Intracellular ice formation in mouse oocytes subjected to interrupted rapid cooling

The formation of ice crystals within cells (IIF) is lethal. The classical approach to avoiding it is to cool cells slowly enough so that nearly all their supercooled freezable water leaves the cell osmotically before they have cooled to a temperature that permits IIF. An alternative approach is to cool the cell rapidly to just above its ice nucleation temperature, and hold it there long enough to permit dehydration. Then, the cell is cooled rapidly to -70 degrees C or below. This approach, often called interrupted rapid cooling, is the subject of this paper. Mouse oocytes were suspended in 1.5M ethylene glycol (EG)/PBS, rapidly cooled (50 degrees C/min) to -25 degrees C and held for 5, 10, 20, 30, or 40 min before being rapidly cooled (50 degrees C/min) to -70 degrees C. In cells held for 5 min, IIF (flashing) occurred abruptly during the second rapid cool. As the holding period was increased to 10 and 20 min, fewer cells flashed during the cooling and more turned black during warming. Finally, when the oocytes were held 30 or 40 min, relatively few flashed during either cooling or warming. Immediately upon thawing, these oocytes were highly shrunken and crenated. However, upon warming to 20 degrees C, they regained most of their normal volume, shape, and appearance. These oocytes have intact cell membranes, and we refer to them as survivors. We conclude that 30 min at -25 degrees C removes nearly all intracellular freezable water, the consequence of which is that IIF occurs neither during the subsequent rapid cooling to -70 degrees C nor during warming.     

Subcellular proteomics of cell differentiation: quantitative analysis of the plasma membrane proteome of Caco-2 cells

Human colorectal carcinoma (Caco-2) cells undergo in culture spontaneous enterocytic differentiation, characterized by polarization and appearance of the functional apical brush border membrane. To provide insights into the biology of differentiation, we have performed a comparative proteomic analysis of the plasma membranes from proliferating cells (PCs) and the apical membranes from differentiated cells (DCs). Proteins were resolved by SDS-PAGE, in-gel digested and analyzed by RP-LC and MS/MS. Alternatively, proteins were digested in solution, and tryptic peptides were labeled with isotopic tags and analyzed by 2-D LC followed by MS/MS. Among the 1125 proteins identified in both proteomes, 76 were found to be significantly increased in the membranes of DCs and 61 were increased in PCs. Majority of the proteins increased in the apical membranes were metabolic enzymes, proteins involved in the maintenance of cellular structure, transmembrane transporters, and proteins regulating vesicular transport. In contrast, majority of the proteins increased in the membranes of PCs were involved in gene expression, protein synthesis, and folding. Both groups contained many novel proteins with yet to be identified functions, which could provide potential new markers of the intestinal cells or of colorectal cancer.     

Selenium-independent epididymis-restricted glutathione peroxidase 5 protein (GPX5) can back up failing Se-dependent GPXs in mice subjected to selenium deficiency

We have previously characterized and cloned a secreted sperm-bound selenium-independent glutathione peroxidase protein (GPX5), the expression of which was found to be restricted to the mouse caput epididymidis. Because of the lack of selenium (Se) in the active site of this enzyme, unlike the other animal GPXs characterized to date, it was suspected that GPX5 does not function in the epididymis as a true glutathione peroxidase in vivo. In the present report, following dietary selenium deprivation which is known to reduce antioxidant defenses and favor oxidative stress in relation with depressed Se-dependent GPX activities, we show that the epididymis is still efficiently protected against increasing peroxidative conditions. In this model, the caput epididymides of selenium-deficient animals showed a limited production of lipid peroxides, a total GPX activity which was not dramatically affected by the shortage in selenium availability and an increase in GPX5 mRNA and protein levels. Altogether, these data strongly suggest that the selenium-independent GPX5 could function as a back-up system for Se-dependent GPXs.     

Factors affecting yield and survival of cells when suspensions are subjected to centrifugation

The goals of the centrifugation of cell suspensions are to obtain the maximum yield of cells with minimum adverse effects of centrifugation. In the case of mechanically sensitive cells such as mouse sperm, the two goals are somewhat contradictory in that g-forces sufficient to achieve high yields are damaging, and g-forces that yield high viability produce low yields. This paper mathematically analyzes the factors contributing to each goal. The total yield of pelleted cells is determined by the sedimentation rate governed by Stokes' Law, and depends on the relative centrifugal force, centrifugation time, size and shape of the cells, density of the cells and medium, viscosity of the medium, and the length of the column of suspension. Because in the situation analyzed the column is short relative to the rotor radius, the analysis considers the centrifugal field to be quasi-homogeneous. The assumption is that cells are not damaged during sedimentation, but that they become injured at an exponential rate once they are pelleted, a rate that will depend on the specific cell type. The behavior is modeled by the solution of coupled differential equations. The predictions of the analysis are in good agreement with experimental data on the centrifugation of mouse sperm.     

Complexity of phenotypes and symbiotic behaviour of <Emphasis Type="Italic">Rhizobium leguminosarum</Emphasis> biovar <Emphasis Type="Italic">trifolii</Emphasis> exopolysaccharide mutants

Quantitative in vitro antibacterial activities, i.e., minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs), of 12 -lactam antibiotics against Agrobacterium tumefaciens strains LBA4404 and EHA101 were examined, in order to identify antibiotics effective in eliminating the bacteria in Agrobacterium-mediated plant genetic transformation. The antibacterial activities of -lactams tested against strain EHA101 were equal to or less than those tested against strain LBA4404. Cefotaxime, cefbuperazone, and meropenem had high activities against strain LBA4404 (MBC <1 mg l^–1). Against strain EHA101, however, only meropenem showed activity comparable to that against strain LBA4404. The production of -lactamase was observed only in strain EHA101.Abbreviations CFU Colony-forming unit - MBC Minimum bactericidal concentration - MIC Minimum inhibitory concentration - PBP Penicillin-binding protein     

Effects of sugars on the kinetics of drying and on the survival of partially dehydrated larvae of Anopheles mosquitoes

The ability to cryopreserve a stage of Anopheles mosquitoes would facilitate the development of strains incapable of transmitting malaria. Cryopreservation requires that the freezable water in cell systems be removed or rendered incapable of undergoing ice formation. The present study was concerned with the rate at which water is removed from lst instar larvae of Anopheles gambiae by air-drying, with the extent of dehydration that the larvae will tolerate, and with the effect of trehalose and sucrose on both drying kinetics and survival. Eighty-one percent of the larvae are water. Air-drying removes 90% of that water in approximately 20 min. Survivals after partial dehydration are highest if the larvae are rehydrated in 1/2x isotonic saline (0.13 osm); they are poorest if rehydrated in water or 0.13 osm sucrose. In the former, about 34% survive the removal of half the water, but next to none survive the loss of >70% initial water. Prior exposure to 0.2 M trehalose for as little as 1 min slows the drying rate and increases the tolerance of the larvae to dehydration. With 30-min exposure, 88% survive the loss of 50% of their water and 63% survive the loss of 75%. Protection is abolished with 0.4 M trehalose. The results are similar with sucrose. It is substantially reduced if sugar-exposed larvae are briefly washed with water prior to drying. The protection appears not to be related to the decreased drying rate. Rather it appears related, by an unknown mechanism, to the presence of sugar on the outer surface of the larvae.     

Peroxynitrite activates K+-Cl- cotransport in human erythrocytes

Peroxynitrite was found to induce the release of K+ via the Na+/Cl- cotransport system, as do other oxidants. Since peroxynitrite is formed in vivo, its presence could contribute to a pathological dehydration of red blood cells.     

Indole-3-acetic acid in the culture medium of two axenic green microalgae

In the view of the facts that algal extracts have been used in agriculture asa source of plant growth stimulating agents and IAA has been shown to bepresent in the extracts, a study was planned to establish whether or notaxenic algae can produce IAA. Evidence is provided for extracellular IAAproduction during culture of two axenic green microalgae. IAAidentification was based on co-chromatography with the standard, analysisof UV and fluorescent spectra, and gas chromatography – selectedion-monitoring mass spectrometry. HPLC analyses showed that underthe experimental conditions the amounts of IAA released to the mediumby Scenedesmus armatus and Chlorella pyrenoidosa weregenerally low. IAA tended to occur in Scenedesmus armatus culturemedium at higher concentrations than in that of Chlorellapyrenoidosa. In fast-growing cultures of Scenedesmus armatus,constantly aerated with CO₂/air mixture, the concentration of IAAcalculated per cell was less than in the slow-growing cultures.