Renal function and Na−K-ATPase in rats after suprarenal ligation of inferior vena cava

To assess the effects of altered renal function on Na?K-ATPase, the following groups of rats were studied:

1. rats with suprarenal vena cava ligation (SVCL), la. DOCA-treated rats with SVCL,
2. rats with infrarenal vena cava ligation (IVCL),
3. rats with glycerol-induced acute renal failure,
4. rats with bilateral ureteric ligation, and
5. K-exalate-treated rats with SVCL.

In group 1, acute renal failure with hyperkalemia developed and medullary Na?K-ATPase increased from 95±5 in control to 155±7 μmol Pi/mg prot//h,P<0.001, DOCA did not prevent the increase of Na?K-ATPase. In group 2, medullary Na?K-ATPase decreased from 130±10 in control to 88±7,P<0.01, in rats with IVCL. In group 3, cortical Na?K-ATPase decreased from 55±5 to 27±6,P<0.02. In group 4, Na?K-ATPase was unchanged. In group 5, maintenance of normokalemia prevented the rise in Na?K-ATPase. These experiments demonstrated a K-dependent activation of medullary Na?K-ATPase after SVCL but not in other forms of renal failure. Because SVCL diminishes drastically GFR per nephron, the present findings imply that increased loads of Na and K per nephron are not a prerequisite for an increase in medullary Na?K-ATPase. Hyperkalemia in presence of increased renal venous pressure seems to be causually related to the rise in medullary Na?K-ATPase activity.     

Biochemical genetics of the mammalian oxidative phosphorylation system: Analysis of the difference in the sensitivity of various Chinese hamster cell lines to inhibitors of the mitochondrial ATP synthase complex

Seven different Chinese hamster cell lines were found to vary greatly in their sensitivity to inhibitors of the mitochondrial ATPase. In plating-efficiency experiments, Chinese hamster lung V79 and bone marrow M3-1 cells were approximately 10,000-fold more resistant to oligomycin, 100-fold more resistant to efrapeptin, and 10-fold more resistant to ossamycin and leucinostatin than were ovary CHO or peritoneal B14 cells. In vitro experiments indicated that the increased resistance of V79 versus CHO cells to these inhibitors was due to an increased resistance of the mitochondrial ATPase. Heat-inactivation experiments indicated that there was a difference in the structure of the mitochondrial ATPase of V79 and CHO cells. Genetic experiments indicated that the difference in the sensitivity of V79 and CHO cells to inhibitors of the ATPase and the difference in the structure of the mitochondrial ATPase of V79 and CHO cells was due to a difference in both a nuclear and a cytoplasmic gene.     

Effect of bradykinin on activity of microsomal Na,K-ATPase of rat kidney and brain

Experiments in vitro showed that bradykinin, in a concentration of 10^?3–10^?12 M, affects neither microsomal Mg- or Na, KATPase from the rat kidney. Activation of microsomal Na, K-ATPase from the kidney and cerebral cortex was activated (by 30–40%) 20 min after injection of bradykinin (8 μg/g body weight) into the caudal vein; the Mg-ATPase activity was unchanged under these conditions.     

Effect of morphine in vitro on oxidative phosphorylation in rat liver mitochondria

Experiments in vitro on rat liver mitochondria showed that morphine inhibits oxidation of substrates in the presence of ADP and reduces the rate of phosphorylation and the activity of dinitrophenol-stimulated ATPase. The inhibition constant for all these reactions is the same, namely 6.5 mM. Respiration of mitochondria in the presence of an uncoupling agent and also the activity of ATP synthetase and ATPase of the submitochondrial particles are not inhibited in the presence of morphine. It is postulated that morphine inhibits the transfer of adenine nucleotides across the mitochondrial membrane.     

Dynamics of changes in rat brain and erythrocyte ATPase activity in hypoxic hypoxia

The metabolic action of hypoxia on the enzyme systems of active ion transport depends on the duration of exposure. The activity of Na, K-ATPase is sharply reduced during short periods of hypoxia (15 min) in the microsomes of the brain and in the erythrocytes. With an increase in the exposure to hypoxia an increase in Na, K-ATPase activity is observed (in the erythrocytes after an exposure of 1.5 h in the pressure chamber, in the microsomes after 2 h). The results are evidence of considerable functional changes in the cell membranes during exposure to acute hypoxic hypoxia.     

Short-term effect of aldosterone on renal sodium transport and tubular Na−K-ATPase in the rat

The short-term effect of one single injection of aldosterone on the renal sodium transport on one hand, and the Na?K-ATPase activity on the other hand, was studied in chronic adrenalectomized rats. Sodium transport was estimated by clearances, and Na?K-ATPase was measured in microdissected fragments of the nephron, according to our microtechnique previously described. Five to eight days after adrenalectomy, only 30% of the initial enzyme activity was recovered in the cortical collecting tubule (CCT). Administration of aldosterone completely restored the ATP-ase activity within three hours. Adrenalectomy also curtailed by 20–45% the activity of other nephron segments but aldosterone had no stimulatory effect on them. Sodium-reabsorption also increased after the hormone injection, following the same time (0.5<t _1/2<1 h) and dose dependencies (0.8<K _1/2<0.9 μg/kg) as those observed for the enzyme activity in the CCT. It is concluded that the short-term stimulation of Na?K-ATPase in the collecting tubule, after an acute administration of aldosterone, may be responsible for the simultaneous increase in sodium transport.     

Na+−K+-adenosinetriphosphatase and certain oxidoreductases in the kidney of rats with spontaneous hypertension

The activity of Na⁺?K⁺-adenosinetriphosphatase (Na?K-ATPase), succinate dehydrogenase (SD), lactate dehydrogenase (LD), and glucose-6-phosphate dehydrogenase (G-6-PD) was studied in the cortex and the outer and inner parts of the medulla of the kidneys of rats with spontaneous hypertension (line SHR) and control Wistar rats. No changes in the activity of the above enzymes compared with the control was found in SHR rats aged 6–8 weeks and in the prehypertensive stage. SHR rats at the age of 16–22 weeks, with persistent hypertension, differed from the control rats in their low specific Na?K-ATPase, SD, and LD activity in the tissue of the outer part of the medulla. This difference may be connected with the reduced intensity of energy metabolism and cessation of active sodium transport in the ascending limb of the loop of Henle in the SHR rats and may be responsible for the phenomenon of exaggerated sodium excretion characteristic of hypertension.     

Changes in the physicochemical and enzymic properties of myosin during ontogeny

During development of the calf fetus two forms of fetal myosin appear. The form of myosin characteristic of the skeletal muscle of the calf fetus at 2.5–6 months of development, not present in the later stages, is salted out with ammonium sulfate in a saturation of up to 25%; it has low ATPase activity and is easily denatured. The cholinesterase activity of the myofibrils is connected with this myosin fraction. The second form of fetal myosin is relatively stable, it has much higher ATPase activity, and it is salted out with ammonium sulfate in saturations of 35–50%. Both forms of myosin are found in the early stages of development of the calf fetus, but only the second form in the later stages. By electrophoresis in polyacrylamide gel marked heterogeneity is found in the region of the heavy components infetal myosin solutions obtained in the early periods of development.     

Use of the enzyme-linked immunosorbent assay for the early diagnosis ofMycoplasma pneumoniae infection

IgM and IgG antibodies toMycoplasma pneumoniae were measured in 147 sera from four groups of patients by means of an indirect enzyme-linked immunosorbent assay (ELISA) and the results compared with those obtained by other methods. A good correlation was demonstrated between the complement fixation test and ELISA-IgM and to a lesser extent ELISA-IgG; for the metabolic inhibition test the reverse was the case. The indirect haemagglutination test appeared to detect mainly IgM antibodies. Low levels of IgM antibodies were dectected by ELISA in 60 sera of children not suffering from pneumonia. However, if only high titres (> 800) were regarded as indicative ofMycoplasma pneumoniae infection, a presumptive diagnosis could have been made in 42 of 73 single acute phase sera from patients. Comparable results were obtained with IHA. The diagnostic level of IgM antibodies was reached during the second week of the disease. It is concluded that examination of a single serum sample by either ELISA-IgM or IHA may assist in early diagnosis ofMycoplasma pneumoniae infection.     

Insulin is involved in transcriptional regulation of NKCC and the CFTR Cl− channel through PI3K activation and ERK inactivation in renal epithelial cells

It is is well known that insulin stimulates glucose transport and epithelial Na⁺ channel (ENaC)-mediated Na⁺ reabsorption; however, the action of insulin on Cl^? secretion is not fully understood. In this study, we investigated the action of insulin on Na⁺–K⁺–2Cl^? cotransporter (NKCC)-mediated Cl^? secretion in epithelial A6 cells. Interestingly, insulin treatment remarkably enhanced the forskolin-stimulated Cl^? secretion associated with an increase in apical Cl^? conductance by upregulating mRNA expression of both CFTR and NKCC, although insulin treatment alone had no effect on the basal Cl^? secretion or apical Cl^? conductance without forskolin application. We next elucidated a role of phosphoinositide 3-kinase (PI3K) in the insulin-induced enhancement of the Cl^? secretion, since insulin actually activated PI3K, resulting in activation of Akt, a downstream molecule of PI3K. LY294002 (a PI3K inhibitor) reduced the Cl^? secretion by suppressing mRNA expression of NKCC, whereas insulin still had a stimulatory action on mRNA expression of CFTR even in the presence of LY294002. On the other hand, we found that a MEK inhibitor (PD98059) further enhanced the insulin-stimulated CFTR mRNA expression and the Cl^? secretion in forskolin-stimulated A6 cells and that insulin induced slight, transient activation of ERK followed by significant inactivation of ERK. These observations suggest that: (1) insulin respectively upregulates mRNA expression of NKCC and CFTR through activation of PI3K and inactivation of ERK; (2) insulin signals on mRNA expression of NKCC and CFTR are not enough to stimulate transepithelial Cl^? secretion, but enhance the stimulatory action of cAMP on transepithelial Cl^? secretion.     

Intravenous immunoglobulin-mediated immunosuppression and the development of an IVIG substitute

Immunoglobulins are glycoproteins produced by the cells of the immune system. Their primary function is to protect the body from pathogenic infection. Moreover, a concentrated polyclonal mixture of immunoglobulin G (IgG), the so-called intravenous IgG (IVIG), has been used to treat various chronic and systemic disorders of the immune system. Studies on the effects of IVIG in autoimmune disease models have revealed that IgG Fc fragments confer protection against various autoimmune diseases. The identification of this IgG Fc immunomodulatory component is important for the development of IVIG substitutes. The focus of this review is to introduce one of the Fc regulatory entities and to provide a summary of the current knowledge of the putative general mechanisms underlying IVIG activity in vivo on the basis of these Fc fragments. We also address the recent insights into several approaches for the development of IVIG substitutes.     

Dependence of cyclic-AMP induced relaxation on Ca2+ and calmodulin in skinned smooth muscle of guinea pigTaenia coli

cAMP (10^?6–10^?4 M) produced a dose-dependent relaxation of Ca²⁺-induced contraction in the guinea-pig taenia coli skinned with 1% Triton X-100. At 0.53 μM Ca²⁺ and 0.05 μM calmodulin (CaM), cAMP (10^?4 M) produced a maximal relaxation of 75% (pH 6.7; 25°C). Increasing Ca²⁺ (0.8 μM) or CaM (0.37 μM) reduced cAMP-induced relaxation to 25 and 5% respectively. At high CaM (5 μM), cAMP-induced relaxation could be completely inhibited by as low as 0.25 μM Ca²⁺. Furthermore, small increases in Ca²⁺ or CaM could effectively reverse the cAMP-induced relaxation in the continuous presence of cAMP. These results demonstrate that small modulations in the Ca²⁺-calmodulin activity have a strong effect on the ability of cAMP to produce a direct relaxing effect on the contractile proteins in skinned fiber. It is suggested that the effects of cAMP on the cellular mechanisms that lower cytoplasmic free Ca²⁺ concentration may act as the important determinants of the extent of the direct inhibitory effect of cAMP on the contractile elements. These two mechanisms may act in concert in this fashion to effect cAMP-induced relaxation in smooth muscle during β-adrenergic stimulation.     

Antibody-dependent cell-mediated cytotoxicity againstEscherichia coli O antigens

Antibodies againstEscherichia coli O antigen from rabbits immunized with formalin-killed bacteria were tested for cytotoxic capacity in an antibody-dependent cell-mediated cytotoxicity (ADCC) assay with human lymphocytes as effector cells and autologous papainized erythrocytes coated with O antigen as target cells. The cytotoxic titres were compared with the titres obtained with three methods of antibody quantitation. It was found that ADCC recorded antibodies with similar sensitivity as the enzyme-linked immunosorbent assay (ELISA) for IgG, but was much more sensitive than the ammonium sulphate precipitation (ASP) and indirect haemagglutination (IHA) usingβ-mercaptoethanol reduced sera. The ADCC titres were found to correlate very well with the titres obtained with ASP, ELISA and IHA for IgG but not for IgM, which is in accordance with a previous notion that ADCC is primarily mediated via IgG antibodies. ADCC should be considered as a possible immunopathologic mechanism in renal parenchymal damage in connection with urinary tract infections.     

Enzyme-linked immunosorbent assay for detection of solubleToxoplasma gondii antigen in acute-phase toxoplasmosis

Sixty-two sera from 51 patients with lymphadenopathy presumed to be due to acute-phase toxoplasmosis were tested for specific IgM class antibodies by both the immunofluorescence antibody toToxoplasma gondii in sera were first dissociated in 3M NaSCN. Antigen attached to the solid phase was detected with enzyme-coupled IgG antibody toToxoplasma antibody toToxoplasma gondii in sera were first dissociated in 3M NaSCN. Antigen attached to the solid phase was detected with enzyme-coupled IgG antibody toToxoplasma gondii. Neither hepatitis B surface antigen nor antigen ofMycoplasma pneumoniae, rubella, cytomegalovirus or herpes simplex virus interfered with this ELISA. Soluble antigen was detected in 13(30%) of 42 IgM-positive acute-phase toxoplasmosis patients and in only one of 20 sera cleared of IgM. None of an additional 44 IgM-negative patients with low IgG titres had a positive result in the antigen ELISA. Follow-up studies in four acute-phase toxoplasmosis patients showed that the soluble antigen cleared in all cases before the specific IgM antibodies. Simultaneous detection of IgM antibodies toToxoplasma gondii and soluble antigen would thus seem to indicate an early stage of the infection.     

Mutational profiling of the RAS,PI3K,MET and b-catenin pathways in cancer of unknown primary: a retrospective study of the Hellenic Cooperative Oncology Group

Cancer of unknown primary origin (CUP) had a poor prognosis, determined by clinico-histological characteristics, partly due to the lack of insights on its biology. We screened tumour DNA from 87 patients with CUP for CTNNB1 (coding exons 2,3,4,5), MET (coding exon 18), PIK3CA (coding exons 9,20), KRAS (coding exons 1,2), BRAF (coding exon 15) gene mutations by using dd-sequencing and evaluated their impact on prognosis. Mutated gene incidences in the 87 CUP cases were: KRAS 11 (12.6 %), BRAF 5 (5.7 %), PIK3CA 8 (9 %), MET 6 (6.7 %) and CTNNB1 18 (20.7 %). Several mutations in the KRAS gene were not the commonly encountered mutations in other solid tumours. Activating mutations were observed in 10.2 % in KRAS, 4.5 % in BRAF, 6.6 % in PIK3CA, 4.5 % in MET, and 19.5 % in CTNNB1. Activating mutations in PIK3CA coding exon 9 were inversely correlated with MET coding exon 18 activating mutations (p = 0.036). MET activating mutations were prognostic for poor Progression-Free Survival (median PFS 5 vs 9 months, p = 0.009) and Overall Survival (median OS 7 vs 20 months, p = 0.005). The complex profile of either CTNNB1 or MET mutations also had an adverse prognostic significance (median OS 11 vs 21 months, p = 0.015). No other gene mutation exhibited prognostic significance. In multivariate analysis, poor performance status, male gender, visceral disease and adenocarcinoma histology, but not gene mutations, were independently associated with poor patient outcome. CTNNB1 gene mutations are frequent, and along with MET mutations have an adverse prognostic effect in patients with CUP.     

An anti-human immunoglobulin M monoclonal antibody for detection of antibodies toToxoplasma gondii

An anti-human μ-chain monoclonal antibody, Tibi 82, was produced and tested for specificity by radioimmunoassay. Its reliability in detecting IgM antibodies toToxoplasma gondii was tested by two reverse immunosorbent methods (IgM-ISAGA and IgM-SPIHA) and the IgM fluorescent antibody test (IgM-IFA) on 400 sera. Whereas the results obtained with Tibi 82 and with two polyclonal reagents were highly correlated, the third commercial polyclonal reagent provided many false negative results. By standardizing IgM binding, Tibi 82 allowed the comparison of IgM-ISAGA with IgM-SPIHA on 100 sera: 17 % of the sera tested showed discrepancies due to the different toxoplasma antigens used. Although Tibi 82 facilitated the reading of results and enhanced sensitivity and specificity of the double-sandwich IgM-IFA method, the latter was still less sensitive than IgM-ISAGA with Tibi 82. Tests with the monoclonal antibody were consistently superior to tests with polyclonal antibodies.     

Action of histamine and 3-isobutyl-1-methylxanthine on cAMP activation of protein kinase in dog gastric mucosa

Dog gastric mucosa was incubated with histamine, IMX and db-cAMP, and the tissue was analyzed for cAMP content and cAMP-dependent protein kinase activity. Results show that in the absence of IMX, histamine does not produce measurable changes in either cAMP content or protein kinase activity ratios. In the presence of 5×10^?5 mol/I IMX histamine elicits a dose-dependent accumulation of cAMP, and this accumulation is reflected in elevated protein kinase activity ratios. When IMX concentration is increased to 5×10^?4 mol/l, the histamine effect is more pronounced. Incubation of gastric mucosa with 10^?6 mol/l db-cAMP results in elevated cAMP tissue levels both in the absence and presence of IMX, but protein kinase activity ratio is significantly elevated only in the presence of 5×10^?4 mol/l IMX. It is concluded that histamlen stimulates cAMP formation and protein kinase activation in dog gastric mucosa, but elevations are detectable only when the phosphodiesterase enzyme is inhibited.     

Searching for the cause of the acquired immune deficiency syndrome

An outbreak of unexplained immune deficiency associated with opportunistic infection and Kaposi's sarcoma is occuring in the USA and other parts of the world. Affected individuals with what has come to be known as the acquired immune deficiency syndrome (AIDS) have a high mortality. Epidemiological features suggest the presence of a transmissable agent, but no responsible agent has yet been identified. Homosexual and bisexual men make up 75 % of these affected individuals. Cytomegalovirus, Epstein Barr and herpes simplex viruses, organisms that commonly affect male homosexuals, may produce some features of AIDS. Individually or collectively, however, they can not account for the emergence of a previously unrecognized clinical syndrome. Hepatitis B is prevalent in patients with AIDS and may play a role as a co-factor in the disease. The properties of a number of other known viruses may provide a model for the pathogenesis of some features of the AIDS immunodeficiency. Newly described simian acquired immune deficiency syndrome (SAIDS) is the best available animal model. In man, the retrovirus, human T-cell leukemia virus (HTLV) may play a role in AIDS. However, HTLV or any other known virus cannot yet be assumed to cause AIDS. It is likely that an as yet unrecognized agent is the key causative agent of AIDS.     

Subcellular fractions in rubella immunoassays

Rubella virus-infected cells were fractionated by differential and sucrose gradient centrifugations. Rubella virus antigens distributed into all fractions but particulate material in the 100, 000×g pellet was shown to be enriched about two-fold for rubella virus antigen. Similarly, sucrose gradient fractions for rough endoplasmic reticulum and smooth cellular membranes were enriched for rubella virus antigens. The 100, 000 ×g pellet and the isolated cellular membranes proved to be useful when different fractions were used in solid-phase immunoassays for rubella virus-specific IgG or IgM. These fractions were equal in quality of the semipurified rubella virus preparations in the IgG assays but inferior to those in the IgM assays. However, simultaneous use of 35/25 % sucrose fractions from infected and non-infected cells reveals non-specific binding of IgM to the antigens and renders the IgM tests more specific for rubella virus.