HLA-A, -B and -DR antigens in IgA nephropathy

Fifty-six Irish patients with IgA Nephropathy were typed for HLA-A and -B antigens. Forty-six were typed for HLA-DR antigens. The frequencies of these antigens did not differ significantly from the frequency in the general population. The influence of genetic factors in the pathogenesis of this disease remains uncertain.     

Cloning of nucleoplasmin from Xenopus laevis oocytes and analysis of its developmental expression

Nucleoplasmin is the most abundant protein in the nucleus of Xenopus laevis oocytes. We cloned a cDNA coding for nucleoplasmin from an expression library of immature Xenopus laevis oocytes. The deduced amino acid sequence shows that the carboxyl terminus is very hydrophilic and contains an unusual stretch of 12 glutamic acid residues, which is consistent with one of the proposed functions of nucleoplasmin--that of promoting chromatin assembly. The last 50 amino acids are lysine- and alanine-rich and contain short stretches of homology to histone H1. These regions could be involved in interactions with nucleosomes. The levels of nucleoplasmin mRNA and protein during oogenesis and embryogenesis were investigated using Northern blots, Western blots, and in situ hybridization to oocyte sections. The mRNA is detected during oogenesis but not during embryogenesis, suggesting that nucleoplasmin may be an exclusively maternally expressed gene. However, the protein is present throughout embryogenesis and undergoes pronounced changes in its level of phosphorylation during maturation of the oocyte and just after midblastula transition. These results support the notion that nucleoplasmin is not only important in oocytes but also plays a major role during the rapid cleavages of early embryogenesis.     

NH4 + conductance in Xenopus laevis oocytes

 Current-clamp and voltage-clamp techniques were used to study the effects of NH₄ ⁺ on the cell membrane conductance in Xenopus laevis oocytes. Superfusing the oocytes with NH₄Cl resulted in a depolarization of the oocyte’s cell membrane potential and, at a clamp potential of –70 mV, in an inward current. The magnitude of the inward current was proportional to the NH₄Cl concentration in the extracellular solution and on membrane potential. The reversal potential, E _rev , was –35.5 ± 11.6 mV under control conditions and –3.1 ± 11.0 mV (n = 19) in the presence of NH₄Cl (10 mmol/l). Superfusion of the oocytes with nominally Ca²⁺-free solution affected the NH₄Cl-evoked response only marginally. Replacement of extracellular Na⁺ by N-methyl-D-glucamine⁺ markedly reduced, but did not eliminate, the NH₄Cl-sensitive current and shifted the reversal potential to more negative potentials. The NH₄Cl-induced current was substantially inhibited by 0.1 mmol/l flufenamate, and was less affected by blockers of the endogenous K⁺ conductance, Ba²⁺ and isosorbiddinitrate (ISDN). The results are compatible with the activation of a conductance by NH₄Cl for Na⁺ and NH₄ ⁺. The mechanism by which NH₄Cl activates the conductance remains unknown. Received: 9 December 1996 / Received after revision: 10 March 1997 / Accepted: 13 March 1997     

NH4 + conductance in Xenopus laevis oocytes

 Superfusing Xenopus laevis oocytes with NH₄Cl (10 mmol/l, pH 7.5) resulted in an inward current at a clamp potential of –70 mV. In paired experiments (n=22), the NH₄Cl-induced peak current was –293±94 nA, under control conditions (osmolality: 240 mosmol/kg), and rose to –523±196 nA when osmolality was reduced to 144 mosmol/kg. In parallel with the rise in NH₄Cl-induced inward current, membrane conductance at –70 mV doubled and the zero-current potential changed from +3.3±9.4 mV to –22.0±8.0 mV (n=22) in the presence of NH₄Cl during exposure to a hypoosmolar solution. In the absence of NH₄Cl, oocytes responded to hypoosmolality with a shift in zero-current potential to more negative values and an increased conductance which became partially sensitive to isosorbiddinitrate (ISDN), suggesting the activation of a volume-sensitive K⁺ channel. Membrane conductance in the presence of NH₄Cl was decreased by ISDN to similar extents under isoosmolal and hypoosmolal conditions, indicating that NH₄ ⁺ enters the oocytes through a volume-sensitive conductance separate from the ISDN-sensitive K⁺ channel. Received: 20 July 1998 / Received after revision and accepted: 19 October 1998     

SGK1 increases Na,K-ATP cell-surface expression and function in Xenopus laevis oocytes

The Na⁺-retaining hormone aldosterone increases the cell-surface expression of the luminal epithelial sodium channel (ENaC) and the basolateral Na⁺ pump (Na,K-ATPase) in aldosterone-sensitive distal nephron cells in a coordinated fashion. To address the question of whether aldosterone-induced serum and glucocorticoid-regulated kinase-1 (SGK1) might be involved in mediating this regulation of Na,K-ATPase subcellular localization, similar to that of the epithelial Na⁺ channel (ENaC), we co-expressed the Na,K-ATPase (rat 1- and Xenopus laevis 1-subunits) and Xenopus SGK1 in Xenopus oocytes. Measurements of the Na⁺ pump current showed that wild-type SGK1 increases the function of exogenous Na,K-ATPase at the surface of Xenopus oocytes. This appeared to be secondary to an increase in Na,K-ATPase cell-surface expression as visualized by Western blotting of surface-biotinylated proteins. In contrast, the functional surface expression of two other exogenous transporters, the heterodimeric amino acid transporter LAT1-4F2hc and the Na⁺/phosphate cotransporter NaPi-IIa, was not increased by SGK1 co-expression. The total pool of exogenous Na,K-ATPase was increased by the co-expression of SGK1, and similarly also by ENaC co-expression. This latter effect depended on the [Na⁺] of the buffer and was not additive to that of SGK1. When the total Na,K-ATPase was increased by ENaC co-expression, SGK1 still increased Na,K-ATPase cell-surface expression. These observations in Xenopus oocytes suggest the possibility that SGK1 induction and/or activation could participate in the coordinated regulation of Na,K-ATPase and ENaC cell-surface expression in the aldosterone-sensitive distal nephron.     

Expression of HLA-A and -B antigens on differentiating U-937 cells

Cells from the human immature monocytoid cell line U-937 were induced with 12-O-Tetradecanoyl-phorbol-13-acetate (TPA) to differentiate towards macrophage-like cells. The expression of HLA-antigens during differentiation was examined with a panel of monoclonal antibodies directed against monomorphic and polymorphic determinants. Class II antigens could be detected neither on uninduced nor on TPA-induced U-937 cells. While the expression of HLA-A3 did not change significantly during differentiation, the "supertypic" specificities HLA-Bw4 and Bw6 as well as the "private" specificity HLA-B18 could be detected only on a drastically decreased number of cells after 4 days of exposure to TPA. This may imply a selective loss of HLA-B molecules from the cell membrane and therefore a separate regulatory control of HLA-A and -B antigens.     

Voltage clamping of Xenopus laevis oocytes utilizing agarose-cushion electrodes

Two-electrode voltage clamping of expressed ion channels in intact oocytes of the South African clawed frog Xenopus laevis has been refined to allow stable, low-resistance electrical access to the cytosol (50–800 k). Glass microelectrodes were filled with a cushion of 1 % agarose at their tips to prevent KC1 leakage (agarose-cushion electrodes). Insertion of these electrodes into X. laevis oocytes yielded stable preparations for periods of more than l h with a stable input resistance of 1–4M. Furthermore, a simple modification of the voltage-clamp circuit (charging compensator) is described that increases the flexibility of arrangements for differential recording of the membrane potential in order to subtract voltage drops across a series resistance. The result is a considerable increase in the practically attainable speed of the voltage clamp with the conventional two-electrode arrangement. The performance of the charging compensator was tested on an equivalent circuit that simulates the oocyte and electrodes. In addition, the combination of agarose-cushion electrodes and the charging compensator was tested on oocytes expressing Shaker H4 currents. The fidelity of the voltageclamp circuit was also verified by measuring the membrane potential with additional independent microelectrodes connected to a differential amplifier, independent of the two-electrode voltage clamp system. The system described here will be useful for ion channel studies in X. laevis oocytes requiring long-term recordings and/or measurements of large, fast ion currents.     

HLA-A and -B antigens in Japanese patients with congenital hypothyroidism and their parents

The frequency of HLA-A and B antigens were studied in 32 Japanese patients with congenital hypothyroidism due to thyroid dysgenesis, and in their parents. The incidence of the Aw24 antigen was significantly higher in 27 mothers of patients with ectopic thyroid (91.3%, corrected P less than 0.037) and it seemed to be slightly higher in patients (77.7%) than in controls (56.8%). The Aw24 antigen was also found in 4 patients with thyroid hypoplasia and their mothers. No difference was found in the incidences of the antigen in the fathers of patients and in controls. The haplotype frequencies were not significantly different in controls, patients and their parents. These findings suggest that the gene for susceptibility to congenital hypothyroidism due to thyroid dysgenesis is closely linked to the gene for the HLA-A locus of the patients' mothers.     

HLA-A, -B, -DR, -DQ antigens in black North Americans

HLA antigens of black North Americans are not as well-characterized as those of other ethnic groups. We present results from HLA typing of black Americans using the 10th International Histocompatibility Workshop (IHW) sera. 1502 black Americans are typed for HLA-A, -B antigens. 128 black Americans are typed for HLA-DR, -DQ antigens. New specificities and splits established by the 10th IHW are identified in our data. Antigen frequency, gene frequency and haplotype frequency are computed for each phenotype observed. Haplotype A30--Bw42--DRw18--DQw4 and haplotype Aw36--Bw53 in strong linkage disequilibrium is characteristic of this black population. DR and DQ association patterns are similar to those of white North Americans. A major difference is the association of DRw8 with DQw7 in blacks versus an association of DRw8 with DQw4 in whites.     

Citrus exocortis viroid: survey of protein synthesis in Xenopus laevis oocytes following addition of viroid RNA

Citrus exocortis viroid (CEV) RNA was injected into oocytes of Xenopus laevis under conditions which demonstrated translation of bromegrass mosaic virus (BMV) RNA and rabbit hemoglobin mRNA. The viroid RNA was neither translated nor interfered with protein synthesis. These results were not altered by adenylation of the CEV RNA. BMV-directed protein synthesis could be detected following the injection of a total RNA preparation extracted from infected barley. The absence of evidence for a viroid-specified protein in these studies further supports a regulatory role for the viroid RNA.     

Proton transport mechanism in the cell membrane of Xenopus laevis oocytes

Mechanisms of H⁺ transport across the plasma cell membrane of prophase-arrested oocytes of Xenopus laevis were investigated by testing the effect of ion substitutions and inhibitors on cytoplasmic pH (pH_i), membrane potential (V _m) and membrane resistance (R _m). During superfusion with control solution of pH=7.4, pH_i was 7.49±0.12 (n=15), V _m was –61.9±7.8 mV (n=34) (cytoplasm negative), and R _m was 2.9±1.5 M (n=19). These data confirm that H⁺ ions are not distributed at electrochemical equilibrium. By following pH_i during recovery of the oocytes from an acid load (20 mmol/l NH₄Cl) in the presence and absence of extracellular Na⁺ or amiloride (1 mmol/l), a Na/H exchanger was identified. On the basis of the known Na⁺ gradient across the cell membrane, this transporter could suffice to generate the observed H⁺ disequilibrium distribution. Utilizing blockers or ion-concentration-step experiments no evidence was obtained for an ATP-driven H⁺ pump or for passive acid/base transporters such as H⁺ conductances or Na⁺ (HCO ₃ ^– )₃ cotransport. The membrane depolarization observed in response to extracellular acidification appeared to result from a pH-dependent, Ba²⁺-inhibitable K⁺ conductance.     

An endogenous inactivating inward-rectifying potassium current in oocytes of Xenopus laevis

 An endogenous inward-rectifying K⁺ current is described, which is present in native oocytes of some Xenopus laevis donors. Experiments were performed using defolliculated oocytes from donor frogs obtained from two different suppliers. In all oocytes from animals from one source, an inward-rectifying K⁺ current could be elicited with negative pulses from a holding potential of –20 mV in external solutions with a high K⁺ concentration. Increasing external K⁺ concentrations increased the amplitude of this current and shifted the reversal potential towards more positive potentials. In 118 mM KCl, the inward-rectifying K⁺ current partially inactivated between –20 and –80 mV and completely inactivated at more negative membrane potentials; 50% steady-state inactivation occurred near –50 mV. The time course of inactivation of the inward-rectifying current could be well fitted with two exponentials. The slow time constant had values of about 500 ms and was voltage independent. In contrast, the fast time constant and the time to reach the peak inward current decreased with more negative membrane potentials. Ba²⁺, Cs⁺, quinine (all 5 mM) and 50 mM tetraethylammonium partially blocked the inward-rectifying K⁺ current, whereas 10 mM 4-aminopyridine was without blocking effect. The oxidant chloramine-T blocked the inward-rectifying K⁺ current without slowing its inactivation. Received: 21 November 1995 / Received after revison and accepted: 24 May 1996     

HLA-A, -B, and -DR antigens in North African patients with nasopharyngeal carcinoma

Seventy-six North African patients (most from Algeria) affected with nasopharyngeal carcinoma (NPC) have been studied for their HLA-A, -B and -DR phenotypes and compared with a control North African population.
Antigens HLA-A3, HLA-B5 and HLA-Bw15 were found more frequently in the NPC group than in the control group (30.3% vs 17.6%, 38.2% vs 24.4% and 9.2% vs 0.8%, respectively). HLA-Aw33, HLA-B14 and HLA-DR4 were less frequent in the patients than in the controls (3.9% vs 16.8%, 1.3% vs 16% and 13.2% vs 29.1%, respectively). After correction for the number of specificities tested, these differences were not statistically significant. They were, however, more striking when compared to normal Kabyles (Algerian Berbers), a major ethnical population in Algeria, with lower incidences of the HLA-B5 antigen and of the HLA-Aw33-B14 haplotype. This could suggest, in North Africa, either the existence of MHC-linked genes of resistance or susceptibility to NPC, in Berbers especially, or a preferential occurrence of NPC in non-Berbers.
Antibody titers against the Epstein-Barr virus (EBV) associated early antigen (EA) and viral capsid antigen (VCA) have been measured. No correlation was observed between HLA phenotypes and the anti-EBV serological response of the patients.     

Histocompatibility antigens HLA-A, -B, -DR in Greek patients with Kaposi''s sarcoma

Thirty-two Greek patients with histologically documented Kaposi's sarcoma, aged 46 to 82 years, were typed for HLA-A, B and DR antigens. None of them was homosexual and they had not been subjected to any immunosuppressive therapy. The study revealed a significant increase of HLA-DR5 (53.1% vs. 21.4%, R.R. 4.1) and a decreased frequency of HLA-DR1 (3.3% vs. 16.6%, R.R. 0.16). An increased frequency of HLA-B18 was also noted (43.7% vs. 20.7% R.R. 2.96). These results indicate that the same positive association with HLA-DR5 antigen is observed in Greek patients as in other patients of Mediterranean origin and support the view that HLA linked factor(s) may have a role in the development of the disease.     

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.