Independence of bacteriophage N15 lytic and linear plasmid replication from the heat shock proteins DnaJ, DnaK, and GrpE.

The chromosome of the temperate bacteriophage N15 replicates as a linear plasmid with covalently closed ends (or hairpins) when it forms a lysogen. I found that, in contrast to the cases for lambda and the low-copy-number plasmids F and P1, both phage and plasmid replication of N15 are independent of the heat shock proteins DnaJ, DnaK, and GrpE.     

Regulation of phosphoinositide hydrolysis induced by histamine and guanine nucleotides in human HeLa carcinoma cells. Calcium and pH dependence and inhibitory role of protein kinase C

The regulation of phospholipase C has been investigated in both intact and streptolysin-O permeabilized human HeLa carcinoma cells. Stimulation of phospholipase C by histamine and guanosine-5'-O-thiotriphosphate (GTP[S]) requires the presence of at least 10 nM free Ca2+, but is not significantly further increased by raising [Ca2+]i to greater than 10(-6) M. The pH optimum of the inositol phosphate response is at pH 6.8, while small changes in intracellular pH, as occur during hormonal stimulation (0.2-0.4 unit) attenuate the histamine/GTP[S]-induced stimulation of phospholipase C. Increasing cellular cAMP levels, either through addition of cell permeable cAMP analogues to intact cells or by stimulation with isoproterenol, does not affect histamine responsiveness, arguing against cross-talk between both signalling pathways. In contrast, we found that the response to histamine and/or GTP[S] is largely inhibited after brief pretreatment of the cells with phorbol esters or synthetic diacylglycerol prior to permeabilization, suggesting that protein kinase C exerts feedback inhibition at the level of, or downstream from, the putative GTP-binding protein.     

Prognostic value of response after three MOPP cycles in Hodgkin's disease--stage III and IV

Sixty-eight patients with Hodgkin's disease stage III and IV were evaluated after three out of six MOPP cycles. At that time, 46 (68%) were classified as early responders and 22 as slow responders. The criteria of response were: disappearance of B symptoms, decrease in the size of the largest lymph nodes (by more than 50%) and significant reduction (more than 20%) of mediastinal enlargement. Out of 43 early responders, 38 were in complete remission after six MOPP cycles and only five out of 22 slow responders. Such an early response is only related to the absence of B symptoms at the time of diagnosis (p less than 0.05). The survival curves of early responders and slow responders were significantly different (p less than 0.02). A rapid erythrocyte sedimentation rate (ESR) (greater than 50 mm) was the most frequently abnormal sign found in the group not responding after three MOPP cycles (p less than 0.0001). Such a significant prognostic value of early response is observed for stage III but not for stage IV patients. We conclude that early clinical response after three MOPP cycles is a good prognostic factor which must be kept in mind in the formulation of the therapeutic regimen for Hodgkin's disease stage III and IV.     

Rb fluxes in Chinese hamster ovary cells as a function of membrane cholesterol content

Steady-state fluxes of ⁸⁶Rb⁺ (as a tracer for K⁺) were measured in Chinese hamster ovary cells (CHO-K1) and a mutant (CR1) defective in the regulation of cholesterol biosynthesis; the membrane cholesterol content of this mutant was varied by growing it on a range of cholesterol supplements to lipid-free medium (Sinensky, M. (1978) Proc. Natl. Acad. Sci. U.S. 75, 1247–1249).Analogous to previous findings in ascites tumor cells, ⁸⁶Rb⁺ influx in the parent strain was differentiated into a ouabain-inhibitable ‘pump’ flux, furosemide-sensitive, chloride-dependent exchange diffusion, and a residual ‘leak’ flux.On the basis of this flux characterization, ⁸⁶Rb⁺ pump and leak fluxes were measured in the mutant as a function of membrane cholesterol content. Pump and leak fluxes, when expressed per ml cell water, were independent of the cholesterol content of the mutant. Moreover, ⁸⁶Rb⁺ fluxes in the mutant were equal to those in the parent strain. Our data imply that the flux behavior of K⁺ in the steady state is independent of the ordering of membrane lipid acyl chains.     

Current concepts in Bcl-2 family member regulation of female germ cell development and survival

Since the cloning of the bcl-2 gene in 1985, considerable progress has been made in elucidating the function of Bcl-2 and related proteins in controlling apoptosis. Although much of this work initially relied on the ectopic expression of bcl-2 gene family members in cell lines in vitro, a number of genetically manipulated mice have been generated to better understand the in vivo significance of specific family members to organ development and homeostasis. Of the many tissues that exhibit apoptosis at some point during fetal or postnatal life, the female gonads arguably possess one of the highest and most protracted incidences of apoptosis, associated with development and maturation of the germ line. Moreover, female germ cells (oocytes) are, for as-yet poorly understood reasons, extremely vulnerable to a host of pathological insults, such as anti-cancer therapies, that ultimately cause premature ovarian failure and infertility due to accelerated oocyte death. Accordingly, efforts to understand the occurrence and regulation of apoptosis in the ovary are of considerable importance from both biological and clinical perspectives. This review will highlight what is known of apoptosis in the female gonads, and the role that Bcl-2 family members play in regulating this process.     

Differential current decay profiles of epithelial sodium channel subunit combinations in polarized renal epithelial cells

In many epithelial tissues in the body, the rate of Na(+) reabsorption is governed by the activity of the epithelial sodium channel (ENaC). The assembly, trafficking, and turnover of the three ENaC subunits (alpha, beta, and gamma) is complex and not well understood. Recent experiments suggest that ENaC must be proteolytically cleaved for maximal activity and may explain the discrepancies reported in prior biochemical approaches focused on quantitating the trafficking and half-life of full-length subunits. As an alternative approach to examining the dynamics of ENaC subunits, we have generated doxycycline-repressible replication-defective recombinant adenoviruses encoding individual epitope-tagged mouse ENaC subunits and expressed these in polarized MDCK I cells. Co-infection with these viruses encoding all three subunits generates robust amiloride-sensitive currents in polarized MDCK cells. Significant current was also observed in cells expressing alpha- and gamma-mENaC in the absence of beta-mENaC. These currents did not appear to result from association with endogenous canine beta-ENaC. Treatment of alpha beta gamma-expressing cells with cycloheximide (CHX) resulted in the rapid inhibition (within 3 h) of approximately 50-80% of the initial current; however, a sizable fraction of the initial current remained even after 6 h of CHX. By contrast, CHX addition to cells expressing only alpha- and gamma-mENaC resulted in rapid decay in current with no residual fraction. Our data suggest that ENaC channels of differing stoichiometries are differentially trafficked and degraded and provide support for the possibility that noncoordinate trafficking of ENaC subunits may function in vivo as a mechanism to modulate ENaC activity.     

Osmotic swelling-provoked release of organic osmolytes in human intestinal epithelial cells

Human Intestine 407 cells respond to osmotic cell swelling by the activation of Cl(-)- and K(+)-selective ionic channels, as well as by stimulating an organic osmolyte release pathway readily permeable to taurine and phosphocholine. Unlike the activation of volume-regulated anion channels (VRAC), activation of the organic osmolyte release pathway shows a lag time of approximately 30-60 s, and its activity persists for at least 8-12 min. In contrast to VRAC activation, stimulation of organic osmolyte release did not require protein tyrosine phosphorylation, active p21(rho), or phosphatidylinositol 3-kinase activity and was insensitive to Cl(-) channel blockers. Treatment of the cells with putative organic anion transporter inhibitors reduced the release of taurine only partially or was found to be ineffective. The efflux was blocked by a subclass of organic cation transporter (OCT) inhibitors (cyanine-863 and decynium-22) but not by other OCT inhibitors (cimetidine, quinine, and verapamil). Brief treatment of the cells with phorbol esters potentiated the cell swelling-induced taurine efflux, whereas addition of the protein kinase C (PKC) inhibitor GF109203X largely inhibited the response, suggesting that PKC is involved. Increasing the level of intracellular Ca(2+) by using A-23187- or Ca(2+)-mobilizing hormones, however, did not affect the magnitude of the response. Taken together, the results indicate that the hypotonicity-induced efflux of organic osmolytes is independent of VRAC and involves a PKC-dependent step.     

Parenchymal cells purified from Xenopus liver and maintained in primary culture synthesize vitellogenin in response to estradiol-17β and serum albumin in response to dexamethasone

Xenopus liver was disaggregated by perfusion with collagenase. The released cells were separated according to their equilibrium densities on gradients of Metrizamide. Highly purified populations of parenchymal cells, sinusoidal cells, and melanocytes were obtained. All the parenchymal cells in an animal, before, during, and after hormone stimulation, are contained within a single density peak and subpopulations are not observed. Normal female parenchymal cells are less dense than normal male cells and estrogen stimulation causes a decrease in the peak density of the parenchymal cell population. Electron microscopic examination revealed the appearance and disappearance of lipid vacuoles in parenchymal cells following estrogen treatment. We hypothesize that the changing lipid content of these cells accounts for the shift in cell density. Immunofluorescent staining revealed that all liver parenchymal cells contain either vitellogenin or serum albumin, depending upon the prior treatment with hormones in vivo. Primary cultures of purified parenchymal and sinusoidal cells were established and were found to secrete different sets of proteins. The addition of estradiol-17β to cultures of parenchymal cells from either male or female frogs induces the synthesis and secretion of vitellogenin and two other polypeptides. Furthermore, the synthesis and secretion of polypeptides normally produced by parenchymal cells are curtailed as a result of estrogen treatment. Addition of glucocorticoids to a similar population of cells produces an increase in the synthesis and secretion of polypeptides, one of which is serum albumin. These results demonstrate that every parenchymal cell is responsive to two different classes of steroid hormones, estrogens and glucocorticoids, and in response to these hormones the same cells can synthesize and secrete alternate sets of polypeptides.     

Resveratrol Inhibits the Epithelial Sodium Channel via Phopshoinositides and AMP-Activated Protein Kinase in Kidney Collecting Duct Cells

Resveratrol, a naturally occurring phytoalexin, has reported cardioprotective, anti-inflammatory, chemopreventative and antidiabetic properties. Several studies indicate the multiple effects of resveratrol on cellular function are due to its inhibition of class 1A phosphoinositide 3-kinase (PI3K) mediated signaling pathways, but it also activates AMP-activated protein kinase (AMPK). As sodium transport in the kidney via the Epithelial Sodium Channel (ENaC) is highly sensitive to changes in phosphoinositide signaling in the membrane and AMPK, we employed resveratrol to probe the relative effects of phosphatidylinositol species in the plasma membrane and AMPK activity and their impact on ENaC activity in mouse cortical collecting duct (mpkCCD_c14) cells. Here we demonstrate that resveratrol acutely reduces amiloride-sensitive current in mpkCCD_c14 cells. The time course and dose dependency of this inhibition paralleled depletion of the PI(3,4,5)P₃ reporter (AKT-PH) in live-cell microscopy, indicating the early inhibition is likely mediated by resveratrol''s known effects on PI3K activity. Additionally, resveratrol induces a late inhibitory effect (4–24 hours) that appears to be mediated via AMPK activation. Resveratrol treatment induces significant AMPK activation compared with vehicle controls after 4 h, which persists through 16 h. Knockdown of AMPK or treatment with the AMPK inhibitor Compound C reduced the late phase of current reduction but had no effect on the early inhibitory activity of resveratrol. Collectively, these data demonstrate that resveratrol inhibits ENaC activity by a dual effect: an early reduction in activity seen within 5 minutes related to depletion of membrane PIP₃, and a sustained late (4–24 h) effect secondary to activation of AMPK.