Formaldehyde kills spores of Bacillus subtilis by DNA damage and small, acid-soluble spore proteins of the ααααααα/βββββββ-type protect spores against this DNA damage

Killing of wild-type spores of Bacillus subtilis with formaldehyde also caused significant mutagenesis; spores (termed α⁻β⁻) lacking the two major α/β-type small, acid-soluble spore proteins (SASP) were more sensitive to both formaldehyde killing and mutagenesis. A recA mutation sensitized both wild-type and α⁻β⁻ spores to formaldehyde treatment, which caused significant expression of a recA - lacZ fusion when the treated spores germinated. Formaldehyde also caused protein–DNA cross-linking in both wild-type and α⁻β⁻ spores. These results indicate that: (i) formaldehyde kills B. subtilis spores at least in part by DNA damage and (b) α/β-type SASP protect against spore killing by formaldehyde, presumably by protecting spore DNA.     

Production and properties of ααα-glucosidase from the thermotolerant bacteriumThermomonospora curvata

Thermomonospora curvata contains α-1,4-glucosidase that is induced duringgrowth on maltose and starch. Maltose acts as an inducer of α-glucosidase even in thepresence of glucose. An intracellular thermostable α-glucosidase from T. curvata wasdetected in the crude extract on SDS-PAGE by means of modified colour reaction afterrenaturation of the enzyme. The enzyme was purified 59-fold to homogeneity with a yield of17·7% by a combination of ion-exchange and hydrophobic interaction chromatography andgel filtration. The enzyme has an apparent molecular mass of 60±1 kDa and isoelectric point4·1. The α-glucosidase exhibits optimum activity at pH 7·0–7·5 and54°C. The activity is inhibited by heavy metals and is positively affected by Ca²+ andMg²+. The enzyme hydrolyses maltose, sucrose, p-nitrophenyl-α- d -glucopyranoside and maltodextrins from maltotriose up to maltoheptaose with a decreasingefficiency. The K_m for maltose and p-NPG are 12 and 2·3 mmol l⁻¹,respectively.     

Immunohistochemical Localization of G Protein β1, β2, β3, β4, β5, and γ3 Subunits in the Adult Rat Brain

Abstract: The regional distributions of the G protein β subunits (Gβ1–β5) and of the Gγ3 subunit were examined by immunohistochemical methods in the adult rat brain. In general, the Gβ and Gγ3 subunits were widely distributed throughout the brain, with most regions containing several Gβ subunits within their neuronal networks. The olfactory bulb, neocortex, hippocampus, striatum, thalamus, cerebellum, and brainstem exhibited light to intense Gβ immunostaining. Negative immunostaining was observed in cortical layer I for Gβ1 and layer IV for Gβ4. The hippocampal dentate granular and CA1–CA3 pyramidal cells displayed little or no positive immunostaining for Gβ2 or Gβ4. No anti-Gβ4 immunostaining was observed in the pars compacta of the substantia nigra or in the cerebellar granule cell layer and Purkinje cells. Immunoreactivity for Gβ1 was absent from the cerebellar molecular layer, and Gβ2 was not detected in the Purkinje cells. No positive Gγ3 immunoreactivity was observed in the lateral habenula, lateral septal nucleus, or Purkinje cells. Double-fluorescence immunostaining with anti-Gγ3 antibody and individual anti-Gβ1–β5 antibodies displayed regional selectivity with Gβ1 (cortical layers V–VI) and Gβ2 (cortical layer I). In conclusion, despite the widespread overlapping distributions of Gβ1–β5 with Gγ3, specific dimeric associations in situ were observed within discrete brain regions.     

The loop between β-strands β2 and β3 and its interaction with the N-terminal α-helix is essential for biogenesis of α7 nicotinic receptors

Recently, we have shown that the α-helix present at the N-termini of α7 nicotinic acetylcholine receptors plays a crucial role in their biogenesis. Structural data suggest that this helix interacts with the loop linking β-strands β2 and β3 (loop 3). We studied the role of this loop as well as its interaction with the helix in membrane receptor expression. Residues from Asp62 to Val75 in loop 3 were mutated. Mutations of conserved amino acids, such as Asp62, Leu65 and Trp67 abolished membrane receptor expression in Xenopus oocytes. Others mutations, at residues Asn68, Ala69, Ser70, Tyr72, Gly74, and Val 75 were less harmful although still produced significant expression decreases. Steady state levels of wild-type and mutant α7 receptors (L65A, W67A, and Y72A) were similar but the formation of pentameric receptors was impaired in the latter (W67A). Mutation of critical residues in subunits of heteromeric nicotinic acetylcholine receptors (α3β4) also abolished their membrane expression. Complementarity between the helix and loop 3 was evidenced by studying the expression of chimeric α7 receptors in which these domains were substituted by homologous sequences from other subunits. We conclude that loop 3 and its docking to the α-helix is an important requirement for receptor assembly.     

Membrane Proteases and Aminoglycoside Antibiotic Resistance

We present genetic studies that help define the functional network underlying intrinsic aminoglycoside resistance in Pseudomonas aeruginosa. Our analysis shows that proteolysis, particularly that controlled by the membrane protease FtsH, is a major determinant of resistance. First, we examined the consequences of inactivating genes controlled by AmgRS, a two-component regulator required for intrinsic tobramycin resistance. Three of the gene products account for resistance: a modulator of FtsH protease (YccA), a membrane protease (HtpX), and a membrane protein of unknown function (PA5528). Second, we screened mutations inactivating 66 predicted proteases and related functions. Insertions inactivating two FtsH protease accessory factors (HflK and HflC) and a cytoplasmic protease (HslUV) increased tobramycin sensitivity. Finally, we generated an ftsH deletion mutation. The mutation dramatically increased aminoglycoside sensitivity. Many of the functions whose inactivation increased sensitivity appeared to act independently, since multiple mutations led to additive or synergistic effects. Up to 500-fold increases in tobramycin sensitivity were observed. Most of the mutations also were highly pleiotropic, increasing sensitivity to a membrane protein hybrid, several classes of antibiotics, alkaline pH, NaCl, and other compounds. We propose that the network of proteases provides robust protection from aminoglycosides and other substances through the elimination of membrane-disruptive mistranslation products.     

γ-Glutamylcysteine synthetase and γ-glutamyl transferase as differential enzymatic sources of γ-glutamylpeptides in mice

Some γ-glutamylpeptides in blood plasma are putative biomarkers for pathological conditions of the liver. γ-Glutamyltransferase (GGT) and γ-glutamylcysteine synthetase (γ-GCS) are two such potential enzymes that are responsible for the production of γ-glutamylpeptides. GGT produces γ-glutamylpeptides by transferring the γ-glutamyl moiety from glutathione to an amino acid or a peptide. γ-GCS normally catalyzes the production of γ-glutamylcysteine from glutamate and cysteine in the glutathione-synthesizing reaction, but other amino acids can also serve as an acceptor of a γ-glutamyl group, thus resulting in the formation of a variety of γ-glutamylpeptides. Based on liquid chromatography–mass spectrometry analyses, we observed differences in the distribution of γ-glutamylpeptides between the liver and kidney and were able to measure the activities of γ-GCS as well as the GGT reactions by quantifying the resulting γ-glutamylpeptides. The enzymatic characterization of γ-GCS in liver homogenates indicated that several γ-glutamylpeptides including γ-glutamyltaurine are actually produced. Cys showed the lowest Km value (0.06 mM) while other amino acids had much higher Km values (ranging from 21 to 1800 mM). The moderate Km values for these amino acids suggest that they were not the preferred amino acids in this conversion but were utilized as acceptor substrates for the production of the corresponding γ-glutamylpeptides by the γ-GCS reaction under Cys-deficient conditions. Thus, the production of these γ-glutamylpeptides by γ-GCS is directly correlated with a low Cys content, suggesting that their measurement in blood plasma could be useful for predicting the presymptomatic disease state of the liver with a defect in GSH redox balance.     

Effect of Starvation on the Endocytic Pathway in Dictyostelium Cells

Dictyostelium discoideum amoebae have been used extensively to study the structure and dynamics of the endocytic pathway. Here, we show that while the general structure of the endocytic pathway is maintained in starved cells, its dynamics rapidly slow down. In addition, analysis of apm3 and lvsB mutants reveals that the functional organization of the endocytic pathway is profoundly modified upon starvation. Indeed, in these mutant cells, some of the defects observed in rich medium persist in starved cells, notably an abnormally slow transfer of endocytosed material between endocytic compartments. Other parameters, such as endocytosis of the fluid phase or the rate of fusion of postlysosomes to the cell surface, vary dramatically upon starvation. Studying the endocytic pathway in starved cells can provide a different perspective, allowing the primary (invariant) defects resulting from specific mutations to be distinguished from their secondary (conditional) consequences.Dictyostelium discoideum is a widely used model organism for studying the organization and function of the endocytic pathway. In Dictyostelium, the organization of the endocytic pathway is similar to that in higher eukaryotes. The pathway in Dictyostelium can be divided into four steps (see Fig. S1 in the supplemental material): uptake at the plasma membrane of particles and medium, transfer through early acidic endocytic compartments (lysosomes), passage into less acidic postlysosomes (PLs), and finally, exocytosis of undigested materials (17, 20). Thus, Dictyostelium recapitulates many of the functions of the endocytic pathway in mammalian cells, including some features observed in most cell types (lysosome biogenesis) and some observed only in specialized cells (phagocytosis, macropinocytosis, and lysosome secretion).Dictyostelium amoebae live in the soil, where they feed by ingesting and digesting other microorganisms. In addition, axenic laboratory strains can macropinocytose medium to ensure their growth. Accordingly, both in natural situations and in laboratory settings, the endocytic pathway plays a key role in the acquisition of nutrients by Dictyostelium cells. In agreement with this notion, several observations suggest that the physiology of the endocytic pathway is sensitive to nutrient availability. In particular, starvation induces secretion of lysosomal enzymes by an unknown mechanism (11). The morphology of the endocytic pathway is also sensitive to nutritional cues, as shown for example by the observation that formation of multilamellar endosomes is enhanced in cells fed with bacteria (18).Here, we analyzed the effect of starvation on the organization as well as the dynamics of the endocytic pathway. We found that, while the overall organization was not extensively modified in starved cells, the dynamics of endocytic compartments were altered. Moreover, analysis of two specific knockout mutants, the apm3 (6) and lvsB (8) strains, revealed that their phenotype was profoundly altered upon starvation, providing further insight about the role of Apm3 and LvsB in the endocytic pathway.     

β‐1,3‐Glucanases in wheat and resistance to the Russian wheat aphid

The Russian wheat aphid (RWA), Diuraphis noxia (Mordvilko) is one of the most destructive insect pests of cereals world-wide. Although resistant cultivars have been bred, the biochemical mechanism of resistance is unknown. The aim of this work was to gain information on the mechanism of resistance which could contribute to more directed breeding of resistant cultivars in the future. The effect of RWA infestation on the inter- and intracellular β-1,3-glucanase activities was studied in different resistant wheat (Triticum aestivum L.) cultivars containing the Dn-1 gene for RWA resistance and corresponding near-isogenic susceptible cultivars. The activity was determined spectrophotometrically by measuring the release of glucose from laminarin. Infestation differentially induced the intra- and intercellular activities to much higher levels in resistant than susceptible cultivars within 48 h. According to immunological studies induced enzyme activities were due to increased protein levels. The intracellular β-1,3-glucanase contained about 8% exo-activity. The exo-activity made an insignificant contribution to the intercellular activity. The genetic background into which the resistance gene was bred did affect the level of activity that corresponded to the resistance performance. Seven apoplastic isoforms of β-1,3-glucanase, varying from acidic to basic, were resolved by isoelectric focusing. All isoenzymes were equally induced and no specific one could be linked to resistance or susceptibility. The RWA induced β-1,3-glucanase activity in resistant cultivars closely resembles defence responses during pathogenesis and seems to be part of a general defence response like the hypersensitive reaction (HR), which confers resistance to the RWA. This knowledge might be helpful in future to identify genes for RWA resistance. The increased β-1,3-glucanase activity after RWA infestation might serve as an additional measure to biochemically trace resistance in crosses during breeding.     

On the Identification of α- and β-Tubulin Subunits

Abstract: Confusion appears to have arisen in the literature regarding the designation of α-and β-tubulin in polyacrylamide gels. The presence or absence of 8 M-urea in sodium dodecyl sulfate (SDS) polyacrylamide gels leads to different patterns for unalkylated tubulin subunits (and other proteins), making difficult the designation of the α and β subunits by original definition using electrophoretic mobility in the molecular weight dimension. The specific biochemical property of posttranslational tyrosylation of the α subunit has been used to identify further this subunit. Under all conditions tested, the β subunit has been found to be more acidic than the α subunit, with isoelectric point differences that agree with theoretical and published values. If the tubulin subunits are reduced and alkylated, the β subunit migrates more rapidly in SDS polyacrylamide gels, with or without urea present. However, unalkylated tubulin subunits can comigrate or even reverse their relative mobility if 8 M-urea-SDS polyacrylamide gels are used for subunit separation. The results also confirm the earlier reports that the post-translational tyrosylation of protein appears exclusively restricted to α-tubulin and can be demonstrated in an in vivo situation. In addition, the results suggest that only the α₂ subunit of tubulin is tyrosylated.     

The α- and β-Tubulin Genes of Euplotes octocarinatus

ABSTRACT. The α- and the β-tubulin genes of the hypotrichous ciliate Euplotes octocarinatus were isolated from a size-selected macronuclear DNA library. The α-tubulin gene is located on a 1,587 bp macronuclear DNA molecule and the β-tubulin gene on a 1,524 bp macronuclear DNA molecule. Sequencing revealed that all the cysteine residues of the two genes are encoded by the common cysteine codons UGU and UGC and none by an UGA codon. This is in contrast to the genes of E. octocarinatus sequenced so far, where some of the cysteines are encoded by the opal codon UGA. The tubulin genes end like other Euplotes genes with a TAA. They do not contain introns. The last codon for an amino acid in the α-tubulin gene is a GAA which codes for glutamic acid. This is in contrast to what has been reported for most α-tubulin genes, but it supports findings for other hypotrichous ciliates. No evidence for the existence of more than one type of α- and one type of β-tubulin genes could be obtained.     

Schistosoma japonicum Eggs Induce a Proinflammatory,Anti-Fibrogenic Phenotype in Hepatic Stellate Cells

Hepatic fibrosis induced by egg deposition is the most serious pathology associated with chronic schistosomiasis, in which the hepatic stellate cell (HSC) plays a central role. While the effect of Schistosoma mansoni eggs on the fibrogenic phenotype of HSCs has been investigated, studies determining the effect of eggs of S . japonicum on HSCs are lacking. Disease caused by S . japonicum is much more severe than that resulting from S. mansoni infection so it is important to compare the pathologies caused by these two parasites, to determine whether this phenotype is due to the species interacting differently with the mammalian host. Accordingly, we investigated the effect of S . japonicum eggs on the human HSC cell line, LX-2, with and without TGF-β (Transforming Growth Factor beta) co-treatment, so as to determine the impact on genes associated with fibrogenesis, inflammation and matrix re-organisation. Activation status of HSCs was assessed by αSMA (Alpha Smooth Muscle Actin) immunofluorescence, accumulation of Oil Red O-stained lipid droplets and the relative expression of selected genes associated with activation. The fibrogenic phenotype of HSCs was inhibited by the presence of eggs both with or without TGF-β treatment, as evidenced by a lack of αSMA staining and reduced gene expression of αSMA and Col1A1 (Collagen 1A1). Unlike S. mansoni-treated cells, however, expression of the quiescent HSC marker PPAR-γ (Peroxisome Proliferator-Activated Receptor gamma) was not increased, nor was there accumulation of lipid droplets. In contrast, S . japonicum eggs induced the mRNA expression of MMP-9 (Matrix Metalloproteinase 9), CCL2 (Chemokine (C-C motif) Ligand 2) and IL-6 (Interleukin 6) in HSCs indicating that rather than inducing complete HSC quiescence, the eggs induced a proinflammatory phenotype. These results suggest HSCs in close proximity to S . japonicum eggs in the liver may play a role in the proinflammatory regulation of hepatic granuloma formation.     

Sustained Nicotine Exposure Differentially Affects α3β2 and α4β2 Neuronal Nicotinic Receptors Expressed in Xenopus Oocytes

Abstract: To determine whether prolonged exposure to nicotine differentially affects α3β2 versus α4β2 nicotinic receptors expressed in Xenopus oocytes, oocytes were coinjected with subunit cRNAs, and peak responses to agonist, evoked by 0.7 or 7 µ M nicotine for α4β2 and α3β2 receptors, respectively, were determined before and following incubation for up to 48 h with nanomolar concentrations of nicotine. Agonist responses of α4β2 receptors decreased in a concentration-dependent manner with IC₅₀ values in the 10 n M range following incubation for 24 h and in the 1 n M range following incubation for 48 h. In contrast, responses of α3β2 receptors following incubation for 24–48 h with 1,000 n M nicotine decreased by only 50–60%, and total ablation of responses could not be achieved. Attenuation of responses occurred within the first 5 min of nicotine exposure and was a first-order process for both subtypes; half-lives for inactivation were 4.09 and 2.36 min for α4β2 and α3β2 receptors, respectively. Recovery was also first-order for both subtypes; half-lives for recovery were 21 and 7.5 h for α4β2 and α3β2 receptors, respectively. Thus, the responsiveness of both receptors decreased following sustained exposure to nicotine, but α4β2 receptors recovered much slower. Results may explain the differential effect of sustained nicotine exposure on nicotinic receptor-mediated neurotransmitter release.     

α, βI, βII, δ, and ε Protein Kinase C Isoforms and Compound Activity in the Sciatic Nerve of Normal and Diabetic Rats

Abstract: Defective protein kinase C (PKC) has been implicated in impaired Na⁺,K⁺-ATPase activity in the sciatic nerve of streptozotocin-induced diabetic rats. In the present study, α, βI, βII, γ, δ, and ε isoform-specific antibodies were used in parallel to the measurement of compound PKC activity for the characterization of PKC distribution and isoform expression in sciatic nerves of normal and diabetic rats. To distinguish isoform expression between the axonal and glial compartments, PKC isoforms were evaluated in nerves subjected to Wallerian degeneration and in a pure primary Schwann cell culture. α, βI, βII, δ, and ε but no γ isoforms were detected in sciatic nerve. Similar immunoreactivity was observed in degenerated nerves 3–4 days after transection except for diminished βI and ε species; in Schwann cell cultures, only α, βII, δ, and ε were detected. In normal nerves, two-thirds of PKC compound activity was found in the cytosol and 50% of total enzyme activity translocated to the Na⁺,K⁺-ATPase-enriched membrane fraction with phorbol myristate acetate. Similar redistribution patterns were observed for the immunoreactivity of all isoforms with the exception of δ, which did not translocate to the membrane with phorbol myristate acetate. No abnormality in compound PKC activity, in the immunoreactive intensity, or in the distribution of PKC isoforms could be detected in rat sciatic nerve after 6–12 weeks of diabetes. Thus, defective activation rather than decreased intrinsic PKC activity may occur in diabetic neuropathy.     

Distribution of mRNAs Encoding the Peroxisome Proliferator-Activated Receptor α, β, and γ and the Retinoid X Receptor α, β, and γ in Rat Central Nervous System

Abstract: We report the isolation, by RT-PCR, of partial cDNAs encoding the rat peroxisome proliferator-activated receptor (PPAR) isoforms PPARα, PPARβ, and PPARγ and the rat retinoid X receptor (RXR) isoforms RXRα, RXRβ, and RXRγ. These cDNAs were used to generate antisense RNA probes to permit analysis, by the highly sensitive and discriminatory RNase protection assay, of the corresponding mRNAs in rat brain regions during development. PPARα, PPARβ, RXRα, and RXRβ mRNAs are ubiquitously present in different brain regions during development, PPARγ mRNA is essentially undetectable, and RXRγ mRNA is principally localised to cortex. We demonstrate, for the first time, the presence of PPAR and RXR mRNAs in primary cultures of neonatal meningeal fibroblasts, cerebellar granule neurons (CGNs), and cortical and cerebellar astrocytes and in primary cultures of adult cortical astrocytes. PPARα, PPARβ, RXRα, and RXRβ mRNAs are present in all cell types, albeit that PPARα and RXRα mRNAs are at levels near the limit of detection in CGNs. PPARγ mRNA is expressed at low levels in most cell types but is present at levels similar to those of PPARα mRNA in adult astrocytes. RXRγ mRNA is present either at low levels, or below the level of detection of the assay, for all cell types studied.     

PARD3 gene variation as candidate cause of nonsyndromic cleft palate only

Nonsyndromic cleft palate only (NSCP) is a common congenital malformation worldwide. In this study, we report a three‐generation pedigree with NSCP following the autosomal‐dominant pattern. Whole‐exome sequencing and Sanger sequencing revealed that only the frameshift variant c.1012dupG [p. E338Gfs*26] in PARD3 cosegregated with the disease. In zebrafish embryos, ethmoid plate patterning defects were observed with PARD3 ortholog disruption or expression of patient‐derived N‐terminal truncating PARD3 (c.1012dupG), which implicated PARD3 in ethmoid plate morphogenesis. PARD3 plays vital roles in determining cellular polarity. Compared with the apical distribution of wild‐type PARD3, PARD3‐p. E338Gfs*26 mainly localized to the basal membrane in 3D‐cultured MCF‐10A epithelial cells. The interaction between PARD3‐p. E338Gfs*26 and endogenous PARD3 was identified by LC–MS/MS and validated by co‐IP. Immunofluorescence analysis showed that PARD3‐p. E338Gfs*26 substantially altered the localization of endogenous PARD3 to the basement membrane in 3D‐cultured MCF‐10A cells. Furthermore, seven variants, including one nonsense variant and six missense variants, were identified in the coding region of PARD3 in sporadic cases with NSCP. Subsequent analysis showed that PARD3‐p. R133*, like the insertion variant of c.1012dupG, also changed the localization of endogenous full‐length PARD3 and that its expression induced abnormal ethmoid plate morphogenesis in zebrafish. Based on these data, we reveal PARD3 gene variation as a novel candidate cause of nonsyndromic cleft palate only.     

Purification and characterization of ββ-glucosidase from Aspergillus niger strain 322

An extracellular β-glucosidase enzyme was purified from the fungus Aspergillus niger strain 322 . The molecular mass of the enzyme was estimated to be 64 kDa by SDS gel electrophoresis. Optimal pH and temperature for β-glucosidase were 5·5 and 50 °C, respectively. Purified enzyme was stable up to 50 °C and pH between 2·0 and 5·5. The K_m was 0·1 mmol l⁻¹ for cellobiose. Enzyme activity was inhibited by several divalent metal ions.