Plasmid cloning and expression of the E. coli polA + gene in S. cerevisiae

Summary The E. coli polA ⁺ gene has been subcloned from a specialised transducing phage onto a low copy number plasmid. Plasmid-encoded DNA polymerase I was synthesised at 2 to 3 times the wild-type E. coli level, and was biochemically indistinguishable from chromosomally-encoded protein. It was able to counteract the radio sensitivity of polA1, polAex1, polAex2 and polA12 mutants, but no complementation of polA107 mutants occurred, even though the plasmid polA⁺ gene was expressed. S. cerevisiae ars-1 or 2 replicative sequences were introduced into the polA⁺ plasmid. Transformation of yeast with these constructs increased total DNA polymerase levels 2–20 times, depending upon assay conditions. The additional activity was discriminated from yeast DNA polymerases by its ability to use low concentrations of substrate, by its resistance to chemical inhibition, and by co-electrophoresis with pure DNA polymerase I and its proteolytic fragments. The polA⁺ gene was expressed in yeast without the aid of yeast promotor sequences. However, deletion of cloned DNA more than 99 base pairs in front of the structural gene prevented expression in yeast but not in E. coli, indicating that the two organisms use different sequences for expression of the plasmid polA⁺ gene.     

Effect of iodine isotope on the pituitary-thyroid and immune systems of children living on the territories polluted by radionuclides

Examination of thyroid function and immune status of children living on the territories polluted by radionuclides in 1993–1994 revealed¹³¹I-dependent thyroid autoimmune reactions. These data indicate a possible effect of¹³¹I on the pituitary-thyroid and immune systems of children living on the radiation controlled territories. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 126, No. 8, pp. 216–220, August, 1998     

Sodium dependence of the epithelial sodium conductance expressed in Xenopus laevis oocytes

The epithelial Na⁺ conductance was expressed in Xenopus laevis oocytes by injection of size-fractionated mRNA of bovine tracheal epithelium. Fractionation was achieved by sucrose density gradient centrifugation. Successful expression was analysed by recording current/voltage (I/V) curves in the presence and absence of amiloride (10 mol/l). The newly expressed conductance was half-maximally inhibited by 44 nmol/l amiloride and exhibited a selectivity for Na⁺ over K⁺ of 1401. I/V curves obtained at different extracellular Na⁺ concentrations ([Na⁺]_o) were subjected to a Goldman-fit analysis to obtain the relation between Na⁺ permeability (P _Na) and [Na⁺]_o. The data show that decreasing [Na⁺]_o from 85 mmol/l to 0.85 mmol/l increased P _Na by more than threefold, which is thought to reflect Na⁺ channel inhibition by increasing [Na⁺]_o. This effect clearly exceeded what can be attributed to concentration saturation of single Na⁺ channel conductance (Palmer and Frindt (1986) Proc Natl Acad Sci USA 83:2767). No correlation of inhibition with intracellular Na⁺ concentration was observed. Preservation of the [Na⁺]_o-dependent self-inhibition by the newly expressed Na⁺ conductance suggests that it is an intrinsic property of the Na⁺ channel protein, probably mediated by an extracellular Na⁺ binding site.     

Effect of human defensins on the cytoplasmic Ca2+ content in platelets

The effect of the total fraction of human defensins (HNP-1, HNP-2, and HNP-3) on the cytoplasmic Ca²⁺ content ([Ca²⁺]_i) in the platelets of healthy donors was studied. At concentrations of 0.1–40 μg/ml and an incubation time of 10 min defensins have no effect on [Ca²⁺]_i in platelets labeled with Fura-2AM. However, at higher concentrations (100 μg/ml) they increased platelet [Ca²⁺]_i. In addition, defensins (40 μg/ml) inhibited the Ca²⁺ increase in platelets induced by thrombin, adenosine diphosphate, and the lipopolysaccharide ofS. typhimurium endotoxin. The most pronounced inhibitory effect was observed in a suspension of thrombin-stimulated platelets. It is shown that the effect of human defensins on the functional activity of platelets is due to the alterations in the intracellular Ca²⁺. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 118, N ^o 12, pp. 600–603, December, 1994     

Analysis of demographic and clinical factors affecting the outcome of radioiodine therapy in patients with hyperthyroidism

Introduction

The influence of demographic and clinical factors on the outcome of ¹³¹I therapy in hyperthyroid patients has been examined, based on a retrospective evaluation of results obtained in patients submitted to ¹³¹I treatment at the Department of Nuclear Medicine and Oncological Endocrinology, Medical University of Lodz (Province Hospital, Zgierz). The goal of the study was to analyse such factors as the age and sex of patients, disease duration, as well as the hormonal status before ¹³¹I application, which could have an influence on the effects of therapy with radioiodine ¹³¹I.

Material and methods

The study involved 500 randomly selected patients with hyperthyroidism, treated with ¹³¹I radioiodine. The following 3 groups were defined: group 1 – patients with multinodular goitre (MNG), n = 200; group 2 – patients with a single autonomous nodule of the thyroid (AFTN), n = 100; group 3 – patients with Graves’ disease (GD), n = 200. The local ethics committee (in the Polish Mother''s Memorial Hospital – Research Institute, Lodz) approved the study.

Results

The obtained results indicate that the efficacy of therapy with ¹³¹I applied in patients with MNG, AFTN and GD does not depend on either patient sex or patient age. The length of antithyroid treatment before ¹³¹I therapy onset does not appear to have any effect on the therapy outcome, and the baseline thyrotropin concentration seems to be significant only in the case of GD.

Conclusions

The analysed demographic factors do not affect the outcome of ¹³¹I therapy in hyperthyroidism.     

Changed count of CD4+ T-lymphocytes in the bone marrow of aggressive CBA mice

Enhanced immune response of aggressive CBA mice after 10 daily confrontations in sensory contact on day 4 after immunization with sheep red blood cells (5×10⁸) is paralleled by an increase in the count of CD4⁺ T-cells in the bone marrow. Aggressive behavior, weight of the spleen, and count of CD4⁺ T-helpers in the bone marrow (which is increased only in aggressors with a history of at least 3 victories) are correlated. The effect of aggressive behavior on immunity can be caused by changes of the neurochemical status of the brain and determined by an increase in the CD4⁺ T-helper count. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 124, No. 11, pp. 544–546, November, 1997     

Potassium and sodium ions in the nerve tissue of emotionally overstrained rabbits

Measurement of K⁺ and Na⁺ concentrations in samples of individual brain nuclei and in ganglia of the autonomic nervous system from rabbits subjected to severe emotional stress (ES) through aperiodic stimulation of ventromedial hypothalamic nuclei and electrocutaneous stimulation revealed significantly altered levels of these ions in locus ceruleus samples from animals predisposed to ES-induced cardiovascular disorders and in samples of neurons of the caudal part of the brainstem from those resistant to such disorders. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 118, No 8, pp. 129–131, August, 1994 Presented by K. V. Sudakov, Member of the Russian Academy of Medical Sciences     

Microelectrode study of voltage-dependent Ba2+ and Cs+ block of apical K+ channels in the skin of Rana temporaria

The blockage of the apical K⁺ channels in frog species Rana temporaria by Ba²⁺ and Cs⁺ is strongly voltage-dependent. The interaction of both blockers with the K⁺ channels was studied by recording relations between the K⁺ currents (I _K) and the transepithelial and intracellular potential. Mucosal Ba²⁺ and Cs⁺ depress I _K, hyperpolarize the cell and induce pronounced nonlinearities in the current/voltage (I/V) relations. The nonlinearities are caused by the voltage-dependent interaction of Ba²⁺ and Cs⁺ with the binding site. Consequently, the apical membrane resistance not only depends on the blocker concentration but also on the apical membrane potential. Also the fractional resistance, fR _a, and the voltage divider ratio, fV _a, will change with blocker concentration and voltage. Owing to this non-ohmic behaviour, measurements of fV _a in the presence of Ba²⁺ deviate markedly from the expected fR _a values. The inhibitory effect of Ba²⁺ and Cs⁺ was analysed at different transepithelial and apical membrane voltages. The relation between the Michaelis-Menten constants and the voltage could be fitted with equations based on Eyring rate theory with the assumption of a single binding site. With this model we calculated the relative electrical position of the binding site for the blocker (), referred to the extracellular side of the channel. We obtained for Ba²⁺, =0.34±0.05 and for Cs⁺, =0.81±0.01. Comparison of the results from apical and transepithelial I/V relations demonstrates that the analysis of the transepithelial data provides overestimated values of the Hill coefficient and results in an underestimation of .     

Current-voltage relations of Cs+-inhibited K+ currents through the apical membrane of frog skin

The voltage-dependence of the inhibitory effect of mucosal Cs⁺ on the inward K⁺ current through the apical membrane of frog skin (Rana temporaria) was studied by recording transepithelial current-voltage relations. Experiments were performed with skins exposed to NaCl and KCl Ringer solutions on the serosal and mucosal side respectively (contron skins), as well as with tissues incubated with K₂SO₄ Ringer solutions on both sides (depolarized skins). Studies of the dose-depedence of the Cs⁺ block showed that under both experimental conditions the apparent affinity of Cs⁺ increased as the transepithelial potential was clamped at higher mucosal positive voltages. Under control conditions, the concentration of Cs⁺ required to block 50% of the K⁺ current (K_Cs) recorded while the transepithelial voltage was clamped at zero mV was 16 mmol/1. K_Cs decreased exponentially with muscosal positive voltages. The dependence of K_Cs on the membrane potential was analyzed with Eyring rate theory in which Cs⁺ was assumed to block the K⁺ transport by binding to a site within the channel. The analysis showed that this site is located at a relative electrical distance =0.32 of the voltage drop across the apical membrane, measured from the cytosolic side. The Hill coefficient obtained from this analysis wasn=3.1. Experiments with K⁺-depolarized tissues showed that only inward K⁺ currents recorded with positive transepithelial voltages were depressed by external Cs⁺. Also under these conditions K_Cs showed an exponential dependence on the transepithelial potential. The analysis of these data with the rate theory revealed =0.09 andn=1.7. The difference in found in control and depolarized tissues can be explained by the influence of the basolateral membrane resistance on theI–V relations.     

Effect of calcium channel blocker verapamil on DNA synthesis and activity of nucleolar organizer in cardiomyocytes of newborn albino rats

Fivefold administration of L-type Ca²⁺ channel blocker verapamil in a dose of 1.5 mg/kg to newborn rats increased the parameters of DNA-synthetic activity in the myocardium: labeling index and intensity increased 1.24-fold on average. Body weight decreased with simultaneous increase in the absolute and relative heart weight. Morphometric parameters of the nucleus and nucleoli were practically unaffected. Our findings suggest that the Ca²⁺ channel blocker verapamil administered at the early postnatal stages can modulate morphogenesis of the heart Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 127, No. 6, pp. 658–660, June, 1999     

Electrophysiological investigation of microdissected gastric glands of bullfrog II. Basolateral membrane properties in the presence of histamine

Following the technical approach described in the preceding publication we have investigated if, and how, stimulation of gastric HCl secretion affects the basolateral ion transport properties of oxyntopeptic cells of Rana catesbeiana stomach. To this end microdissected gastric glands were punctured with conventional or H⁺-sensitive glass microelectrodes and the effects of changing bath ion concentrations on the cell membrane potential (V _b) and cell pH (pH_i) were determined. Except for a transient alkalinization, histamine (0.5 mmol/l) did not significantly affect V _b or pH_i. The latter averaged 7.18±0.03 (mean±SEM, n=5) under resting conditions (0.1 mmol/l cimetidine) and 7.21±0.07 (n=5) in the presence of histamine. In addition, neither the initial velocity nor the final steady-state value of the cell alkalinization following a 101 reduction of bath Cl^– concentration changed in the presence of histamine, and the same holds true for the cell acidification following a 101 reduction of bath HCO₃ ^– concentration. These observations indicate that the basolateral Cl^–/HCO₃ ^– exchanger was not stimulated by histamine, and that no other base transporters were activated. By contrast, the V _b response to elevation of bath K ⁺ concentration decreased, and so did the initial depolarizing V _b response to bath Cl^– substitution, while the secondary hyperpolarizing response increased. The latter observations are compatible with the notion that stimulation by histamine reduced a pH-insensitive part of the basolateral K⁺ conductance and reduced also the basolateral Cl^– conductance.     

A novel synergistic stimulation of Na+-transport across frog skin (Xenopus laevis) by external Cd2+- and Ca2+-ions

Isolated skin of the clawed frogXenopus laevis was mounted in an Ussing-chamber. The transcellular sodiumcurrent (I _Na) was identified either as amiloride-blockable (10^–3 mol/l) short-circuit current (I _SC), or by correctingI _SC for the shunt-current obtained with mucosal Tris. A dose of 10 mmol/l Cd²⁺ applied to the mucosal side increased the current by about 70%. The half-maximal effect was reached at a Cd²⁺-concentration of 2,6 mmol/l (in NaCl-Ringer). The quick and fully reversible effect of Cd²⁺ could not be seen when 10^–3 mol/l amiloride was placed in the outer, Na⁺-containing solution, nor when Na⁺ was replaced by Tris. This suggests that Cd²⁺ stimulatesI _Na. Cd²⁺ intefered with the Na⁺-current self-inhibition, and therefore with the saturation ofI _Na by increasing the apparent Michaelis constant (K _Na) of this process. The I _Na recline after stepping up mucosal [Na⁺] was much reduced in presence of Cd²⁺. Ca²⁺-ions on the mucosal side had an identical effect to Cd²⁺, and 10 mmol/l Ca²⁺ increaseI _Na by about 100%. The half-maximal effect was obtained with 4.4 mmol/l Ca²⁺. The mechanism ofI _Na-stimulation by Ca²⁺ did not seem to differ from that of Cd²⁺. Thus, although of low Na⁺-transport capacity,Xenopus skin appears to be as good a model for Na⁺-transporting epithelia asRanidae skin, with the exception of the calcium effect which, so far, has not been reported forRanidae.     

Molecular characterization of h − mutants of Schizosaccharomyces pombe

Summary Meade and Gutz (1976) have described mat2:2 mutants of Schizosaccharomyces pombe having various defects in the Plus (P) function; four different classes were distinguished. Of special interest are the class II mutants in which none of the P mating-type functions is expressed. We made Southern analyses of 23 class II strains. In most of these, the P cassette and the K region are deleted as in h ^–s strains, however, some distinct differences were found as to the intensity of the bands in the blots (classes Ila, Ilb 1, and IIb2). The class Ilb mutants have strong bands characteristic of lethal deletions (h ^–L) and mat2:1 ⁰ plasmids. Two class II mutants turned out to have a typical h⁹⁰ mating-type region with an intact P cassette, but they seem to have a completely defective switching signal at matl:1 (new class V). Mutants of classes I, III, and IV yielded band patterns identical to those of an h ⁹⁰ strain; they obviously have point mutations in the P cassette.     

Comparative study of the effects of recombinant tumor necrosis factor and synthetic peptides corresponding to its fragments on phagocytosis mediated by Fc- and MF-receptors

The effects of human recombinant tumor necrosis factor and its peptides on the macrophage-like cells of continuous cell line P388D1 were studied. One of the peptides, 123–131, was found capable of not only mimicking, but also of blocking the effects of recombinant tumor necrosis factor. The results permit us to tentatively identify the 123–131 site of the factor molecule as being responsible for its activating effect on macrophages. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 121, N ^o 3, pp. 304–308, March, 1996 Presented by I. P. Ashmarin, Member of the Russian Academy of Medical Sciences     

Na+/H+ countertransport and calcium exchange in peripheral blood cells of healthy subjects and patients with chronic heart failure

A comparative study is performed of Na⁺/H⁺ exchange and Ca²⁻ mobilization in erythrocytes and platelets of patients with stage I–II chronic heart failure caused by dilative cardiomyopathy and ischemic heart disease. A significant rise in the Na⁺/H⁺ exchange rate is found in the cells of chronic heart failure patients, which correlates with an elevated erythrocyte and platelet concentration of Ca²⁺ and an increased “calcium” response of platelets to inductors. The findings testify to a certain functional relationship between various cation-transporting cellular systems whose change in properties upon chronic heart failure can play an important pathogenic role. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 118, N ^o 12, pp. 572–575, December, 1994     

Promoter Methylations of p16 and p14 Genes in Early and Advanced Gastric Cancer. Correlations of the Modes of Their Occurrence with Histologic Type

The INK4a/ARF locus encodes two cell cycle-regulatory proteins, p16^INK4a and p14^ARF. These share an exon using different reading frames, and act through Rb and p53 pathways. Recently, it has been found that silencing of p16^INK4a and p14^ARF expressions by aberrant methylation of the CpG islands in the promoter regions is an alternative mechanism that inactivates possible tumor suppressor functions in various tumors. To clarify the features of gastric cancers with promoter methylation of p16^INK4a and p14^ARF, we investigated the methylation status in gastric cancer cell lines and primary gastric cancers using methylation-specific PCR (MSP), and correlated the methylation status with microsatellite instability (MSI), DNA ploidy pattern, p53 immunohistochemistry, and various clinicopathologic factors, paying attention to the correlations with the histologic types. Of 10 cell lines studied, silencing of the expression of p16^INK4a and p14^ARF due to promoter methylation was detected by MSP and RT-PCR in six (60%) and two (20%) cell lines, respectively. p14^ARF silencing was detected only in cell lines derived from gastric cancer of the diffuse type, while p16^INK4a silencing was found in cell lines derived from both diffuse and intestinal types. In 59 primary gastric cancers, promoter methylation of p16^INK4a and p14^ARF was found in 10 (17%) and 14 (24%) of the tumors independently, there being an association with DNA diploidy, but not with p53 immunohistochemistry. p16^INK4a methylation was found irrespective of tumor stages and histology. Whereas p14^ARF methylation was found more frequently in intestinal type cancers in an early stage and in diffuse type cancers in an advanced stage, MSI tended to be related especially to p14^ARF methylation in cancers of the intestinal type. Thus, the significance of p14^ARF methylation differed between intestinal and diffuse types, while such a difference was not observed in p16^INK4a methylation.     

Luminal transport system for H+/organic cations in the rat proximal tubule

The efflux of radiolabelled organic cations from the tubular lumen into proximal tubular cells was investigated by using the stop-flow microperfusion method. The efflux rate increased in the sequence: N ¹-methylnicotinamide (NMeN⁺) < cimetidine < tetraethylammonium (TEA⁺) < N-methyl-4-phenylpyridinium (MPP⁺). Preloading the animals by i.v. infusion or pre perfusion of the peritubular capillaries with NMeN⁺ increased the efflux rate of MPP⁺. Luminal efflux was also augmented when the tubular solution was made alkaline with HCO ₃ ^– or phosphate, whereby HCO ₃ ^– is more effective than phosphate. Replacement of Na⁺ by Cs⁺ showed no effect. With i.v. preloading the animals with NMeN⁺ and with 25 mM HCO ₃ ^– in the luminal perfusate the 2-s efflux follows kinetics with a Michaelis constant K _m=0.21 mmol/l and maximal flux J _max=0.42 pmol · cm^–1 · s^–1 and a permeability term with P=37.7 m² · s^–1. Comparing the apparent luminal inhibitory constant values for MPP⁺ with the apparent contraluminal values of substrates of homologous series, it was found that (1) limitation by molecular size occurs at the contraluminal cell side earlier than at the luminal cell side; (2) affinity increases with hydrophobicity of the substrates at the luminal cell side, with a steeper or equal slope than at the contraluminal cell side; (3) affinity increases with basicity (i.e. pKa values) at the luminal cell side with a steeper slope than at the contraluminal cell side. Taken together, substrates with low hydrophobicity and low basicity interact at the luminal cell side more weakly than at the contraluminal cell side. On the other hand large, hydrophobic substrates have, at the luminal cell side, a higher affinity than at the contraluminal cell side. Many substrates, however, have equal affinity at the luminal and contraluminal cell sides.     

The effect of low and high NaCl diets on oral glucose tolerance

Summary The effects of low and high NaCl diets on plasma glucose and insulin responses to glucose ingestion were investigated in 15 patients with essential hypertension. Oral glucose (75 g) tolerance tests were carried out while patients were taking diets with low (2 g/day) and high (20 g/day) NaCl content. Fasting plasma glucose and insulin levels were both significantly lower during ingestion of the high NaCl diet (p<0.05). After glucose ingestion, the incremental areas under the two hour plasma glucose and insulin curves were significantly smaller during ingestion of the high NaCl diet (glucosep<0.005 and insulinp<0.025). These findings that low NaCl diets increase the glycemic response to glucose loads suggest that use of NaCl restriction for the treatment of essential hypertension may not always be desirable.Abbreviations Na⁺, K⁺-ATPase sodium, potassium adenosinetriphosphatase - SEM standard error of the mean     

Effect of parathyroid hormone on the connecting tubule from the rabbit kidney: biphasic response of transmural voltage

The effect of parathyroid hormone (PTH) on ion transport was examined by observing transmural (V _T) and basolateral membrane voltage (V _B) in the in vitro perfused rabbit connecting tubule. Addition of 10 nmol/l PTH to the bath induced a biphasic response of V _T, with hyperpolarization followed by depolarization. Chlorophenylthioadenosine cyclic 3,5-monophosphate mimicked the effect of PTH, which did not change the V _B in the connecting tubule cell, but mainly caused changes in the apical membrane voltage. The V _T of distal convoluted tubule and the cortical collecting duct were not affected by PTH. Elimination of Na⁺ from the lumen abolished the PTH-induced V _T responses in the connecting tubule. In the presence of 10 mol/l amiloride, PTH caused an initial hyperpolarization but did not induce the late depolarization. The same was seen in the absence of luminal Ca²⁺. Either addition of 0.1 mmol/l ouabain to the bath or elimination of bath Na⁺ completely abolished the PTH-induced V _T changes. The presence of 5 mmol/l Ba²⁺ in the lumen did not affect the response to PTH. These findings indicate that the initial hyperpolarization may be caused by an increase in Na⁺ influx across the luminal membrane through an amiloride-insensitive Na⁺ conductive pathway and that the late depolarization may be caused by the decrease in Na⁺ influx through the amiloride-sensitive Na⁺ conductive pathway. Luminal Ca²⁺ is necessary for the late depolarization caused by PTH. On the basis of these observations, we suggest that PTH initially increase influxes of both Na⁺ and Ca²⁺ across the luminal membrane and that an increase in intracellular Ca²⁺ in turn suppresses Na⁺ entry through the luminal amiloride-sensitive Na⁺ channel.     

Murine CD5+, CD8+ Normal Fetal Liver Cells Enhance an Immune Response: Benzo(α) Pyrene-Exposed CD5+ Fetal Liver Cells are Inhibitors

The effects of 150 ug benzo(α)pyrene/gm body weight given intraperitoneally to the pregnant mouse at mid-gestation leads to long-lasting (> 18 months post-birth) immune deficiency in the progeny. Although the progeny are immunodeficient their T cell subsets induced marked enhancement and/or inhibition of cell proliferation in an immune response. Earlier we saw a striking increase (up to 7-fold) in CD8⁺ cells in the 18 day gestation liver of benzo(α)pyrene-exposed fetuses. We hypothesized that immune deficiency in carcinogen-exposed progeny could be attributed to an increase in classical CD8⁺ suppressor T-cells. Surprisingly, however, we found that CD8⁺ and CD5⁺ (Lyt 1⁺) cells of normal fetal liver enhance cell proliferation in an immune response. However, liver CD5⁺ cells from benzo(α)pyrene-exposed fetuses led to a dramatic reduction of the enhancing effect. Thus, as a novel finding, it appears that the profile of CD5⁺ cells, under the ontogenic influence of benzo(α)pyrene, transforms from cells that normally augment cell proliferation in an immune response to cells that are inhibitors. On the other hand the functional status of CD8⁺ cells is not affected. This change in physiological status of CD5⁺ cells may have important implications on T and B cell interactions, and the role of CD5⁺cells in T cell receptor signaling.