The β1-Subunit of the MaxiK Channel Associates with the Thromboxane A2 Receptor and Reduces Thromboxane A2 Functional Effects

The large conductance voltage- and Ca²⁺-activated K⁺ channel (MaxiK, BK_Ca, BK) is composed of four pore-forming α-subunits and can be associated with regulatory β-subunits. One of the functional roles of MaxiK is to regulate vascular tone. We recently found that the MaxiK channel from coronary smooth muscle is trans-inhibited by activation of the vasoconstricting thromboxane A₂ prostanoid receptor (TP), a mechanism supported by MaxiK α-subunit (MaxiKα)-TP physical interaction. Here, we examined the role of the MaxiK β1-subunit in TP-MaxiK association. We found that the β1-subunit can by itself interact with TP and that this association can occur independently of MaxiKα. Subcellular localization analysis revealed that β1 and TP are closely associated at the cell periphery. The molecular mechanism of β1-TP interaction involves predominantly the β1 extracellular loop. As reported previously, TP activation by the thromboxane A₂ analog U46619 caused inhibition of MaxiKα macroscopic conductance or fractional open probability (FP_o) as a function of voltage. However, the positive shift of the FP_o versus voltage curve by U46619 relative to the control was less prominent when β1 was coexpressed with TP and MaxiKα proteins (20 ± 6 mV, n = 7) than in cells expressing TP and MaxiKα alone (51 ± 7 mV, n = 7). Finally, β1 gene ablation reduced the EC₅₀ of the U46619 agonist in mediating aortic contraction from 18 ± 1 nm (n = 12) to 9 ± 1 nm (n = 12). The results indicate that the β1-subunit can form a tripartite complex with TP and MaxiKα, has the ability to associate with each protein independently, and diminishes U46619-induced MaxiK channel trans-inhibition as well as vasoconstriction.     

Poly(gamma-glutamylcysteinyl)glycine Synthesis in Datura innoxia and Binding with Cadmium : Role in Cadmium Tolerance

The effects of Cd on poly(γ-glutamylcysteinyl)glycine [(γEC)_nG] biosynthesis and formation of (γEC)_nG:Cd complexes were measured in two cell lines of Datura innoxia with differing Cd tolerance. In addition, RNA synthesis, protein synthesis, and GSH concentrations were measured during a 48 hour exposure to Cd. Exposure to 250 micromolar CdCl₂ was toxic to the sensitive line, whereas the tolerant line survived and grew in its presence. Cd-sensitive cells synthesized the same amount of (γEC)_nG as tolerant cells during an initial 24 hour exposure to 250 micromolar CdCl₂. However, rates of (γEC)_nG:Cd complex formation differed between the two cell lines with the sensitive cells forming complexes later than tolerant cells. In addition, the complexes formed by sensitive cells were of lower molecular weight than those of tolerant cells and did not bind all of the cellular Cd. Pulse-labeling of cells with l-[³⁵S]cysteine resulted in equivalent rates of incorporation into the (γEC)_nG of both cell lines during the initial 24 hours after Cd. Rates of protein and RNA synthesis were similar for both cell lines during the initial 8 hours after Cd but thereafter declined rapidly in sensitive cells. This was reflected by a decline in viability of sensitive cells. The GSH content of both cell lines declined rapidly upon exposure to Cd but was higher in sensitive cells throughout the experiment. These results show that the biosynthetic pathway for (γEC)_nG synthesis in sensitive cells is operational and that relative overproduction of (γEC)_nG is not the mechanism of Cd-tolerance in a Cd-tolerant cell line of D. innoxia. Rapid formation of (γEC)_nG:Cd complexes that bind all of the cellular Cd within 24 hours appears to correlate with tolerance in these cells.     

Amino Acid Composition of the Host-specific Toxin of Helminthosporium carbonum

The host-specific toxin of Helminthosporium carbonum (C₃₂H₅₀N₆O₁₀) was hydrolyzed by 6 n HCl to yield a number of α-amino acids. The common amino acids, proline and alanine, occurred in a ratio of 1:2. Two other unstable α-amino acids that produced lower color values with ninhydrin were also produced. One of these was tentatively identified as 2-amino-2,3-dehydro-3-methylpentanoic acid by electrolytic reduction to isoleucine. Additional ninhydrin-reacting substances were produced in low yield and probably represented secondary hydrolysis products of the unstable amino acids. The finding of an α,β-unsaturated linkage in H. carbonum toxin explains the instability of the compound and may also account for its specific toxicity.     

The epithelial sodium channel in the Australian lungfish,Neoceratodus forsteri (Osteichthyes: Dipnoi)

Epithelial sodium channel (ENaC) is a Na⁺-selective, aldosterone-stimulated ion channel involved in sodium transport homeostasis. ENaC is rate-limiting for Na⁺ absorption in the epithelia of osmoregulatory organs of tetrapods. Although the ENaC/degenerin gene family is proposed to be present in metazoans, no orthologues or paralogues for ENaC have been found in the genome databases of teleosts. We studied full-length cDNA cloning and tissue distributions of ENaCα, β and γ subunits in the Australian lungfish, Neoceratodus forsteri, which is the closest living relative of tetrapods. Neoceratodus ENaC (nENaC) comprised three subunits: nENaCα, β and γ proteins. The nENaCα, β and γ subunits are closely related to amphibian ENaCα, β and γ subunits, respectively. Three ENaC subunit mRNAs were highly expressed in the gills, kidney and rectum. Amiloride-sensitive sodium current was recorded from Xenopus oocytes injected with the nENaCαβγ subunit complementary RNAs under a two-electrode voltage clamp. nENaCα immunoreactivity was observed in the apical cell membrane of the gills, kidney and rectum. Thus, nENaC may play a role in regulating sodium transport of the lungfish, which has a renin–angiotensin–aldosterone system. This is interesting because there may have been an ENaC sodium absorption system controlled by aldosterone before the conquest of land by vertebrates.     

Encystment and Polymer Production by Azotobacter vinelandii in the Presence of β-Hydroxybutyrate

Cells of Azotobacter vinelandii encysted in Burk's nitrogen-free liquid media which had been supplemented with n-butyl alcohol, β-hydroxybutyrate, or crotonate. Butyraldehyde and butyrate did not influence the extent of encystment. In the absence of glucose, β-hydroxybutyrate enhanced the rate and extent of encystment. In the presence of glucose, it promoted abortive encystment, which was manifested by the disorganization of the exine and the release of a highly viscous material into the medium. The soluble, viscous polymer was separated from the medium by a series of ethyl alcohol precipitations and identified as a mucopeptide. It was cleaved by treatment with lysozyme and lysostaphin with a concomitant increase in reducing power. It contained 13.9% N; 56% amino acids, as alanine (alanine, lysine, and glutamic acids); and 42% hexosamines. The polymer appeared to be similar to a noncross-linked peptidoglycan.     

Decarboxylation of α-Keto Acids by Streptococcus lactis var. maltigenes

Decarboxylation rates for a series of C-3 to C-6 α-keto acids were determined in the presence of resting cells and cell-free extracts of Streptococcus lactis var. maltigenes. The C-5 and C-6 acids branched at the penultimate carbon atom were converted most rapidly to the respective aldehydes in the manner described for α-carboxylases. Pyruvate and α-ketobutyrate did not behave as α-carboxylase substrates, in that O₂ was absorbed when they were reacted with resting cells. The same effect with pyruvate was noted in a nonmalty S. lactis, accounting for CO₂ produced by some “homofermentative” streptococci. Mixed substrate reactions indicated that the same enzyme was responsible for decarboxylation of α-ketoisocaproate and α-ketoisovalerate, but it appeared unlikely that this enzyme was responsible for the decarboxylation of pyruvate. Ultrasonic disruption of cells of the malty culture resulted in an extract inactive for decarboxylation of pyruvate in the absence of ferricyanide. Dialyzed cell-free extracts were inactive against all keto acids and could not be reactivated.     

Potential Application of N-Carbamoyl-β-Alanine Amidohydrolase from Agrobacterium tumefaciens C58 for β-Amino Acid Production

An N-carbamoyl-β-alanine amidohydrolase of industrial interest from Agrobacterium tumefaciens C58 (βcar_At) has been characterized. βcar_At is most active at 30°C and pH 8.0 with N-carbamoyl-β-alanine as a substrate. The purified enzyme is completely inactivated by the metal-chelating agent 8-hydroxyquinoline-5-sulfonic acid (HQSA), and activity is restored by the addition of divalent metal ions, such as Mn²⁺, Ni²⁺, and Co²⁺. The native enzyme is a homodimer with a molecular mass of 90 kDa from pH 5.5 to 9.0. The enzyme has a broad substrate spectrum and hydrolyzes nonsubstituted N-carbamoyl-α-, -β-, -γ-, and -δ-amino acids, with the greatest catalytic efficiency for N-carbamoyl-β-alanine. βcar_At also recognizes substrate analogues substituted with sulfonic and phosphonic acid groups to produce the β-amino acids taurine and ciliatine, respectively. βcar_At is able to produce monosubstituted β²- and β³-amino acids, showing better catalytic efficiency (k_cat/K_m) for the production of the former. For both types of monosubstituted substrates, the enzyme hydrolyzes N-carbamoyl-β-amino acids with a short aliphatic side chain better than those with aromatic rings. These properties make βcar_At an outstanding candidate for application in the biotechnology industry.     

Metabolism of delta-Aminolevulinic Acid in Red and Blue-Green Algae

δ-Aminolevulinic acid was incorporated in vivo into C-phycocyanin and B-phycoerythrin in two species of the Rhodophyta (Cyanidium caldarium, Porphyridium cruentum) and three species of the Cyanophyta (Anacystis nidulans, Plectonema boryanum, Phormidium luridum). Amino acid analysis of phycocyanin-¹⁴C from C. caldarium cells which had been incubated with δ-aminolevulinate-4-¹⁴C showed that 84% of the radioactivity incorporated was present in the phycocyanobilin chromophore and less than 16% of the radioactivity cochromatographed with amino acids. These results indicate that δ-aminolevulinate is utilized predominantly via the porphyrin pathway in C. caldarium. Conversely, analysis of phycocyanin-¹⁴C prepared from cells of A. nidulans, P. boryanum, and P. luridum which had been incubated with radiolabeled δ-aminolevulinate demonstrated that 85%, 81%, and 93%, respectively, of the radioactivity incorporated cochromatographed with amino acids. The ratio of incorporated radioactivity in amino acids and phycoerythrobilin was 40:60 in P. cruentum phycoerythrin obtained from cells which had been incubated with δ-aminolevulinate-4-¹⁴C. Succinate-2-3-¹⁴C appeared to be as good a carbon source of amino acids as did C₄ and C₅ of δ-aminolevulinate. These data demonstrate a major alternate route (other than the porphyrin pathway) of δ-aminolevulinate metabolism in red and blue-green algae. The factors responsible for the extent to which δ-aminolevulinate is utilized for synthesis of porphyrins and their derivatives and routes of δ-aminolevulinate catabolism in the organisms employed are discussed.     

Carbohydrate-mediated Recognition of Larval Mosquito Hosts by Romanomermis culicivorax

Proteases, glycosidases, and lectins were tested and the results supported a role in host recognition for glycoproteins containing β-glucose and α-mannose on the cuticular surface of host and parasite. Carbohydrates containing α-glucose, galactose, fucose, or N-acetylglucosamine residues apparently are not involved in nematode attachment. Chitin or a related N-acetylglucosamine polymer was found in R. culicivorax preparasites. Treatment of preparasites with neuraminidase, which hydrolyzes sialic acids, increased nematode attachment to Anopheles freeborni larvae.     

The Zinc Transporter Slc30a8/ZnT8 Is Required in a Subpopulation of Pancreatic α-Cells for Hypoglycemia-induced Glucagon Secretion

SLC30A8 encodes a zinc transporter ZnT8 largely restricted to pancreatic islet β- and α-cells, and responsible for zinc accumulation into secretory granules. Although common SLC30A8 variants, believed to reduce ZnT8 activity, increase type 2 diabetes risk in humans, rare inactivating mutations are protective. To investigate the role of Slc30a8 in the control of glucagon secretion, Slc30a8 was inactivated selectively in α-cells by crossing mice with alleles floxed at exon 1 to animals expressing Cre recombinase under the pre-proglucagon promoter. Further crossing to Rosa26:tdRFP mice, and sorting of RFP⁺: glucagon⁺ cells from KO mice, revealed recombination in ∼30% of α-cells, of which ∼50% were ZnT8-negative (14 ± 1.8% of all α-cells). Although glucose and insulin tolerance were normal, female αZnT8KO mice required lower glucose infusion rates during hypoglycemic clamps and displayed enhanced glucagon release (p < 0.001) versus WT mice. Correspondingly, islets isolated from αZnT8KO mice secreted more glucagon at 1 mm glucose, but not 17 mm glucose, than WT controls (n = 5; p = 0.008). Although the expression of other ZnT family members was unchanged, cytoplasmic (n = 4 mice per genotype; p < 0.0001) and granular (n = 3, p < 0.01) free Zn²⁺ levels were significantly lower in KO α-cells versus control cells. In response to low glucose, the amplitude and frequency of intracellular Ca²⁺ increases were unchanged in α-cells of αZnT8KO KO mice. ZnT8 is thus important in a subset of α-cells for normal responses to hypoglycemia and acts via Ca²⁺-independent mechanisms.     

Specific Inhibition of Histone Deacetylase 8 Reduces Gene Expression and Production of Proinflammatory Cytokines in Vitro and in Vivo

ITF2357 (generic givinostat) is an orally active, hydroxamic-containing histone deacetylase (HDAC) inhibitor with broad anti-inflammatory properties, which has been used to treat children with systemic juvenile idiopathic arthritis. ITF2357 inhibits both Class I and II HDACs and reduces caspase-1 activity in human peripheral blood mononuclear cells and the secretion of IL-1β and other cytokines at 25–100 nm; at concentrations >200 nm, ITF2357 is toxic in vitro. ITF3056, an analog of ITF2357, inhibits only HDAC8 (IC₅₀ of 285 nm). Here we compared the production of IL-1β, IL-1α, TNFα, and IL-6 by ITF2357 with that of ITF3056 in peripheral blood mononuclear cells stimulated with lipopolysaccharide (LPS), heat-killed Candida albicans, or anti-CD3/anti-CD28 antibodies. ITF3056 reduced LPS-induced cytokines from 100 to 1000 nm; at 1000 nm, the secretion of IL-1β was reduced by 76%, secretion of TNFα was reduced by 88%, and secretion of IL-6 was reduced by 61%. The intracellular levels of IL-1α were 30% lower. There was no evidence of cell toxicity at ITF3056 concentrations of 100–1000 nm. Gene expression of TNFα was markedly reduced (80%), whereas IL-6 gene expression was 40% lower. Although anti-CD3/28 and Candida stimulation of IL-1β and TNFα was modestly reduced, IFNγ production was 75% lower. Mechanistically, ITF3056 reduced the secretion of processed IL-1β independent of inhibition of caspase-1 activity; however, synthesis of the IL-1β precursor was reduced by 40% without significant decrease in IL-1β mRNA levels. In mice, ITF3056 reduced LPS-induced serum TNFα by 85% and reduced IL-1β by 88%. These data suggest that specific inhibition of HDAC8 results in reduced inflammation without cell toxicity.     

Characterization of Poly-γ-Glutamate Hydrolase Encoded by a Bacteriophage Genome: Possible Role in Phage Infection of Bacillus subtilis Encapsulated with Poly-γ-Glutamate

Some Bacillus subtilis strains, including natto (fermented soybeans) starter strains, produce a capsular polypeptide of glutamate with a γ-linkage, called poly-γ-glutamate (γ-PGA). We identified and purified a monomeric 25-kDa degradation enzyme for γ-PGA (designated γ-PGA hydrolase, PghP) from bacteriophage ΦNIT1 in B. subtilis host cells. The monomeric PghP internally hydrolyzed γ-PGA to oligopeptides, which were then specifically converted to tri-, tetra-, and penta-γ-glutamates. Monoiodoacetate and EDTA both inhibited the PghP activity, but Zn²⁺ or Mn²⁺ ions fully restored the enzyme activity inhibited by the chelator, suggesting that a cysteine residue(s) and these metal ions participate in the catalytic mechanism of the enzyme. The corresponding pghP gene was cloned and sequenced from the phage genome. The deduced PghP sequence (208 amino acids) with a calculated M_r of 22,939 was not significantly similar to any known enzyme. Thus, PghP is a novel γ-glutamyl hydrolase. Whereas phage ΦNIT1 proliferated in B. subtilis cells encapsulated with γ-PGA, phage BS5 lacking PghP did not survive well on such cells. Moreover, all nine phages that contaminated natto during fermentation produced PghP, supporting the notion that PghP is important in the infection of natto starters that produce γ-PGA. Analogous to polysaccharide capsules, γ-PGA appears to serve as a physical barrier to phage absorption. Phages break down the γ-PGA barrier via PghP so that phage progenies can easily establish infection in encapsulated cells.     

A New Interpretation of the Dynamic Changes of the Potassium Conductance in the Squid Giant Axon

The solutions, n(t), of the differential equation dn/dt = α (1 - n) n (4 - 6n + 4n² - n³) - βn² (4 - 6n + 4n² - n³) in which α and β are instantaneous functions of membrane potential, are shown to fit with good accuracy the time courses of the rise of potassium conductance during depolarizing steps in clamp potential, found experimentally by Hodgkin and Huxley and by Cole and Moore. The equation is derived by analysing the dynamic behaviour of a system consisting of a square array of interacting pores. The possible role of Ca⁺⁺ ions in this system is discussed.     

Acetolactate metabolism and the presence of a dehydroxy acid dehydratase in micro-organisms

1. The growth characteristics of nine micro-organisms on complex broth and defined media, usually with a single nitrogen source (other than vitamins), were examined as a necessary step before growth of cells for enzyme assays. Six of these bacteria gave a positive colour test with a creatine–potassium hydroxide reagent, indicating the presence of acetoin, which other investigators have shown is formed via the intermediate, α-acetolactate. 2. Cell-free extracts of exponential-phase cells of Bacillus subtilis, Staphylococcus aureus, Proteus morganii, Acetobacter rancens (two strains), A. kuetzingianus, A. acetosus, Acetomonas (Acetobacter) melanogenus and Acetomonas (Acetobacter) suboxydans (A.T.C.C. no. 621) were found to contain the enzyme, dihydroxy acid dehydratase (2,3-dihydroxy acid hydro-lyase). 3. The specific activity of the dehydratase from organisms grown on valine- and isoleucine-deficient media was greater than those grown on a complex broth or media containing complete amino acid mixtures. The omission of valine plus isoleucine from a medium containing 19 amino acids caused an increase in the dehydratase specific activity of Staphylococcus aureus and Proteus morganii. 4. The rate of keto acid formation from αβ-dihydroxyisovalerate by extracts of six of the above-named organisms was faster than, but somewhat proportional to, the similar rate from αβ-dihydroxy-β-methyl-n-valerate as substrate. 5. These findings may be related to acetolactate synthesis, acetoin formation and valine–isoleucine biosynthesis in the above-mentioned micro-organisms.     

Degradation of Hydrocarbons by Members of the Genus Candida II. Oxidation of n-Alkanes and 1-Alkenes by Candida lipolytica

Candida lipolytica ATCC 8661 was grown in a mineral-salts hydrocarbon medium. n-Alkanes and 1-alkenes with 14 through 18 carbon atoms were used as substrates. Ether extracts of culture fluids and cells obtained from cultures grown on the various substrates were analyzed by thin-layer and gas-liquid chromatography. Analyses of fluids from cultures grown on n-alkanes indicated a predominance of fatty acids and alcohols of the same chain length as the substrate. In addition, numerous other fatty acids and alcohols were present. Analyses of saponifiable and nonsaponifiable material obtained from the cells revealed essentially the same products. The presence of primary and secondary alcohols, as well as fatty acids, of the same chain length as the n-alkane substrate suggested that attack on both the methyl and α-methylene group was occurring. The significance of these two mechanisms in the degradation of n-alkanes by this organism was not evident from the data presented. Analyses of fluids from cultures grown on 1-alkenes indicated the presence of 1,2-diols, as well as ω-unsaturated fatty acids, of the same chain length as the substrate. Alcohols present were all unsaturated. Saponifiable and nonsaponifiable material obtained from cells contained essentially the same products. The presence of 1,2-diols and ω-unsaturated fatty acids of the same chain length as the substrate from cultures grown on 1-alkenes indicated that both the terminal methyl group and the terminal double bond were being attacked.     

Switching between polymerase and exonuclease sites in DNA polymerase ε

The balance between exonuclease and polymerase activities promotes DNA synthesis over degradation when nucleotides are correctly added to the new strand by replicative B-family polymerases. Misincorporations shift the balance toward the exonuclease site, and the balance tips back in favor of DNA synthesis when the incorrect nucleotides have been removed. Most B-family DNA polymerases have an extended β-hairpin loop that appears to be important for switching from the exonuclease site to the polymerase site, a process that affects fidelity of the DNA polymerase. Here, we show that DNA polymerase ε can switch between the polymerase site and exonuclease site in a processive manner despite the absence of an extended β-hairpin loop. K967 and R988 are two conserved amino acids in the palm and thumb domain that interact with bases on the primer strand in the minor groove at positions n−2 and n−4/n−5, respectively. DNA polymerase ε depends on both K967 and R988 to stabilize the 3′-terminus of the DNA within the polymerase site and on R988 to processively switch between the exonuclease and polymerase sites. Based on a structural alignment with DNA polymerase δ, we propose that arginines corresponding to R988 might have a similar function in other B-family polymerases.     

Oxidation of Alkyl-substituted Cyclic Hydrocarbons by a Nocardia during Growth on n-Alkanes

Nocardia 107-332, a soil isolate, oxidizes short-chain alkyl-substituted cyclic hydrocarbons to cyclic acids while growing on n-alkanes. Cyclic acids are produced also from relatively long-chain alkyl-substituted cyclics such as n-nonylbenzene or n-dodecylbenzene which alone support growth in a mineral-salts medium. ω-Oxidation of the alkyl substituents is followed by β-oxidation. It is of particular interest that cyclic acids such as cyclohexaneacetic and phenylacetic with C₂ residual carboxylic acid substituents are resistant to further oxidation by the nocardia but cyclic acids with C₁ or C₃ substituents are readily oxidized and utilized for growth.

The specificity of microbial oxidations is demonstrated by the conversion of p-isopropyltoluene (p-cymene) to p-isopropylbenzoic acid in n-alkane, growth-supported nocardia cultures.

     

Fatty Acid Production from Amino Acids and α-Keto Acids by Brevibacterium linens BL2

Low concentrations of branched-chain fatty acids, such as isobutyric and isovaleric acids, develop during the ripening of hard cheeses and contribute to the beneficial flavor profile. Catabolism of amino acids, such as branched-chain amino acids, by bacteria via aminotransferase reactions and α-keto acids is one mechanism to generate these flavorful compounds; however, metabolism of α-keto acids to flavor-associated compounds is controversial. The objective of this study was to determine the ability of Brevibacterium linens BL2 to produce fatty acids from amino acids and α-keto acids and determine the occurrence of the likely genes in the draft genome sequence. BL2 catabolized amino acids to fatty acids only under carbohydrate starvation conditions. The primary fatty acid end products from leucine were isovaleric acid, acetic acid, and propionic acid. In contrast, logarithmic-phase cells of BL2 produced fatty acids from α-keto acids only. BL2 also converted α-keto acids to branched-chain fatty acids after carbohydrate starvation was achieved. At least 100 genes are potentially involved in five different metabolic pathways. The genome of B. linens ATCC 9174 contained these genes for production and degradation of fatty acids. These data indicate that brevibacteria have the ability to produce fatty acids from amino and α-keto acids and that carbon metabolism is important in regulating this event.