In vitro binding of the CcpA protein of Bacillus megaterium to cis-acting catabolite responsive elements (CREs) of Gram-positive bacteria

Abstract Using DNA band migration retardation assays, specific binding of the CcpA protein of Bacillus megaterium to the ds-acting catabolite responsive element (CRE) of the xyl operon of B. subtilis has been demonstrated. Binding of CcpA was specifically inhibited by addition of unlabeled DNA fragments containing CREs of other operons but not by DNA fragments lacking a CRE. Binding was stimulated by high concentrations of phosphate, pyrophosphate, and organic phosphate esters and specifically inhibited by serine phosphorylated HPr and its conformational analogue, S46D HPr. This report therefore documents the specific binding of CcpA to a target CRE and defines its regulation by HPr(ser-P) and phosphorylated metabolites.     

Immunological crossreactivity to the catabolite control protein CcpA from Bacillus megaterium is found in many Gram-positive bacteria

Abstract The catabolite control protein CcpA from Bacillus megaterium was overproduced as a fusion protein to a 6xhis affinity tag and purified to homogeneity. Polyclonal antibodies of high affinity and specificity were raised against the purified protein. The serum did not crossreact with purified Lac repressor despite the fact that CcpA and LacI belong to the same protein family. Using this antiserum we identified proteins that share antigenic determinants with CcpA in many Gram-positive bacteria, including bacilli, staphylococci, streptococci, lactic acid bacteria, and some actinomycetes.     

Catabolite repression of inositol dehydrogenase and gluconate kinase syntheses in Bacillus subtilis

The regulation of induction of inositol dehydrogenase (EC 1.1.1.18) and gluconate kinase (EC 2.7.1.12) was studied in Bacillus subtilis. Inositol dehydrogenase is induced by myo-inositol and gluconate kinase is induced by D-gluconate. Both inductions were strongly repressed by rapidly metabolizable carbohydrates such as D-glucose, D-mannose, D-fructose and glycerol (D-glucose had the strongest repressive effect) but they were weakly repressed by slowly metabolizable carbohydrates. Although each carbohydrate exerted a stronger effect on the induction of inositol dehydrogenase than that of gluconate kinase, it showed a similar tendency with respect to the degree of repression of each induction. This catabolite repression could not be diminished by addition of cyclic AMP to medium. In addition, non-metabolizable D-glucose analogues had no or weak repressive effects. On the assumption that rapidly metabolizable carbohydrates might be metabolized to repress both inductions, it was investigated whether several mutants blocked in the Embden-Meyerhof pathway could produce metabolite(s) (repressor) to repress them. A phosphoglycerate kinase (EC 2.7.2.3) deficient mutant could produce the repressor from D-glucose, D-mannose, D-fructose and glycerol but other mutants could not produce it from carbohydrates unable to be metabolized ineach mutant. Thus, catabolite repression of both enzyme inductions seemed to be under similar regulation. The identification of the possible repressor of the induction of inositol dehydrogenase and gluconate kinase in vivo was discussed.     

An X-ray study of the cytoplasmic membranes of two Gram-positive bacteria

X-ray diffraction diagrams of partially disordered one-dimensional lattices of isolated bacterial cytoplasmic membranes are described and they provide a basis for suggesting possible molecular structures of bacterial membranes.Biochemical and electron microscope evidence points towards a lipid bilayer with a high degree of fluidity. The protein molecules are in a disordered configuration in the membrane.     

The mechanism of sugar-mediated catabolite repression of the propionate catabolic genes in Escherichia coli

Carbon catabolite repression (CCR) is a well-known phenomenon that involves the preferential utilization of glucose as a carbon source. Cyclic adenosine monophosphate (cAMP) and the cAMP receptor protein (CRP) mediate CCR. Recently, a second CCR hierarchy that leads to the preferential consumption of arabinose over xylose, mediated by arabinose-bound AraC, has been identified. In this study, we report yet another CCR hierarchy that causes the preferential utilization of sugars (arabinose, galactose, glucose, mannose, and xylose) over a short-chain fatty acid (propionate). Expression of the propionate catabolic (prpBCDE) genes is down-regulated in the presence of these sugars. Sugar-mediated repression of the propionate catabolic genes is independent of sugar-specific regulators such as AraC and dependent on global regulators of sugar transport such as the cAMP-CRP complex and the Phosphotransferase System (PTS). Inhibition of the prpBCDE promoter is encountered during rapid sugar uptake and metabolism. This unique regulatory crosstalk between sugar metabolism and fatty acid metabolism may help provide new insights into CRP-dependent catabolite repression acting in conjunction with non-carbohydrate metabolism.     

Distribution of vesicular glutamate transporter 1 (VGLUT1) in the mouse esophagus

In rat and mouse esophagus, vesicular glutamate transporter 2 (VGLUT2) has been demonstrated to identify vagal intraganglionic laminar endings (IGLEs); this has recently also been shown for VGLUT1 in rat esophagus. In this study, we have investigated the distribution of VGLUT1 in the mouse esophagus and compared these results with the recently published data from the rat esophagus. Unexpectedly, we have discovered that VGLUT1 mostly fails to identify IGLEs in the mouse esophagus. This is surprising, since the distribution of VGLUT2 shows comparable results in both species. Confocal imaging has revealed substantial colocalization of VGLUT1 immunoreactivity (-ir) with cholinergic and nitrergic/peptidergic markers within the myenteric neuropil and in both cholinergic and nitrergic myenteric neuronal cell bodies. VGLUT1 and cholinergic markers have also been colocalized in fibers of the muscularis mucosae, whereas VGLUT1 and nitrergic markers have never been colocalized in fibers of the muscularis mucosae, although this does occur in fibers of the muscularis running to motor endplates. Thus, VGLUT1 is contained in the nitrergic innervation of mouse esophageal motor endplates, another difference from the rat esophagus. VGLUT1-ir is therefore present in extrinsic and intrinsic innervation of the mouse esophagus, but the significant differences from the rat indicate species variations concerning the distribution of VGLUTs in the peripheral nervous system. This study was supported by the Johannes und Frieda Marohn-Stiftung, Erlangen.     

Evidence for a Functional O-Linked N-Acetylglucosamine (O-GlcNAc) System in the Thermophilic Bacterium Thermobaculum terrenum

Post-translational modification of proteins is a ubiquitous mechanism of signal transduction in all kingdoms of life. One such modification is addition of O-linked N-acetylglucosamine to serine or threonine residues, known as O-GlcNAcylation. This unusual type of glycosylation is thought to be restricted to nucleocytoplasmic proteins of eukaryotes and is mediated by a pair of O-GlcNAc-transferase and O-GlcNAc hydrolase enzymes operating on a large number of substrate proteins. Protein O-GlcNAcylation is responsive to glucose and flux through the hexosamine biosynthetic pathway. Thus, a close relationship is thought to exist between the level of O-GlcNAc proteins within and the general metabolic state of the cell. Although isolated apparent orthologues of these enzymes are present in bacterial genomes, their biological functions remain largely unexplored. It is possible that understanding the function of these proteins will allow development of reductionist models to uncover the principles of O-GlcNAc signaling. Here, we identify orthologues of both O-GlcNAc cycling enzymes in the genome of the thermophilic eubacterium Thermobaculum terrenum. The O-GlcNAcase and O-GlcNAc-transferase are co-expressed and, like their mammalian orthologues, localize to the cytoplasm. The O-GlcNAcase orthologue possesses activity against O-GlcNAc proteins and model substrates. We describe crystal structures of both enzymes, including an O-GlcNAcase·peptide complex, showing conservation of active sites with the human orthologues. Although in vitro activity of the O-GlcNAc-transferase could not be detected, treatment of T. terrenum with an O-GlcNAc-transferase inhibitor led to inhibition of growth. T. terrenum may be the first example of a bacterium possessing a functional O-GlcNAc system.     

Distribution of P2X<Subscript>3</Subscript> receptor immunoreactivity in myenteric ganglia of the mouse esophagus

Intraganglionic laminar endings (IGLEs) represent the major vagal afferent terminals throughout the gut. Electrophysiological experiments revealed a modulatory role of ATP in the IGLE-mechanotransduction process and the P2X₂-receptor has been described in IGLEs of mouse, rat and guinea pig. Another purinoceptor, the P2X₃-receptor, was found in IGLEs of the rat esophagus. These findings prompted us to investigate occurrence and distribution of the P2X₃-receptor in the mouse esophagus. Using multichannel immunofluorescence and confocal microscopy, P2X₃-immunoreactivity (-iry) was found colocalized with the vesicular glutamate transporter 2 (VGLUT2), a specific marker for IGLEs, on average in three-fourths of esophageal IGLEs. The distribution of P2X₃ immunoreactive (-ir) IGLEs was similar to that of P2X₂-iry and showed increasing numbers towards the abdominal esophagus. P2X₃/P2X₂-colocalization within IGLEs suggested the occurrence of heteromeric P2X_2/3 receptors. In contrast to the rat, where only a few P2X₃-ir perikarya were described, P2X₃ stained perikarya in ~80% of myenteric ganglia in the mouse. Detailed analysis revealed P2X₃-iry in subpopulations of nitrergic (nNOS) and cholinergic (ChAT) myenteric neurons and ganglionic neuropil of the mouse esophagus. We conclude that ATP might act as a neuromodulator in IGLEs via a (P2X₂)-P2X₃ receptor-mediated pathway especially in the abdominal portion of the mouse esophagus.     

Chemical coding of neurons in the myenteric plexus and external muscle of the small and large intestine of the mouse

Immunohistochemical techniques were used to examine the presence and co-localisation of a range of putative neurotransmitters and other neuronal markers in the myenteric plexus of the small and large intestine of the mouse. Distinct sub-populations of myenteric neurons were identified, based on the combinations of substances they contained and the distribution of their fibres. In the small intestine, there were two major classes of circular muscle motor neurons; one class was characterised by the presence of nitric oxide synthase, vasoactive intestinal peptide plus neuropeptide Y (NOS/VIP/NPY), and the second class contained calretinin plus substance P (CalR/SP). There were seven classes of neurons that innervated myenteric ganglia; these contained NOS, VIP, NOS/VIP, NPY, CalR/calbindin (CalB), SP or 5-HT. In the large intestine, there were five major classes of motor neurons that contained NOS, NOS/VIP, GABA, SP, or CalR/SP, and seven major classes of neurons that innervated myenteric ganglia and contained NOS, VIP, CalR/CalB, CalR, SP, GABA or 5-HT. Although some aspects of the patterns of co-localisation are similar to those in other species, this study re-inforces recent analyses that indicate significant species differences in neurochemical patterns in the enteric neurons of different species. Received: 28 August 1995 / Accepted: 30 November 1995     

Validation of MALDI-TOF MS for rapid classification and identification of lactic acid bacteria, with a focus on isolates from traditional fermented foods in Northern Vietnam

Aims: To evaluate the potential use of MALDI-TOF MS for fast and reliable classification and identification of lactic acid bacteria (LAB) from traditional fermented foods. Methods and Results: A total of 119 strains of LAB from fermented meat (nem chua) were analysed with both (GTG)(5) -PCR fingerprinting and MALDI-TOF MS. Cluster analysis of the profiles revealed five species represented by a single isolate both in (GTG)(5) -PCR and in MALDI-TOF MS; five species grouped alike for (GTG)(5) -PCR and for MALDI-TOF MS; however, differences in minimal similarity between the delineated (GTG)(5) -PCR and MALDI-TOF MS clusters could be observed; three species showed more heterogeneity in their MALDI-TOF MS profiles compared to their (GTG)(5) -PCR profiles; two species, each represented by a single MALDI-TOF cluster, were subdivided in the corresponding (GTG)(5) -PCR dendrogram. As proof of the identification potential of MALDI-TOF MS, LAB diversity from one fermented mustard sample was analysed using MALDI- TOF MS. PheS gene sequencing was used for validation. Conclusions: MALDI-TOF MS is a powerful, fast, reliable and cost-effective technique for the identification of LAB associated with the production of fermented foods. Significance and Impact of the Study: Food LAB can be identified using MALDI-TOF MS, and its application could possibly be extended to other food matrices and/or other food-derived micro-organisms.     

Control of carbon partitioning and photosynthesis by the triose phosphate/phosphate translocator in transgenic tobacco plants (Nicotiana tabacum L.). I. Comparative physiological analysis of tobacco plants with antisense repression and overexpression of the triose phosphate/phosphate translocator

 The physiological properties of transgenic tobacco plants (Nicotiana tabacum L.) with decreased or increased transport capacities of the chloroplast triose phosphate/phosphate translocator (TPT) were compared in order to investigate the extent to which the TPT controls metabolic fluxes in wild-type tobacco. For this purpose, tobacco lines with an antisense repression of the endogenous TPT (αTPT) and tobacco lines overexpressing the TPT gene isolated from the C₄ plant Flaveria trinervia (FtTPT) were used. The F. trinervia TPT expressed in yeast cells exhibited transport characteristics identical to the TPT from C₃ plants. Neither antisense TPT plants nor FtTPT overexpressors showed a phenotype when grown in a greenhouse in air. Contents of starch and soluble sugars in upper source leaves were similar in TPT underexpressors and FtTPT overexpressors compared to the wild type at the end of the photoperiod. The FtTPT overexpressors incorporated more ¹⁴CO₂ in sucrose than the wild type, indicating that the TPT limits sucrose biosynthesis in the wild type. There were only small effects on labelling of amino acids and organic acids. The mobilisation of starch was enhanced in αTPT lines but decreased in FtTPT overexpressors compared to the wild type. Enzymes involved in starch mobilisation or utilisation, such as α-amylase or hexokinase were increased in αTPT plants and, in the case of amylases, decreased in FtTPT overexpressors. Moreover, α-amylase activity exhibited a pronounced diurnal variation in αTPT lines with a maximum activity after 8 h in the light. These changes in starch hydrolytic activities were confirmed by activity staining of native gels. Activities of glucan phosphorylases were unaffected by either a decrease or an increase in TPT activity. There were also effects of TPT activities on steady-state levels of phosphorylated intermediates as well as total amino acids and malate. In air, there was no or little effect of altered TPT transport activity on either rates of photosynthetic electron transport and/or CO₂ assimilation. However, in elevated CO₂ (1500 μl · l⁻¹) and low O₂ (2%) the rate of CO₂ assimilation was decreased in the αTPT lines and was slightly higher in FtTPT lines. This shows that the TPT limits maximum rates of photosynthesis in the wild type. Received: 26 March 1999 / Accepted: 21 August 1999     

c-kit-Dependent development of interstitial cells and electrical activity in the murine gastrointestinal tract

In vivo injection of a neutralizing, monoclonal antibody (ACK2) to the receptor tyrosine kinase (c-kit) disrupts the normal motility patterns of the mouse small intestine. Immunohistochemical studies showed that cells expressing c-kit-like immunoreactivity (c-kit-LI) decreased in numbers in response to ACK2, but the identity of these cells is unknown. We investigated the identity and development of the cells that express c-kit-LI in the mouse small intestine and colon. Cells in the region of the myenteric plexus and deep muscular plexus of the small intestine and in the subserosa, in the myenteric plexus region, within the circular and longitudinal muscle layers, and along the submucosal surface of the circular muscle in the colon were labeled with ACK2. The distribution of cells that express c-kit-LI was the same as that of interstitial cells (ICs). In whole-mount preparations cells with c-kit-LI were interconnected, forming a netword similar to the network formed by cells that stained with methylene blue, which has been used as a marker for ICs in the mouse gastrointestinal tract. Immunocytochemistry verified that ICs were labeled with ACK2. Multiple injections of animals with ACK2 between days 0 and 8 post partum (pp) caused a dramatic reduction in the number of ICs compared to control animals. From an ultrastructural point of view, the proliferation and development appeared to be suppressed in some classes of ICs, while others displayed an altered course of development. Functional studies showed that the decrease in ICs was accompanied by a loss of electrical rhythmicity in the small intestine and reduced neural responses in the small bowel and colon. Morphological experiments showed that c-kit-positive cells are ICs, and physiological evidence reinforced the concept that ICs are involved in generation of rhythmicity and translation of neural inputs in gastrointestinal smooth muscles. Controlling the development of ICs provides a powerful new tool for the investigation of the physiological role of these cells.     

Developmental expression of MNAR mRNA in the mouse brain

During the development of the central nervous system, estrogen influences cellular differentiation and determines the functional connectivity of distinct neural networks. Estrogens generally act through nuclear estrogen receptors (ERs). Recent research has additionally revealed rapid estrogen effects requiring the binding of estrogen to membrane/cytoplasmic ERs and the activation of intracellular signaling systems such as the Src/MAPK cascade. The scaffold protein MNAR/PELP1 appears to be the designated functional mediator of such non-genomic estrogen effects between non-nuclear ERs and Src/MAPKs. In this study, we demonstrate the expression and differential regulation of MNAR mRNA in the developing male and female mouse brain by quantitative polymerase chain reaction. In the midbrain and hypothalamus, a gradual decline in MNAR mRNA levels has been observed prenatally with the highest values at embryonic day 15 and lowest at postnatal day 15. In the cortex, mRNA levels do not fluctuate until postnatal day 7 but decrease thereafter. No differences in MNAR expression between sexes have been detected. Analysis of neuronal and astroglia-enriched cell cultures has revealed the presence of MNAR in both cell types.     

The translation of the messenger for the poly(A)-binding protein-associated with translated mRNA is suppressed. A case of cytoplasmic repression in duck erythroblasts

In vivo protein synthesis in duck erythroblasts was compared to in vitro translation of polyribosomal and free cytoplasmic mRNA. The in vivo study showed the absence of de novo synthesis of the Mr 73 000 poly(A)-binding protein found associated with all polyribosomal mRNA. In vitro translation demonstrated that the mRNA for this protein is absent from the polyribosomal mRNA fraction but constitutes a medium frequency messenger among the repressed free mRNA. This result confirms the existence of a qualitative translational control in terminal differentiating duck erythroblasts leading eventually to the arrest of the protein synthesizing machinery.     

Different functions of HOPS isoforms in the cell

Hepatocyte odd protein shuttling (HOPS) moves between nucleus and cytoplasm. HOPS overexpression leads to cell cycle arrest in G₀/G₁, and HOPS knockdown causes centrosome alterations, with subsequent abnormal cell division. Recently, we demonstrated that HOPS acts as a functional bridge in NPM-p19^Arf interactions. Here we show that HOPS is present in 3 different isoforms that play distinct intracellular functions. Although HOPS is a transmembrane ubiquitin, an isoform with intermediate molecular weight is cleaved from the membrane and released into the cytosol, to act as the shuttling protein. We identified a signal peptide peptidase structure in N-terminal membrane-bound HOPS that allows the regulated intramembrane proteolysis (RIP) system to control the relative amounts of the released, shuttling isoform capable of binding NPM. These results argue for distinct, isoform-specific functions of HOPS in the nucleolus, nucleus, and cytoplasm and provide insight into the dynamics of HOPS association with NPM, whose mutation and subsequent delocalization is found in 30% of acute myeloid leukemia patients.     

Carbon monoxide mediates heme oxygenase 1 induction via Nrf2 activation in hepatoma cells

Carbon monoxide (CO) and nitric oxide (NO) are two gas molecules which have cytoprotective functions against oxidative stress and inflammatory responses in many cell types. Currently, it is known that NO produced by nitric oxide synthase (NOS) induces heme oxygenase 1 (HO1) expression and CO produced by the HO1 inhibits inducible NOS expression. Here, we first show CO-mediated HO1 induction and its possible mechanism in human hepatocytes. Exposure of HepG2 cells or primary hepatocytes to CO resulted in dramatic induction of HO1 in dose- and time-dependent manner. The CO-mediated HO1 induction was abolished by MAP kinase inhibitors (MAPKs) but not affected by inhibitors of PI3 kinase or NF-kappaB. In addition, CO induced the nuclear translocation and accumulation of Nrf2, which suppressed by MAPKs inhibitors. Taken together, we suggest that CO induces Nrf2 activation via MAPKs signaling pathways, thereby resulting in HO1 expression in HepG2 cells.     

Developmental distribution of CaM kinase II in the antennal lobe of the sphinx moth <Emphasis Type="Italic">Manduca sexta</Emphasis>

The antennal lobe (primary olfactory center of insects) is completely reorganized during metamorphosis. This reorganization is accompanied by changing patterns of calcium signaling in neurons and glial cells. In the present study, we investigated the developmental distribution of a major calcium-dependent protein, viz., calcium/calmodulin-dependent protein kinase II (CaM kinase II), in the antennal lobe of the sphinx moth Manduca sexta by using a monoclonal antibody. During synaptogenesis (developmental stages 6–10), we found a redistribution of CaM kinase II immunoreactivity, from a homogeneous distribution in the immature neuropil to an accumulation in the neuropil of the glomeruli. CaM kinase II immunoreactivity was less intense in olfactory receptor axons of the antennal nerve and antennal lobe glial cells. Western blot analysis revealed a growing content of CaM kinase II in antennal lobe tissue throughout metamorphosis. Injection of the CaM kinase inhibitor KN-93 into pupae resulted in a reduced number of antennal lobe glial cells migrating into the neuropil to form borders around glomeruli. The results suggest that CaM kinase II is involved in glial cell migration.This work was supported by the DFG LO779/2.     

Caspase-8 acts as a key upstream executor of mitochondria during justicidin A-induced apoptosis in human hepatoma cells

Justicia procumbens is a traditional Taiwanese herbal remedy used to treat fever, pain, and cancer. Justicidin A, isolated from Justicia procumbens, has been reported to suppress in vitro growth of several tumor cell lines as well as hepatoma cells. In this study, justicidin A activated caspase-8 to increase tBid, disrupted mitochondrial membrane potential (Delta psi(m)), and caused the release of cytochrome c and Smac/DIABLO in Hep 3B and Hep G2 cells. Justicidin A also reduced Bcl-x(L) and increased Bax and Bak in mitochondria. Caspase-8 inhibitor (Z-IETD) attenuated the justicidin A-induced disruption of Delta psi(m). Growth of Hep 3B implanted in NOD-SCID mice was suppressed significantly by oral justicidin A (20 mg/kg/day). These results indicate that justicidin A-induced apoptosis in these cells proceeds via caspase-8 and is followed by mitochondrial disruption.