Different susceptibility of red cell membrane proteins to calpain degradation.

The presence of low levels of calpastatin activity in erythrocytes of hypertensive rats affects regulation of calpain activity so it is highly susceptible to activation within physiological fluctuations in [Ca2+]. Under identical conditions, in red cells of normotensive rats, calpain activation is efficiently controlled by the high levels of calpastatin activity, and a progressive increase in proteinase activity can only be observed in parallel with a decrease in the level of calpastatin. In intact erythrocytes from hypertensive rats exposed to small variations in [Ca2+], degradation of anion transport protein (band 3) and Ca(2+)-ATPase appears as a primary event indicating that these two transmembrane proteins are probably early recognized as targets of intracellular calpain activity. Furthermore, band 3 protein seems to be structurally modified in erythrocytes from hypertensive rats, as indicated by its increased susceptibility to degradation in the presence of 10-50 microM Ca2+. In addition, when exposed to progressive and limited increases in [Ca2+], erythrocytes from hypertensive rats, but not those from normotensive rats, show a high degree of fragility that can be restored to normal values by inhibition of calpain. These results indicate that, within fluctuations in [Ca2+] close to physiological values, regulation of calpain activity is efficiently accomplished in normal erythrocytes but is completely lost in cells from hypertensive animals. Regulation is of critical importance in maintaining normal structural and functional properties of selective red cell membrane and cytoskeletal proteins, among which band 3 and Ca(2+)-ATPase appear to be the substrates with highest susceptibility to digestion by calpain.     

Activators of protein kinase C depolarize insulin-secreting cells by closing K+ channels.

Carbohydrate stimuli of insulin secretion depolarize the pancreatic B cell and the B-cell line RINm5F by inhibiting ATP-sensitive K+ channels. We examined the possibility that this effect is mediated by activation of protein kinase C. In RINm5F cells, the triose D-glyceraldehyde evoked a rapid increase of the mass of 1,2-diacylglycerol, the endogenous activator of protein kinase C. This effect is mainly due to de novo synthesis of the lipid from glycolytic intermediates, as glyceraldehyde carbon was incorporated into 1,2-diacylglycerol within 1 min of exposure to 14C-labelled glyceraldehyde. The effects of two exogenous activators of kinase C, 4-beta-12-phorbol-myristate 13-acetate (PMA) and 1,2-didecanoylglycerol (DC10) on single K+ channel currents were examined in RINm5F cell-attached membrane patches. Both PMA and DC10 depolarized the cells and decreased the open-state probability of the ATP-sensitive K+ channels. These actions were not due to changes in cellular ATP content, since PMA, like glyceraldehyde, failed to alter cellular ATP. As is the case for glyceraldehyde, PMA and DC10 raised cytosolic free Ca2+ [( Ca2+]i) and stimulated insulin secretion. Both of these effects are inhibited in the absence of external Ca2+. This, and the attenuation of the [Ca2+]i rise by verapamil, suggest that all three stimuli raise [Ca2+]i by promoting Ca2+ influx through voltage-gated channels in turn leading to insulin secretion. As the exogenous activators of protein kinase C mimic the effects of glyceraldehyde, it is proposed that the carbohydrate-mediated production of 1,2-diacylglycerol constitutes the link between metabolism and membrane depolarization.(ABSTRACT TRUNCATED AT 250 WORDS)     

Quantitative determination of methanogenic bacteria in the feces of different mammals

In this research on fresh human, cattle, swine, and rabbit feces, methanogenic bacteria were found in all samples examined, at the following concentrations per gram dry weight: swine, 10⁸; human, 10⁷; cattle, 10⁶; and rabbit, 10⁴. Anaerobic heterotrophic bacteria were found in the following concentrations per gram dry weight: human, 10¹¹; swine, 10¹¹; cattle, 10¹¹; and rabbit, 10¹⁰. The total number of O₂-intolerant was higher than that of O₂-tolerant bacteria: about 10–100 times for methanogenic and 100–1000 times for anaerobic heterotrophic bacteria.     

Rapidly exchanging Ca2+ stores of non-muscle cells

The rapid and transient redistribution of calcium from intracellular stores is a key event of cell activation. The nature and molecular composition of intracellular Ca2+ stores of non-muscle cells are the object of intense investigation. In this paper, we review: (a) the experimental evidence in favor of the existence of intracellular, membrane-bound compartments specialized for uptake, storage and release of calcium, (b) the main protein components of rapidly exchanging Ca2+ stores, i.e. Ca2+ pump, intralumenal Ca2+ binding proteins (calsequestrin, calreticulin, etc.) and Ca2+ channels sensitive to either inositol 1,4,5-trisphosphate or Ca2+, caffeine and ryanodine, and (c) the relationship between Ca2+ stores and the endoplasmic reticulum.     

Antioxidants and redox regulation: Changing notions in a changing world

The concepts of antioxidants and redox regulation are reconsidered in the light of recent findings, and some new future challenges for redox biology are outlined. It is suggested that antioxidants, thioredoxin-mediated redox regulation, and signal transduction mediated by reactive oxygen and nitrogen species, are all part of the same broad mechanism. The integration of different redox inputs, by affecting reversible thiol-disulfide dynamics in a set of target proteins, could result in the regulation of key processes such as proteolysis, gene expression and the functioning of selected metabolic pathways. Most interestingly, redox regulation is not just based on a binary “yes or no” response, and is therefore a convenient way to achieve graded control over the continuum of environmental variables.     

Mechanism of active shrinkage in mitochondria. I. Coupling between weak electrolyte fluxes.

1. A passive penetration of (NH4)2 HPO4 or of K2HPO4+nigericin occurs in respiratory-inhibited liver mitochondria. Addition of succinate at the end of the passive swelling initiates a shrinkage phase which leads to restoration of the initial mitochondrial volume. The rate and time of onset of the active shrinkage depend on the degree of stretching of the mitochondrial membrane. The rate of active shrinkage increases proportionally to the concentration of nigericin while it is strongly inhibited by valinomycin. 2. A number of SH inhibitors such as N-ethylmaleimide, p-chloromercuribenzoate, p-chloromercuriphenylsulphonate, dithiobisnitrobenzoate, exert a marked enhancing effect on the rate of shrinkage. The enhancing effect parallels titration of the phosphate carrier and inhibition of the passive phosphate efflux. In contrast, mersalyl is a powerful inhibitor of the rate of active shrinkage. The inhibition parallels that on phosphate passive efflux and requires higher mersalyl concentrations in respect to inhibition of phosphate influx. 3. The active shrinkage is discussed in terms of (a) a mechanoenzyme, (b) an electrogenic proton pump and (c) a proton-driven Pi pump.     

Isolation of stem-like cells from spontaneous feline mammary carcinomas: phenotypic characterization and tumorigenic potential

Current carcinogenesis theory states that only a small subset of tumor cells, the cancer stem cells or tumor initiating cells (TICs), are responsible for tumor formation and progression. Human breast cancer-initiating cells have been identified as CD44-expressing cells, which retain tumorigenic activity and display stem cell-like properties. Spontaneous feline mammary carcinoma (FMC) is an aggressive cancer, which shows biological similarities to the human tumor counterpart. We report the isolation and phenotypic characterization of FMC-derived stem/progenitor cells, showing in vitro self-renewal, long-lasting proliferation and in vivo tumorigenicity. Twenty-one FMC samples were collected, histologically classified and characterized for the expression of Ki67, EGFR, ER-α and CD44, by immunohistochemistry. By culture in stem cell permissive conditions, we isolated, from 13 FMCs, a CD44-positive subpopulation able to survive and proliferate in vitro as mammospheres of different sizes and morphologies. When injected in NOD/SCID mice, FMC stem-like cells initiate tumors, generating cell heterogeneity and recapitulating the original histotype. In serum-containing medium, spheroid cells showed differentiation properties as shown by morphological changes, the loss of CD44 expression and tumorigenic potential. These data show that stem-defined culture of FMC enriches for TICs and validate the use of these cells as a suitable model for comparative oncology studies of mammary biology and testing therapeutic strategies aimed at eradicating TICs.     

5-Aryl-4-carboxamide-1,3-oxazoles: potent and selective GSK-3 inhibitors

5-Aryl-4-carboxamide-1,3-oxazoles are a novel, potent and selective series of GSK-3 inhibitors. The optimization of the series to yield compounds with cell activity and brain permeability is described.