Stretch-activated ion channels modulate the resting membrane potential during early embryogenesis

By using the patch-clamp technique, stretch-activated ionic channels were found in the membrane of cleaving freshwater fish embryos at the early stages of embryogenesis (2-256 cells). The application of negative pressure to the pipette increased the frequency of activation and the duration of bursts. This type of channel has a preferential K+ selectivity. When bathed on both membrane surfaces with 140 mM KCl the channel conductance was 71 pS. The kinetic behaviour did not depend markedly on either membrane potential (in the range from -70 to +70 mV) or calcium concentration on the cytoplasmic side of the membrane. On continuous recording, the probability of the channel being open was found to change periodically over a 5- to 20-fold range for different cells. These variations correlated with changes in resting potential and membrane conductance during the cell cycle. These results suggest that the oscillation of resting potential within the cell cycle is associated with the operation of stretch-activated ion channels.     

A quantitative study of enterotoxin production by sheep milk staphylococci

Of 124 staphylococcal strains isolated from sheep milk, 78 produced enterotoxin A, B, C, or D when evaluated by an enzyme-linked immunosorbent assay. Enterotoxins A and D, elaborated by 44 and 43 strains, respectively, showed the highest incidence. Enterotoxin production by coagulase-negative strains (one Staphylococcus cohnii, three S. epidermidis, five S. haemolyticus, and four S. xylosus) was detected. Linear and logarithmic-logarithmic regressions of optical density on enterotoxin concentration yielded the best-fitting equations for enterotoxin quantitation. A significantly higher incidence of enterotoxin producers and significantly higher levels of enterotoxins produced were recorded for coagulase-positive, thermostable nuclease-positive, hemolysis-positive, or mannitol-positive strains. Mannitol utilization was the best test for discriminating between enterotoxigenic and nonenterotoxigenic staphylococci.     

Nutrient dynamics within amazonian forests

Summary Relationships between fine root growth, rates of litter decomposition and nutrient release were analysed in a mixed forest on Tierra Firme, a Tall Amazon Caatinga and a Low Bana on podsolized sands near San Carlos de Rio Negro. Fine root growth in the upper soil layers (root mat+10 cm upper soil) was considerably higher in the Tierra Firme forest (1117 g m^-2 yr^-1) than in tall Cattinga (120) and Bana (235). Fine root growth on top of the root mat was stimulated significantly by added N in Tall Caatinga and Low Bana forests, by P in Tierra Firme and Bana forests, and by Ca only in the Tierra Firme forest. Rate of fine root growth in Tierra Firme forest on fresh litter is strongly correlated with the Mg and Ca content of litter. Rate of litter decomposition was inversely related to % lignin and the lignin/N ratio of litter. Litter contact with the dense root mat of the Tierra Firme increased rates of disappearance for biomass, Ca and Mg as compared with litter permanently separated or lifted weekly from the root mat to avoid root attachment. Nitrogen concentration of decomposing litter increased in all forests, net N released being observed only in Caryocar glabrum and Aspidosperma megalocarpum of the Tierra Firme forest after one year of exposure. Results emphasize the differences in limiting nutrients in amazonian forest ecosystems on contrasting soil types: Tierra Firme forests are particularly limited by Ca and Mg, while Caatinga and Bana forests are limited mainly by N availability.     

Benzodiazepines in the brain

Great progress has been made in the last 5 yr in demonstrating the presence of benzodiazepines (BDZs) in mammalian tissues, in beginning studies on the origin of these natural compounds, and in elucidating their possible biological roles. Many unanswered questions remain regarding the sources and biosynthetic pathways responsible for the presence of BDZs in brain and their different physiological and/or biochemical actions. This essay will focus on recent findings supporting that: (1) BDZs are of natural origin; (2) mammalian brain contains BDZs in concentrations ranging between 5.10⁻¹⁰–10⁻⁸ M; (3) dietary source of BDZs might be a plausible explanation for their occurrence in animal tissues, including man; (4) the formation of BDZ-like molecules in brain is a possibility, experimentally supported; (5) BDZ-like molecules including diazepam andN-desmethyldiazepam are elevated in hepatic encephalopathy; and (6) natural BDZs in the brain are involved in the modulation of memory processes. Future studies using the full range of biochemical, physiological, behavioral, and molecular biological techniques available to the neuroscientist will hopefully continue to yield exciting and new information concerning the biological roles that BDZs might play in the normal and pathological functioning of the brain.     

Levels and 75Se-labeling of specific proteins as a consequence of dietary selenium concentration in mice and rats

Selenium-labeled proteins (SLP) distinct from glutathione peroxidase (GSH-PX) recently have been purified and partially characterized. Antisera to two SLP, a 56-kDa and a 14-kDa protein, were generated in rabbits and used to examine expression of these proteins as a consequence of dietary selenium concentration (0.02, 0.2, 2.0 ppm) in mice and rats. Additionally, the kinetics of 75Se labeling in plasma, liver, kidney, and mammary gland were examined over a 40-hr time period as a function of dietary selenium concentration. A plasma 57-kDa protein was labeled by 30 min after 75Se injection and reached maximum labeling by 4 hr. The cellular 56-kDa and 14-kDa proteins, as well as GSH-Px, labeled progressively over 40 hr starting between 1 and 4 hr after injection. In general, the 56-kDa and GSH-Px followed similar labeling patterns, whereas the 14-kDa protein was labeled less and was not labeled in discernible quantities until 40 hr. The extent of labeling of all proteins was inversely proportional to the dietary selenium concentration and was probably a reflection of different endogenous selenium body pools. The most important observation was generated by the immunoblot data. The amount of 56-kDa and 14-kDa proteins as detected and measured on immunoblots was not a function of dietary selenium concentration. This result suggests that the synthesis and maintenance of the 56-kDa and 14-kDa proteins are not selenium dependent, a characteristic which distinguishes the two proteins from GSH-Px. The single exception to the above results was the 40% decrease of liver 14-kDa protein concentration in carcinogen-treated rats fed 2.0 ppm of selenium. An organic selenium compound, selenobetaine, did not lead to a decrease under similar conditions. In 15 rat mammary tumors induced by 7,12-dimethylbenzanthracene and analyzed on immunoblots, the SLP-56 was undetected in 5 cases and appeared as two bands (56,000 Da, 50,000 Da) in 10 cases. This latter result raises the possibility that the expression of SLP-56 may be altered in mammary tumors as compared with normal mammary gland.     

Regulation of potassium conductance in the cellular membrane at early embryogenesis.

At the early stages of development of the fresh water fish loach (Misgurnus fossilis) the resting membrane potential (Er) of cleaving cells oscillates periodically with an amplitude of 8-12 mV. Er oscillation correlates with the cell cycle and is accompanied by changes of K+ conductivity. Two types of K(+)-selective ionic channels with conductance of approximately 70 and 25 pS in symmetrical (150 mM KCl) solution were observed in the membrane of cleaving loach embryos. 'High' conductance and 'low' conductance channels were recorded in approximately 90% and 10% of patches investigated (n = 275), respectively? The activity of 'high' conductance channels was regulated by the application of pressure to the membrane, ie these channels were stretch-activated (SA). The activity of SA channels changes dramatically during the cell-cleavage cycle. At the beginning of interphase the probability of SA channels being in the open state (P0) was minimal, while at prometaphase the probability was increased 10-100-fold. Application of ATP to the cytoplasmic inside-out patches induced a reversible elevation of stretch sensitivity of the SA channels in 50% of the patches, while the non-hydrolyzable analogue of ATP was not effective. Combined application of ATP, cAMP and cAMP-dependent protein kinase (PK) induced a reversible elevation in the SA channel activity while inhibitors of PK prevented its activating effects. Phosphatase inhibitors prolonged the activating effect of PK on SA channels. We propose that oscillations of the resting potential during the cell-cleavage cycle arise due to modulation of SA channel sensitivity to stretch through cAMP-dependent phosphorylation.     

A clonal derivative of mammary epithelial cell line COMMA-D retains stem cell characteristics of unique morphological and functional heterogeneity

The COMMA-D cell line derived from mammary epithelial cells of midpregnant mice was shown previously to be heterogeneous as determined by phase-contrast microscopy, immunocytochemical staining, DNA content, and oncogenic potential (K.D. Danielson et al. (1984) Proc. Natl. Acad. Sci. USA 81, 3756; D. Medina et al. (1986) J. Natl. Cancer Inst. 76, 1143). Clonal subpopulations of COMMA-D cells have now been isolated by both transfection and selection using a dominant-selectable gene transfer vector and by limiting dilution. Despite their clonal origin, these subpopulations in many cases retained the heterogeneity of the parental COMMA-D line. Of 18 clonal lines assayed, only 5 were able to express beta-casein mRNA. Pooled populations of G418-resistant cells expressed substantially higher levels of beta-casein mRNA than the clonal lines. One of the expressing clonal lines, BNW-7, was characterized further, using immunocytochemical techniques. Approximately 10% of BNW-7 cells expressed casein under the appropriate hormonal and cell-substratum conditions by indirect immunofluorescent staining. Casein immunoperoxidase staining of BNW-7 cells on floating collagen gels revealed that casein-producing cells were localized in small alveolar structures, which were formed in a non-hormone-dependent fashion. The cells in these alveolar structures were cuboidal with basally located nuclei, expressed keratin intermediate filament proteins preferentially, and comprised approximately 18% of the total cells. Cells elsewhere on the surface of the gel displayed a flattened morphology, and expressed vimentin intermediate filament proteins preferentially. A proportion of COMMA-D cells, therefore, appeared to have some of the characteristics of mammary stem cells, and retained the ability to differentiate and form phenotypically heterogeneous cell populations in vitro.     

Heterogeneity of Benzodiazepine Receptors: Experimental Differences Between [3H]Flunitrazepam and [3H]Ethyl-β-carboline-3-carboxylate Binding Sites in Rat Brain Membranes

Abstract: This study was designed to analyze possible differences in the binding of [³H]flunitrazepam ([³H]FNZP) and [³H]ethyl - β - carboline - 3 - carboxylate ([³H]β-CCE), to rat brain membranes, in various experimental conditions. In cerebral cortex, hippocampus, cerebellum, and orain stem the number of binding sites for [³H]β-CCE was higher than for [³H]FNZP; both were displaced by clonazepam. Until the 7th day of postnatal brain development the numbers of [³H]FNZP and [³H]β-CCE sites were equivalent; but later on, the β-carboline sites increased to a higher level. Noradrenergic denervation by 6-hydroxydopamine was followed in the hippocampal formation. Already after 2 days, there was a decrease in [³H]FNZP sites, which reached 70% of control after 14 days. Similar results were obtained with DSP-4 denervation. This change was only in B_max and not in K_D, In contrast, the [³H]β-CCE sites did not change with denervation. Neonatal injection of l - 2,4,5 - trihydroxyphenylalamine or DSP-4 produced in the adult a decrease in [³H]FNZP sites in the cerebral cortex, in parallel with the noradrenergic denervation. On the other hand, there was an increase in the cerebellum and brain stem, in correspondence with the hyperinnervation by sprouting. In these rats, the number of sites for [³H]β-CCE did not change in the different brain regions. With 0.1% Triton X-100, applied to synaptosomal membranes, [³H]FNZP binding was reduced by 35%, while that of [³H]β-CCE was not significantly changed. These results suggest that there is heterogeneity of binding sites for benzodiazepine receptors in rat brain. A tentative interpretation of the experiments involving noradrenergic denervation and hyperinnervation, as well as those with Triton X-100, is that [³H]FNZP binds to pre- and postsynaptic receptors, while [³H]β-CCE binds mainly to postsynaptic benzodiazepine receptors.     

Stage specificity of selenium-mediated inhibition of mouse mammary tumorigenesis

The experiments reported herein examined the inhibitory role of selenium in chemical carcinogen-induced mouse mammary tumorigenesis. The results from four different experiments are presented herein and are summarized briefly. First, the results demonstrated that relatively low doses of dietary selenium (0.5–2.0 ppm) inhibited 7,12-dimethylbenzanthracene (DBMA)-induced mouse mammary tumorigenesis. At 2 ppm Se, the mammary tumor incidence was reduced from 56 to 15%. Second, the results suggested that the later stages of mammary tumorigenesis (preneoplastic to neoplastic transformation and tumor growth) are not as sensitive to selenium-mediated inhibition as the early stages, i.e., the induction and/or expression of mammary preneoplastic lesions. Finally, the results demonstrated that selenium markedly inhibited mammary tumorigenesis (from 42 to 8%) even when the mice were exposed to selenium only after the carcinogen treatments had been concluded. The results from these experiments are discussed from the viewpoint that selenium-mediated inhibition is a result of a direct block of DNA synthesis.