A convenient pH-gradient method for the determination of the maximum and minimum pH for microbial growth

Previously existing methods for determining the pH limits for the growth of microorganisms have involved (1), the setting up of individual cultures, each having a specific pH; (2), the pH gradient plate technique devised by Sacks (1956) in which a continuous pH gradient is established in a Petri dish by means of a buffer system; and (3), the pH gradient plate technique of Zak (unpublished), in which a continuous pH gradient is established by means of an electric current. The discontinuous pH gradient technique described here provides a convenient method of determining the maximum and minimum pH at which a microorganism can grow. The technique can be used aerobically or anaerobically, and has a precision of about ± 0.1 pH unit. Data are given for several yeasts and forSerratia marcescens. In all cases, the organisms tested continued to metabolize at pH values beyond those representing the limits for growth, sometimes by as much as 0.5 pH unit. The results suggest that pH limits are unsuitable criteria in microbial classification.     

Localization and cation dependence of a Ca2+- or Mg2+-ATPase from electrocytes of Electrophorus electricus, L

Electrocyte membranes of Electrophorus electricus exhibit high ATPase activity, as demonstrated by cytochemical and biochemical techniques. This activity is visualized as electron-dense deposits in electron micrographs, and appears to be localized only at the innervated face of the electrocyte. ATP hydrolysis can be detected cytochemically or biochemically only in the presence of calcium or magnesium. The effects of Ca or Mg on ATPase activity can be described by Michaelis-like functions with similar apparent Km values for Ca and Mg (0.41 mM and 0.23 mM, respectively). Vmax, however, is fivefold higher in the presence of Mg. The effects of the two cations are not additive, and pH dependence of ATP hydrolysis is identical in the presence of Ca or Mg (maximal at pH 8-9). Therefore, it can be concluded that Ca and Mg activate the same enzyme, the differences in Vmax being attributable to influences in kcat.     

Involvement of the Dihydropyridine Receptor and Internal CaStores in Myoblast Fusion

The process of myoblast fusion during skeletal myogenesis is calcium regulated. Both dihydropyridine receptor and ryanodine receptor are already present on muscle precursors, at the prefusional stage, before they are required for excitation–contraction coupling. Previous pharmacological studies have shown the need for a special pool of Ca²⁺associated with the membrane for the fusion process to occur. We hypothesized that this pool of Ca²⁺is mobilized via a machinery similar to that involved in excitation–contraction coupling. The process of fusion in rat L6 muscle precursors was either totally or partially abolished in the presence of the L-type calcium channel inhibitors SR33557 and nifedipine (half inhibition towards 2 μM), respectively. The inhibition was reversible and dose-dependent. Drugs able to deplete internal calcium stores (caffeine, ryanodine, and thapsigargin) were also tested on the fusion. Both caffeine and thapsigargin drastically inhibited fusion whereas ryanodine had no effect. This suggests that fusion may be controlled by internal pools of Ca²⁺but that its regulation may be insensitive to ryanodine. We presumed that an early form of the ryanodine receptor may exist, with different pharmacological properties than the adult forms. Indeed, Western blot analysis of pre- and postfusional L6 cells demonstrated the presence, at the prefusional stage, of a transient form of the ryanodine receptor protein with an apparent molecular weight slightly different from those of the classical skeletal and cardiac forms. Taken together, these results support the hypothesis that the fusion process is driven by a mechanism involving both the dihydropyridine receptor (α1 subunit of the L-type Ca²⁺channel) and the internal stores of Ca²⁺. The machinery underlying this mechanism might consist of slightly different forms of the classic molecules that in adult muscle ensure excitation–contraction coupling. It remains to be seen, however, whether the mobilization of the internal pool of Ca²⁺is triggered by the type of mechanism already described in skeletal muscle.     

NAD Glycohydrolases: A possible function in calcium homeostasis

NAD glycohydrolases are the longest known enzymes that catalyze ADP-ribose transfer. The function of these ubiquitous, membrane-bound enzymes has been a long standing puzzle. The NAD glycohydrolase are briefly reviewed in light of the discovery by our laboratory that NAD glycohydrolases are bifunctional enzymes that can catalyze both the synthesis and hydrolysis of cyclic ADP-ribose, a putative second messenger of calcium homeostasis.Abbreviations NADase nicotinamide adenine dinucleotide glycohydrolase - NAD nicotinamide adenine dinucleotide - ADP-ribose adenosine diphosphoribose - cADPR cyclic adenosine diphosphoribose     

Validation of the cumulative or replicate NAA method for the determination of trace elements in biological materials

Biological Trace Element Research - To make the best use of time and facilities, a neutron activation system, fully automatic, including spectrum and data processing, to be used with short-lived...     

Bafilomycin A1 Inhibits the Action of Tetanus Toxin in Spinal Cord Neurons in Cell Culture

Abstract: Tetanus toxin (TeNT) is one of the clostridial neurotoxins that act intracellularly to block neurotransmitter release. However, neither the route of entry nor the mechanism by which these toxins gain access to the neuronal cytoplasm has been established definitively. In murine spinal cord cell cultures, release of the neurotransmitter glycine is particularly sensitive to blockade by TeNT. To test whether TeNT enters neurons through acidic endosomes or is routed through the Golgi apparatus, toxin action on potassium-evoked glycine release was assayed in cultures pretreated with bafilomycin A1 (baf A1) or brefeldin A (BFA). baf A1, which inhibits the vacuolar-type H⁺-ATPase responsible for endosome acidification, diminishes the staining of acidic compartments and interferes with the action of TeNT in a dose-dependent manner. TeNT blockade of evoked glycine release is inhibited by 50 and 90% in cultures pretreated with 50 and 100 n M baf A1, respectively, compared with cultures treated with the inhibitor alone. The effects of baf A1 are fully reversible. In contrast, BFA, which disrupts Golgi function, has no effect on TeNT action. These findings provide evidence that TeNT enters the neuronal cytoplasm through baf A1-sensitive acidic compartments and that TeNT is not trafficked through the Golgi apparatus before its translocation into the neuronal cytosol.     

A scanning electron microscope study of normal and vitrified leaves from Datura insignis plantlets cultured in vitro

The surface anatomy of normal and vitreous leaves of plantlets obtained from Datura insignis Barb Rodr nodal segment cultures was compared using scanning electron microscopy. Normal and vitrified leaves are similar in several ways. They are both amphistomatic, and have similar distributions of glandular and non-glandular trichomes. Stomata have similar length, diameter and distribution in normal and vitreous plants. Immature stomata, which have closed pores, and plugged stomata, which contain an amorphous material between their guard cells, occur in both normal and vitrified leaves. Normal and vitreous leaves differ in the frequency of normal and abnormal stomata. Normal stomata have kidney-shaped guard cells and resemble closely those found in field-grown plants, whereas abnormal stomata have deformed guard cells. Normal stomata represent approximately 80% of the total number of stomata in normal leaves, but only 7% of the total number of stomata in vitreous leaves. Abnormal stomata represent 90% of the total number in vitreous leaves. The deformation of guard cells could possibly be a mechanical impediment to stomatal function.     

Characterization of hemocytes from the marine amphipod Parhyale hawaiensis (Dana 1853): Setting the basis for immunotoxicological studies

Hemocytes are circulating blood cells that play a crucial function in amphipods and other crustacean immune systems. The hemocytes of the marine tropical amphipod Parhyale hawaiensis have been used for the evaluation of DNA damage and micronuclei, but they have not been characterized in the scientific literature. The aim of this study was to describe the hemolymph cells of P. hawaiensis and study their phagocytotic activity. Basic dyes were used to differentiate the cell types and the presence of lipids. The total hemocyte counts (THCs) and the proportion and sizes of the hemocyte types were determined. Hemolymph was exposed to Escherichia coli for verification of the presence of phagocytosis. Three cell types, all containing lipids, were identified in P. hawaiensis: granulocytes (oval shape, 13.4 × 7.6 μm), semi-granulocytes (oval shape, 14.1 × 7.2 μm), and hyalinocytes (round shape, 9.6 × 7.2 μm). Those three cell types were found in different percentages in males (64.8%, 31.1%, and 4.2%) and females (70.1%, 28.2%, and 1.7%). THCs for males were 9007 ± 3800 cells per individual and 4695 ± 1892 cells per individual for females. The cells of E. coli were phagocytized by the hemocytes. Our findings increased the knowledge of hemocytes in P. hawaiensis and is a step forward in using hemocyte-based immune responses as an endpoint in ecotoxicology.