Dopamine Release via Protein Kinase C Activation in the Fish Retina

Calcium-dependent phospholipid-sensitive protein kinase [protein kinase C (PKC)] was partially purified from the carp (Cyprinus carpio) retina through DE 52 ion exchange and Cellulofine gel filtration chromatography. The phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) activated PKC in the nanomolar range. A major 38-kDa protein in the retinal supernatants (105,000 g) was phosphorylated in vitro by PKC during a short period (3 min). Other phosphoproteins also appeared during a further prolonged period (greater than 15 min). Rod-bipolar and dopamine (DA) interplexiform cells in the fish retina were immunoreactive to a monoclonal antibody to PKC (alpha/beta-subtype). The PKC antibody recognized a 78-kDa native PKC enzyme by means of an immunoblotting method. Subsequently, the effects of two kinds of PKC activators were investigated on [3H]DA release from retinal cell fractions containing DA cells that had been preloaded with [3H]DA. A phorbol ester (TPA) induced a calcium- and dose-dependent [3H]DA release during a short period (2 min), with the minimal effective dose being approximately 1 nM. Other phorbols having no tumor-promoting activity, such as 4 beta-phorbol and 4 alpha-phorbol 12,13-didecanoate, were ineffective on [3H]DA release. A synthetic diacylglycerol [1-oleoyl-2-acetylglycerol (OAG)], which is an endogenous PKC activator, was also able to induce a significant release of [3H]DA. Furthermore, TPA was found to release endogenous DA from isolated fish retina by a highly sensitive HPLC with electrochemical detection method. The OAG- or TPA-induced [3H]DA or DA release was completely blocked by inhibitors of PKC, such as 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7) and staurosporine.(ABSTRACT TRUNCATED AT 250 WORDS)     

GABAergic Inhibition on Dopamine Cells of the Fish Retina: A [3H]Dopamine Release Study with Isolated Cell Fractions

Inner retinal cells including dopamine (DA) cells were isolated and fractionated from the carp (Cyprinus carpio) retina by an enzyme cell dissociation and metrizamide gradient centrifugation method. When gamma-aminobutyric acid (GABA) antagonists (bicuculline and picrotoxin) were added into the perfusate over such a cell fraction, they stimulated the release of [3H]DA which had been preloaded in the cell fraction. The action of GABA antagonists was dose and Ca2+ dependent. Their minimal effective concentration was very low (0.5 microM). A similar action was elicited by high K+. In the presence of excess GABA, this stimulatory action of GABA antagonists and high K+ on [3H]DA release was completely abolished. To interpret the action of GABA antagonists on DA cells, isolated cell fractions were preincubated with GABAse. After such a treatment, the stimulatory effects of GABA antagonists and high K+ on [3H]DA release were differentiated from each other; the former disappeared whereas the latter remained unchanged. The data strongly suggest that GABA inhibits the DA release from retinal DA cells and thus the GABA antagonists affect [3H]DA release from cell fractions not by a direct membrane action but by a disinhibition mechanism via GABA receptors on the DA cell bodies.     

Synthesis and biological evaluation of aminothiazoles against Histoplasma capsulatum and Cryptococcus neoformans

The design and synthesis of a library of forty novel 2-aminoazole analogues as well as their evaluation as antifungal compounds against Histoplasma capsulatum and Cryptococcus neoformans is described. These structures were derived from N-[5-(1-naphthalenylmethyl)-2-thiazolyl]cyclohexanecarboxamide (41F5), a fungistatic agent previously identified through phenotypic screening (Antimicrob Agents Chemother. 2013;57:4349). Modifications to improve potency and water-solubility of 41F5 focused primarily on the 5-naphthalenyl group, the thiazole core, and the methylene linker between these two structural elements. In general, compounds with lipophilic [5+6] bicyclic ring systems, such as the 7-benzothiophenyl- and 4-indanyl groups, at the 5-position were 2–3 times more active against both fungal species as compared to 41F5. Also, introduction of a carbonyl group at the methylene linker of 41F5 resulted in a 2–3-fold increase in potency. These highly active compounds also showed generally low toxicities against murine P388D1 macrophages resulting in selectivity indices ranging from 63 to >200. Compounds that were highly active against fluconazole-sensitive C. neoformans strains had almost identical activity against fluconazole-resistant variants of this fungus indicating that 14α-demethylase is not their molecular target. Highly active compounds also retained activity against H. capsulatum phagocytosed into P388D1 macrophages.     

<Emphasis Type="Italic">Lecophagus vermicola</Emphasis> sp. nov., a nematophagous hyphomycete with an unusual hunting strategy

Lecophagus vermicola sp. nov. is described and illustrated as a predacious (carnivorous) hyphomycete living in bark fissures of living trees of Platanus and other angiosperm and gymnosperm trees, recorded in Hungary, Luxembourg and France. The fungus captures nematodes unlike other Lecophagus species, which are predators of rotifers and tardigrades. The morphology of the sessile, adhesive knobs differ from all previously described species of the genus which form adhesive pegs. Molecular data confirms that the new species belongs to the Lecophagus clade but without matching existing sequences. The fungus captures victims with adhesive knobs and colonizes its prey with a mycelium of rather broad hyphae on which, again, adhesive knobs are formed which penetrate the cuticule of the victim. Clusters of colonized nematodes form a network utilized to capture more prey. The fungus lives in the xeric, ephemerally aquatic habitat of bark fissures of standing, living or dead, corticated trunks and branches. The genus Haptocara is compared, which has similar adhesive knobs capturing nematodes and similar broad hyphae, but for which no molecular data was available.     

Interactions between Sox10, Edn3 and Ednrb during enteric nervous system and melanocyte development

The requirement for SOX10 and endothelin-3/EDNRB signalling pathway during enteric nervous system (ENS) and melanocyte development, as well as their alterations in Waardenburg-Hirschsprung disease (hypopigmentation, deafness and absence of enteric ganglia) are well established. Here, we analysed the genetic interactions between these genes during ENS and melanocyte development. Through phenotype analysis of Sox10;Ednrb and Sox10;Edn3 double mutants, we show that a coordinate and balanced interaction between these molecules is required for normal ENS and melanocyte development. Indeed, double mutants present with a severe increase in white spotting, absence of melanocytes within the inner ear, and in the stria vascularis in particular, and more severe ENS defects. Moreover, we show that partial loss of Ednrb in Sox10 heterozygous mice impairs colonisation of the gut by enteric crest cells at all stages observed. However, compared to single mutants, we detected no apoptosis, cell proliferation or overall neuronal or glial differentiation defects in neural crest cells within the stomach of double mutants, but apoptosis was increased in vagal neural crest cells outside of the gut. These data will contribute to the understanding of the molecular basis of ENS, pigmentation and hearing defects observed in mouse mutants and patients carrying SOX10, EDN3 and EDNRB mutations.     

Superdormant Spores of Bacillus Species Germinate Normally with High Pressure,Peptidoglycan Fragments,and Bryostatin

Superdormant spores of Bacillus cereus and Bacillus subtilis germinated just as well as dormant spores with pressures of 150 or 500 MPa and with or without heat activation. Superdormant B. subtilis spores also germinated as well as dormant spores with peptidoglycan fragments or bryostatin, a Ser/Thr protein kinase activator.Spores of Bacillus species are formed in sporulation, a process that is generally triggered by starvation for one or more nutrients (13, 19). These spores are metabolically dormant and extremely resistant to a large variety of environmental stresses, including heat, radiation, and toxic chemicals, and as a consequence of these properties, these spores can remain viable in their dormant state for many years (13, 18, 19). However, spores are constantly sensing their environment, and if nutrients return, the spores can rapidly return to growth through the process of spore germination (17). Spore germination is generally triggered by specific nutrients that bind to nutrient germinant receptors, with this binding alone somehow triggering germination. However, spore germination can also be triggered by many non-nutrient agents, including cationic surfactants such as dodecylamine, a 1:1 complex of Ca²⁺ with pyridine-2,6-dicarboxylic acid (dipicolinic acid [DPA], a major spore small molecule), very high pressures, specific peptidoglycan fragments, and bryostatin, an activator of Ser/Thr protein kinases (17, 19, 20). For nutrient germinants in particular, spore germination is also potentiated by a prior sublethal heat treatment termed heat activation (17).While normally the great majority of spores in populations germinate relatively rapidly in response to nutrient germinants, a small percentage of spores germinate extremely slowly. These spores that are refractory to nutrient germination have been termed superdormant spores and are a major concern for the food industry (8). Recently superdormant spores of three Bacillus species have been isolated by repeated germination of spore populations with specific nutrient germinants and isolation of remaining dormant spores (5, 6). These superdormant spores germinate extremely poorly with the nutrient germinants used in superdormant spore isolation, as well as with other nutrient germinants. All of the specific defects leading to spore superdormancy are not known, although an increased level of receptors for specific nutrient germinants decreases levels of superdormant spores obtained with the nutrients that are ligands for these receptors (5). Superdormant spores also have significantly higher temperature optima for heat activation of nutrient germination than the spore population as a whole (7).In contrast to the poor germination of superdormant spores with nutrient germinants, superdormant spores germinate normally with dodecylamine and Ca-DPA (5, 6). This is consistent with possible roles of nutrient germinant receptor levels and/or heat activation temperature optima in affecting spore superdormancy, since neither dodecylamine nor Ca-DPA triggers Bacillus spore germination through nutrient germinant receptors, and germination with these agents is also not stimulated by heat activation (11, 15, 17). However, the effects of high pressures, peptidoglycan fragments, and bryostatin, all of which almost certainly trigger spore germination by mechanisms at least somewhat different than triggering of germination by nutrients, dodecylamine, and Ca-DPA (2, 3, 11, 15, 20, 22, 23), have not been tested for their effects on superdormant spores. Consequently, we have compared the germination of dormant and superdormant spores of two Bacillus species by high-pressures, peptidoglycan fragments, and bryostatin.The spores used in this work were from Bacillus subtilis PS533 (16), a derivative of strain 168 that also carries plasmid pUB110, providing resistance to kanamycin (10 μg/ml), and Bacillus cereus T (originally obtained from H. O. Halvorson). Spores of these strains were prepared and purified as described previously (6, 10, 12). Superdormant spores of B. subtilis were prepared by germination following heat activation at 75°C for 30 min by two germination treatments at 37°C with 10 mM l-valine for 2 h, followed by isolation of remaining dormant spores, all as described previously (5, 10, 12). These superdormant spores germinated extremely poorly with 10 mM valine at 37°C, giving ≤10% germination in 2 h at 37°C, while the initial spore population exhibited >95% germination under the same conditions (data not shown). Superdormant B. cereus spores were isolated similarly, although heat activation was at 65°C for 30 min and the germinant was 5 mM inosine as described previously (6). These superdormant B. cereus spores exhibited <5% germination with inosine in 2 h at 37°C compared to the >95% germination of the initial dormant spores under the same conditions (data not shown).     

Rapid depletion of mutant eukaryotic initiation factor 5A at restrictive temperature reveals connections to actin cytoskeleton and cell cycle progression

Eukaryotic initiation factor 5A (eIF5A) is the only protein in nature that contains hypusine, an unusual amino acid derived from the modification of lysine by spermidine. Two genes, TIF51A and TIF51B, encode eIF5A in the yeast Saccharomyces cerevisiae. In an effort to understand the structure–function relationship of eIF5A, we have generated yeast mutants by introducing plasmid-borne tif51A into a double null strain where both TIF51A and TIF51B have been disrupted. One of the mutants, tsL102A strain (tif51A L102A tif51aΔ tif51bΔ) exhibits a strong temperature-sensitive growth phenotype. At the restrictive temperature, tsL102A strain also exhibits a cell shape change, a lack of volume change in response to temperature increase and becomes more sensitive to ethanol, a hallmark of defects in the PKC/WSC cell wall integrity pathway. In addition, a striking change in actin dynamics and a complete cell cycle arrest at G1 phase occur in tsL102A cells at restrictive temperature. The temperature-sensitivity of tsL102A strain is due to a rapid loss of mutant eIF5A with the half-life reduced from 6 h at permissive temperature to 20 min at restrictive temperature. Phenylmethyl sulfonylfluoride (PMSF), an irreversible inhibitor of serine protease, inhibited the degradation of mutant eIF5A and suppressed the temperature-sensitive growth arrest. Sorbitol, an osmotic stabilizer that complement defects in PKC/WSC pathways, stabilizes the mutant eIF5A and suppresses all the observed temperature-sensitive phenotypes.