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gradient is established in a Petri dish by means of a buffer system; and (3), the pH gradient plate technique of Zak (unpublished), in which a continuous pH gradient is established by means of an electric current.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.     

Evidence of a Monoaminergic-Cholinergic Imbalance Related to Visual Hallucinations in Lewy Body Dementia

Senile dementia of Lewy body type is characterized clinically by a relatively acute onset of fluctuating memory loss and confusion, frequently accompanied by visual hallucinations. Neurochemical analyses of temporal cortex has revealed a distinction between hallucinating and nonhallucinating patients in both cholinergic and monaminergic transmitter activities. In contrast with the cholinergic enzyme choline acetyltransferase, which was more extensively reduced in hallucinating individuals, serotonergic S2 receptor binding and both dopamine and serotonin metabolites were significantly decreased in nonhallucinating cases. These results suggest that an imbalance between monaminergic and cholinergic transmitters is involved in hallucinogenesis in the human brain.     

Modulation of ζ-Protein Kinase C by Cyclic AMP in PC12 Cells Occurs Through Phosphorylation by Protein Kinase A

Abstract: Although cyclic AMP (cAMP) has been reported to cross talk with the protein kinase C (PKC) system, effects of elevated intracellular cAMP on the activities of specific PKC isoforms have not been studied. We report findings from a permeabilized cell assay that was used to examine changes in the activity of the atypical PKC isoforms brought about by exposure of PC12 cells to agents that elevate intracellular cAMP. We found that increases in intracellular cAMP led to rapid stimulation of atypical PKC activity, 40–70% above control, for a sustained period of time, a response that occurred independent of the phorbol 12-myristate 13-acetate (PMA)-sensitive PKC isoforms. Changes in intracellular cAMP levels resulted in a dose-dependent redistribution of ζ-PKC to the cytoplasm with a concomitant increase in the phosphorylation state of the enzyme. Incubation of purified ζ-PKC with increasing concentrations of PKA likewise caused a twofold increase in the phosphorylation state of ζ-PKC. In contrast to the positive effect that PKA-mediated phosphorylation had on the activity of ζ-PKC, the enzyme displayed reduced binding to ras when phosphorylated. Taken together, these findings are consistent with the hypothesis that protein phosphorylation of PKC acts as a positive effector of its enzyme activity and may serve as a negative modulator for interaction with other proteins.     

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     

Coordinate and independent regulation of αB-crystallin and HSP27 expression in response to physiological stress

α-Crystallins share structural and functional properties with the stress protein hsp27. These polypeptides are expressed at low constitutive levels in many tissues including brain, and αB-crystallin and hsp27 can accumulate in central nervous system glia in a variety of neurological conditions. We report here that heat shock and exposure to transition metals result in an increase in the steady state mRNA level of αB-crystallin and hsp27 in primary cultures of rat forebrain astrocytes. Both exposure to tumour necrosis factor-α and hypertonic conditions result in αB-crystallin mRNA accumulation but no change in the hsp27 mRNA level. Under some of these conditions increased synthesis and accumulation of αB-crystallin and hsp27 protein are also evident. We are unable to detect αA-crystallin mRNA in resting or stressed astrocytes. A novel phenomenon involving a transitory change in stress protein mRNA mobility in Northern blots during induction is reported, which is stress type and cell type independent. The results demonstrate multiple stress regulation of αB-crystallin and hsp27 in cultured astrocytes, suggesting that they can legitimately be regarded as stress proteins in the central nervous system. © 1994 wiley-Liss, Inc.     

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.     

Linkage map of seven polymorphic markers on rat Chromosome 18

A genetic linkage map of seven polymorphic markers was created with F₂ intercross progeny of F344/N and LEW/N rats and assigned to rat Chromosome (Chr) 18. Five of the markers described were defined by simple sequence length polymorphisms (SSLPs) associated with five genes: transthyretin (TTR), trypsin inhibitor-like protein (TILP), 2 adrenergic receptor (ADRB2), olfactory neuron-specific G protein (OLF), and gap junction protein (GJA1). One marker was defined by a restriction fragment length polymorphism (RFLP) detected with a probe for the human colony stimulating factor 1 receptor (CSF1R) gene. The D18N1R locus was defined by an anonymous DNA fragment amplified by the randomly amplified polymorphic DNA (RAPD) technique with a single short primer. These seven DNA loci formed a single genetic linkage group 30.4 cM in length with the following order: TTR-6.8 cM-D18N1R-9.1 cM-TILP-4.3 cM-CSF1R-0 cM-ADRB2-10.2 cM-OLF-0 cM-GJA1. The five SSLP markers were highly polymorphic. In a total of 13 inbred rat strains analyzed (F344/ N, LEW/N, LOU/MN, WBB1/N, WBB2/N, MR/N, MNR/N, ACI/N, SHR/N, WKY/N, BN/SsN, BUF/N, and LER/N), three to six alleles were detected for each marker. Remarkable linkage conservation was detected between the region of rat Chr 18 mapped and a region of mouse Chr 18. However, genes associated with these markers have been mapped to three different human chromosomes (Chrs 5, 6, and 18). The markers described here should be useful for genetic mapping studies and genetic monitoring of inbred rat strains.