 100 microM appreciably bound to gelsolin at a physiological salt concentration. Then, the effects of divalent metal ions on the ATP binding to plasma gelsolin were examined. Gelsolin bound to ATP with Kd = 2.4 x 10(-6) M in a solution containing 2 mM MgCl2, whereas micromolar free Ca2+ concentrations inhibited ATP binding. Furthermore, addition of Ca2+ rapidly reversed the preformed nucleotide binding to gelsolin, suggesting that Ca2+ binding to gelsolin leads to a conformational change which disrupts a nucleotide binding fold in the protein molecule.


Methanol and ethanol oxidase respiratory chains of the methylotrophic acetic acid bacterium, Acetobacter methanolicus. Acetobacter methanolicus is a unique acetic acid bacterium which has a methanol oxidase respiratory chain, as seen in methylotrophs, in addition to its ethanol oxidase respiratory chain. In this study, the relationship between methanol and ethanol oxidase respiratory chains was investigated. The organism is able to grow by oxidizing several carbon sources, including methanol, glycerol, and glucose. Cells grown on methanol exhibited a high methanol-oxidizing activity and contained large amounts of methanol dehydrogenase and soluble cytochromes c. Cells grown on glycerol showed higher oxygen uptake rate and dehydrogenase activity with ethanol but little methanol-oxidizing activity. Furthermore, two different terminal oxidases, cytochrome c and ubiquinol oxidases, have been shown to be involved in the respiratory chain; cytochrome c oxidase predominates in cells grown on methanol while ubiquinol oxidase predominates in cells grown on glycerol. Both terminal oxidases could be solubilized from the membranes and separated from each other. The cytochrome c oxidase and the ubiquinol oxidase have been shown to be a cytochrome co and a cytochrome bo, respectively. Methanol-oxidizing activity was diminished by several treatments that disrupt the integrity of the cells. The activity of the intact cells was inhibited with NaCl and/or EDTA, which disturbed the interaction between methanol dehydrogenase and cytochrome c. Ethanol-oxidizing activity in the membranes was inhibited with 2-heptyl-4-hydroxyquinoline N-oxide, which inhibited ubiquinol oxidase but not cytochrome c oxidase. Alcohol dehydrogenase has been purified from the membranes of glycerol-grown cells and shown to reduce ubiquinone-10 as well as a short side-chain homologue in detergent solution.(ABSTRACT TRUNCATED AT 250 WORDS)


Different stability of ligand-receptor complex formed with two endothelin receptor species, ETA and ETB. There are at least two types of endothelin receptors, ETA and ETB, present in various tissues. We found that although biotinylated ET-1 could bind to both ETA and ETB receptors, the stability of the formed ligand-receptor complexes was different. When the preformed complexes of receptor (solubilized from canine brain and lung membranes) and biotinylated ET-1 were subjected to avidin agarose column chromatography, most of the ETA activity was recovered in the pass-through fraction and the remainder was recovered in the 0.5 M KSCN eluate as ligand-free forms. On the other hand, the ETB activity bound firmly to the avidin agarose column was eluted with 1.5 M KSCN. The detection of the complex of 125I-ET-1 and its receptor by SDS-PAGE run at a low temperature was only possible with the ETB fractions and the complex of 125I-ET-1 and ETA was unstable during the separation. These results suggest that the conformation of the ligand binding sites of canine ETA and ETB as well as the stability of their ligand-receptor complexes to SDS are significantly different. Similar observations were also obtained for human ETA and ETB receptors.


Deblocking and subsequent microsequence analysis of N alpha-blocked proteins electroblotted onto PVDF membrane. A method was developed for direct microsequencing of N alpha-acetylated proteins electroblotted onto polyvinylidene difluoride membranes from polyacrylamide gels. N alpha-Acetylated proteins (greater than 32 pmol), including horse heart cytochrome c, five mutants of yeast cytochrome c, and bovine erythrocyte superoxide dismutase, were separated by SDS-PAGE and electroblotted onto polyvinylidene difluoride membranes. The portions of the membrane carrying the bands were cut out and treated with 0.5% polyvinylpyrrolidone in acetic acid solution at 37 degrees C for 30 min. The protein was digested on the membrane with 5-10 micrograms of trypsin at 37 degrees C for 24 h. During tryptic digestion, the resultant peptides were released from the membrane and the N-terminal peptide was efficiently deblocked with 50 mU of acylamino acid-releasing enzyme at 37 degrees C for 12 h. Picomole levels of the deblocked proteins could be sequenced directly by use of a gas-phase protein sequencer.


Retinoic acid induces myogenin synthesis and myogenic differentiation in the rat rhabdomyosarcoma cell line BA-Han-1C. Two clonal rat rhabdomyosarcoma cell lines BA-Han-1B and BA-Han-1C with different capacities for myogenic differentiation have been examined for the expression of muscle regulatory basic helix-loop-helix (bHLH) proteins of the MyoD family. Whereas cells of the BA-Han-1C subpopulation constitutively expressed MyoD1 and could be induced to differentiate with retinoic acid (RA), BA-Han-1B cells did not express any of the myogenic control factors and appeared to be largely differentiation-defective. Upon induction with RA, BA-Han-1C cells expressed also myogenin, in contrast to BA-Han-1B cells which never activated any of the genes encoding muscle bHLH factors. The onset of myogenin transcription in BA-Han-1C cells required de novo protein synthesis and DNA replication suggesting that RA probably did not act directly on the myogenin gene. Although MyoD1 was expressed in proliferating BA-Han-1C myoblasts, muscle-specific reporter genes were not activated indicating that MyoD was biologically inactive. However, transfections with plasmid expressing additional MyoD1 protein resulted in the transactivation of muscle genes even in the absence of RA. mRNA encoding the negative regulatory HLH protein Id was expressed in proliferating BA-Han-1C cells and disappeared later after RA induction which suggested that it may be involved in the regulation of MyoD1 activity. The myogenic differentiation of malignant rhabdomyosarcoma cells strictly correlated with the activation of the myogenin gene. In fact, stable transfections of BA-Han-1C cells with myogenin expressing plasmids resulted in spontaneous differentiation. Together, our results suggest that the transformed and undifferentiated phenotype of BA-Han-1C rhabdomyosarcoma cells is associated with the inactivation of the myogenic factor MyoD1 as well as lack of myogenin expression. RA alleviates the inhibition of myogenic differentiation, probably by activating MyoD protein and myogenin gene transcription. BA-Han-1B cells did not respond to RA and the differentiated phenotype could not be restored by overexpression of MyoD1 or myogenin.


Cooperation between PDGF and FGF converts slowly dividing O-2Aadult progenitor cells to rapidly dividing cells with characteristics of O-2Aperinatal progenitor cells. We have shown previously that oligodendrocyte-type-2 astrocyte (O-2A) progenitor cells isolated from adult rat optic nerves can be distinguished in vitro from their perinatal counterparts on the basis of their much slower rates of division, differentiation, and migration when grown in the presence of cortical astrocytes or PDGF. This behavior is consistent with in vivo observations that there is only a modest production of oligodendrocytes in the adult CNS. As such a behavior is inconsistent with the likely need for a rapid generation of oligodendrocytes following demyelinating damage to the mature CNS, we have been concerned with identifying in vitro conditions that allow O-2Aadult progenitor cells to generate rapidly large numbers of progeny cells. We now provide evidence that many slowly dividing O-2Aadult progenitor cells can be converted to rapidly dividing cells by exposing adult optic nerve cultures to both PDGF and bFGF. In addition, these O-2Aadult progenitor cells appear to acquire other properties of O-2Aperinatal progenitor cells, such as bipolar morphology and high rate of migration. Although many O-2Aadult progenitor cells in cultures exposed to bFGF alone also divide rapidly, these cells are multipolar and migrate little in vitro. Oligodendrocytic differentiation of O-2Aadult progenitor cells, which express receptors for bFGF in vitro, is almost completely inhibited in cultures exposed to bFGF or bFGF plus PDGF. As bFGF and PDGF appear to be upregulated and/or released after injury to the adult brain, this particular in vitro response of O-2Aadult progenitor cells to PDGF and bFGF may be of importance in the generation of large numbers of new oligodendrocytes in vivo following demyelination.