Inhibition of MAP kinase activity moderates changes in expression and function of Cx32 but not claudin-1 during DNA synthesis in primary cultures of rat hepatocytes

The signal transduction pathways and activation of the MAP kinase or PI3 kinase signaling cascade regulate a variety of cellular processes, including proliferation and differentiation in hepatocytes. To elucidate the mechanisms of signal transmission required for the regulation of gap and tight junctions during DNA synthesis in rat hepatocytes, we determined changes of expression and function of gap and tight junctions of cells grown in primary culture, using inhibitors of signaling pathways for MAP kinase (PD98059) and PI3 kinase (LY294002). During the stimulation of DNA synthesis induced by epidermal growth factor (EGF), immunoreactivity and mRNAs of gap junction protein Cx32 and of tight junction protein claudin-1 markedly decreased with reduction of gap junctional intercellular communication (GJIC) and the fence function of tight junctions. In Western blots, whole-cell lysate of claudin-1 protein decreased and phosphorylated Cx32 protein in the insoluble fraction of Triton X-100 increased during the stimulation of DNA synthesis. During reinhibition of DNA synthesis, the changes of Cx32 and claudin-1 returned to control levels, as did both functions. In treatment with the inhibitors before DNA synthesis, PD98059 inhibited the changes of expression and function of Cx32, but not claudin-1, without inhibition of cell growth, whereas LY294002 completely inhibited cell growth. These findings indicate that the PI3 kinase pathway rather than the MAP kinase pathway plays an important role for EGF-induced proliferation of rat hepatocytes, and that changes of Cx32 in hepatocytes during the stimulation of DNA synthesis may be in part controlled through MAP kinase. Furthermore, Cx32, but not claudin-1, protein may be a target of activated MAP kinase in hepatocytes.     

Antibodies against the extracellular enveloped virus B5R protein are mainly responsible for the EEV neutralizing capacity of vaccinia immune globulin

In the event of smallpox bioterrorism, widespread vaccination may be required. Vaccinia immune globulin (VIG) has been used to treat complications from the smallpox vaccine. While the potency of VIG was defined by its ability to neutralize intracellular mature virus, a second form of vaccinia called the extracellular enveloped virus (EEV) is critical for virus spread in the host. The B5R-protein is one of many EEV-specific proteins. Immunoprecipitation and ELISA revealed that VIG recognizes the B5R-protein. An EEV plaque-reduction assay using a recombinant vaccinia that lacks the majority of the extracellular domain of B5R showed that the ability of VIG to neutralize EEV is principally directed at B5R. In addition, absorbing out the anti-B5R antibody present in VIG through the addition of recombinant B5R protein abrogated VIG's ability to significantly neutralize wild-type EEV. This work demonstrates the prominent role of B5R as a target of EEV-neutralizing activity of human antibodies.     

Aromatic residues affecting permeation and gating in dSlo BK channels

 Structural determinants of permeation in large unit conductance calcium-activated potassium channels (BK channels) were investigated. Y293 and F294 in the P-region of dSlo were substituted by tryptophans. Compared to wild-type channels, Y293W channels displayed reduced inward unitary currents while F294W channels exhibited normal inward current amplitudes but flickery kinetics. Both mutations produced changes in current/voltage relations under bi-ionic conditions. Sensitivity to block by external tetraethylammonium (TEA) was affected in both channels, and the voltage dependence of TEA block was increased in F294W channels. Both mutations also affected gating by shifting the half-maximal activation voltage of macroscopic conductance/voltage relations to more positive potentials, and eliminating a slow component of deactivation. The double mutant did not produce ionic currents. These data are consistent with a model in which Y293 contributes to a potassium-binding site close to the outer mouth of the dSlo pore, while F294 contributes to an energy barrier near this site. Received: 16 September 1997 / Received after revision: 20 November 1997 / Accepted: 21 November 1997     

Implication of the common {gamma} chain of the IL-7 receptor in intrathymic development of pro-T cells

The effects of IL-7 on the growth and differentiation of thymocyteswere analyzed using murine fetal thymua organ cultures (FTOC)in the presence of mAbs specific for the conventional IL-7 receptor(1L-7R) and for the common (c) chain. In FTOC, the developmentof CD4^–CD8^– double-negative thymocytes to CD4+CD8+double-positive (DP) and CD4+ or CD8+ single-positive (SP) cellswas not completely blocked by adding these mAbs, although cellgrowth was reduced by the treatment. To define a developingstage sensitive to the mAbs, most immature thymocytes, Pgp-1⁺c-kit cells, were cultured in the 2-deoxyguartosine treatedfetal thymus. In the presence of both mAbs in the culture, neitherDP nor SP thymocytes developed whereas either of the mAbs partiallyblocked their development. These results indicate that the Cchain is involved in early T cell development as an indispensablesubunlt of the functional IL-7 receptor complex.     

Amiloride-sensitive Na+ transport across cultured renal (A6) epithelium: evidence for large currents and high NaK selectivity

Electrical techniques were used to determine the NaK selectivity of the amiloride-sensitive pathway and to characterize cellular and paracellular properties of A6 epithelium. Under control conditions, the mean transepithelial voltage (V _T) was –57±5 mV, the short-circuit current (I _sc) averaged 23±2 A/cm² and the transepithelial resistance (R _T) was 2.8±0.3 kcm² (n=13). V _T and I _sc were larger than reported in previous studies and were increased by aldosterone. The conductance of the amiloride-sensitive pathway (G _amil) was assessed before and after replacement of Na⁺ in the mucosal bath by K⁺, using two independent measurements: (1) the slope conductance (G _T), determined from current-voltage (I-V) relationships for control and amiloride-treated tissues and (2) the maximum amiloride-sensitive conductance (G _max) calculated from the amiloride dose-response relationship. The ratio of G _amil in mucosal Na⁺ solutions to G _amil for mucosal K⁺ solutions was 22±6 for G _T measurements and 15±2 for G _max data. Serosal ion replacements in tissues treated with mucosal nystatin indicated a potassium conductance in the basolateral membrane. Equivalent circuit analyses of nystatin and amiloride data were used to resolve the cellular (E _c) and paracellular (R _j) resistances (5 kcm² and 8–9 kcm², respectively). Analysis I-V relationships for tissues depolarized with serosal K⁺ solutions revealed that the amiloride-sensitive pathway could be described as a Na⁺ conductance with a permeability coefficient (P _Na)=1.5±0.2× 10^–6 cm/s and the intracellular Na⁺ concentration (Na_i)=5±1 mM (n=5), similar to values from other tight epithelia. We conclude that A6 epithelia are capable of expressing large amiloride-sensitive currents which are highly Na⁺ selective.     

Auditory event-related potentials during stage 2 NREM sleep in humans

SUMMARY  Event-related potential (ERP) recordings were used to investigate the nature of auditory stimulus evaluation during stage 2 sleep. Frequent and rare stimuli, differing in intensity and frequency, were presented to six adult subjects while awake and asleep. The latency and voltage distribution of one of the long-latency components evoked during sleep resembled the P3 component evoked while awake. However, it was attenuated in voltage and superimposed on N3, a large late negative component, most probably the slow potential of the K complex. The identification of a P3-like potential during sleep suggests that the P3 potential is not solely a marker of active cognitive processes, but contains a small component which reflects automatic, pre-attentive evaluation of deviant stimuli.     

Morphological variability, lectin binding and Na+,K+-activated adenosine triphosphatase activity of isolated Müller (glial) cells from the rabbit retina

Rabbit retinal Müller cells were isolated by means of papaine and mechanical dissociation. These cells were shown to have a well preserved morphology and to preserve viability for many hours. Intense wheat germ agglutinin binding occurs on the photoreceptor side of Müller cells, especially in the microvillous region. Rabbit retinal Müller cells have a Na+,K+-activated adenosine triphosphatase activity in the same order of magnitude as brain astroglial cells.     

A controlled study of a specific MAO A reversible inhibitor (R011-1163) and amitriptyline in depressive illness

A double blind comparative study of amitriptyline and a new reversible MAO A inhibitor R011-1163 was conducted in 25 depressed inpatients over 4 weeks. Response to treatment was assessed with the Hamilton depression rating scale, the Carroll depression self rating scale and the Visual analogue scale. Both drugs produced significant changes in depressive symptomatology (P less than 0.01, MANOVA) and there were no statistically significant differences between drugs (P greater than 0.05 MANOVA). Side effects were of mild to moderate severity with dry mouth the most commonly reported side effect of amitriptyline and vague, generalised headache in patients, treated with R011-1163.