Evidence for titratable gating charges controlling the voltage dependence of the outer mitochondrial membrane channel,VDAC

Summary A voltage-dependent anion-selective channel, VDAC, is found in outer mitochondrial membranes. VDAC's conductance is known to decrease as the transmembrane voltage is increased in either the positive or negative direction. Charged groups on the channel may be responsible for this voltage dependence by allowing the channel to respond to an applied electric field. If so, then neutralization of these charges would eliminate the voltage dependence. Channels in planar lipid bilayers which behaved normally at pH 6 lost much of their voltage dependence at high pH. Raising the pH reduced the steepness of the voltage dependence and raised the voltage needed to close half the channels. In contrast, the energy difference between the open and closed state in the absence of a field was changed very little by the elevated pH. The groups being titrated had an apparent pK of 10.6. From the pK and chemical modification, lysine epsilon amino groups are the most likely candidates responsible for VDAC's ability to respond to an applied electric field.     

Characterization of a human homologue of the murine peripheral lymph node homing receptor

Lymphocyte trafficking is a fundamental aspect of the immune system that allows B and T lymphocytes with diverse antigen recognition specificities to be exposed to various antigenic stimuli in spatially distinct regions of an organism. A lymphocyte adhesion molecule that is involved with this trafficking phenomenon has been termed the homing receptor. Previous work (Lasky, L., T. Yednock, M. Singer, D. Dowbenko, C. Fennie, H. Rodriguez, T. Nguyen, S. Stachel, and S. Rosen. 1989. Cell. 56:1045-1055) has characterized a cDNA clone encoding a murine homing receptor that is involved in trafficking of lymphocytes to peripheral lymph nodes. This molecule was found to contain a number of protein motifs, the most intriguing of which was a carbohydrate binding domain, or lectin, that is apparently involved in the adhesive interaction between murine lymphocytes and peripheral lymph node endothelium. In this study, we have used the murine cDNA clone to isolate a human homologue of this peripheral lymph node-specific adhesion molecule. The human receptor was found to be highly homologous to the murine receptor in overall sequence, but showed no sequence similarity to another surface protein that may be involved with human lymphocyte homing, the Hermes glycoprotein. The extracellular region of the human receptor contained an NH2 terminally located carbohydrate binding domain followed by an EGF-like domain and a domain containing two repeats of a complement binding motif. Transient cell transfection assays using the human receptor cDNA showed that it encoded a surface glycoprotein that cross reacted with a polyclonal antibody directed against the murine peripheral lymph node homing receptor. Interestingly, the human receptor showed a high degree of sequence homology to another human cell adhesion glycoprotein, the endothelial cell adhesion molecule ELAM.     

The effects of methyltestosteone on reproductive function in male greyhounds

Oral administration of methyltestosterone (MT) at 50 mg/dog/day to intact adult male greyhounds for 90 d resulted in decreased (P < 0.05) mean daily sperm output and mean testicular length. Additionally, the mean diameter of seminiferous tubules in MT-treated dogs tended to decrease (P = 0.08). Mean concentrations of luteinizing hormone (LH) and follicle stimulating hormone (FSH) and concentrations of testosterone in serum were also decreased or tended to decrease (P = 0.0003 to 0.059) at various sampling periods during MT treatment, suggesting alterations in spermatogenesis resulted from decreased serum concentrations of gonadotropins and steroids. Mean daily sperm output, mean testicular length, mean seminiferous tubule diameter and mean concentrations of FSH in serum were not decreased (P > 0.05) at the end of a 90-d recovery period. However, mean concentrations of serum LH and concentrations of testosterone were still lower (P < 0.05) during five of six and one of six sampling times, respectively, during the recovery period than the pretreatment levels, suggesting a prolonged effect of MT treatment on the pituitary/gonadal axis.     

Electrical Energy Changes Conductivity and Determines Optimal Electrotransformation Frequency in Gram-Negative Bacteria

In many bacterial electrotransformation protocols, pulse time is related to the time constant for a capacitor discharging across a sample of fixed resistance. Using an electroporator which controls pulse time independently of the capacitor time constant, we found that the resistance of bacterial suspensions fluctuates widely during capacitor discharge. With three gram-negative species of bacteria, electrotransformation frequency and survival could be more simply related to the electrical energy delivered in each pulse than to component parameters, such as initial field strength, capacitance, and pulse time. In each case, the number of transformants per survivor increased exponentially and leveled off when more than 0.5 to 1.0 J of electrical energy was delivered. An inverse log-linear relationship between survival and energy delivered was also observed for all three species.     

Polymorphic Admixture Typing in Human Ethnic Populations

A panel of 257 RFLP loci was selected on the basis of high heterozygosity in Caucasian DNA surveys and equivalent spacing throughout the human genome. Probes from each locus were used in a Southern blot survey of allele frequency distribution for four human ethnic groups: Caucasian, African American, Asian (Chinese), and American Indian (Cheyenne). Nearly all RFLP loci were polymorphic in each group, albeit with a broad range of differing allele frequencies (δ). The distribution of frequency differences (δ values) was used for three purposes: (1) to provide estimates for genetic distance (differentiation) among these ethnic groups, (2) to revisit with a large data set the proportion of human genetic variation attributable to differentiation within ethnic groups, and (3) to identify loci with high δ values between recently admixed populations of use in mapping by admixture linkage disequilibrium (MALD). Although most markers display significant allele frequency differences between ethnic groups, the overall genetic distances between ethnic groups were small (.066–.098), and <10% of the measured overall molecular genetic diversity in these human samples can be attributed to “racial” differentiation. The median δ values for pairwise comparisons between groups fell between .15 and .20, permitting identification of highly informative RFLP loci for MALD disease association studies.     

Protein Synthesis and Breakdown during Heat Shock of Cultured Pear (Pyrus communis L.) Cells

Cultured pear (Pyrus communis L. cv Passe Crassane) cells were subjected to temperatures of 39, 42, and 45[deg]C. Heat-shock protein (hsp) synthesis was greater at 30[deg]C than at temperatures above 40[deg]C and continued for up to 8 h. Both cellular uptake of radiolabeled methionine and total protein synthesis were progressively lower as the temperature was increased. Polysome levels decreased immediately when cells were placed at 39 or 42[deg]C, although at 39[deg]C the levels began to recover after 1 h. In cells from both temperatures, reassembly occurred after transfer of cells to 25[deg]C Four heat-shock-related mRNAs[mdash]hsp17, hsp70, and those of two ubiquitin genes[mdash]all showed greatest abundance at 39[deg]C and decreased at higher temperatures. Protein degradation increased with time at 42 and 45[deg]C, but at 39[deg]C it increased for the first 2 h and then decreased. In the presence of cycloheximide, which prevented hsp synthesis, protein degradation at 39[deg]C was as great as that at 45[deg]C in the absence of cycloheximide. The data suggest that hsps may have a role in protecting proteins from degradation at the permissive temperature of 39[deg]C. At temperatures high enough to inhibit hsp synthesis, protein degradation was enhanced. Although ubiquitin may play a role in specific protein degradation, it does not appear to be involved in increased protein degradation occurring above 40[deg]C.