Vibrio vulnificus causes fatal infections in susceptible individuals. Group 1 capsular polysaccharide (CPS) operon is responsible for CPS expression, which plays an essential role in the pathogenesis of this pathogen. Cyclic AMP (cAMP) and cAMP receptor protein (crp) complex, which responds to glucose availability and functions as a global regulator, has been known to affect CPS production in this pathogen. This study was undertaken to experimentally verify whether cAMP-Crp directly or indirectly affects CPS production. A mutation in cyaA encoding adenylate cyclase, which is required for cAMP biosynthesis, inhibited V. vulnificus growth and changed opaque colonies to translucent colonies, and these changes were recovered by complementing cyaA or by adding exogenous cAMP. A mutation in crp encoding Crp also inhibited V. vulnificus growth and changed opaque colonies to translucent colonies, and these changes were recovered by complementing crp. Moreover, the crp or cyaA mutation decreased the susceptibility of V. vulnificus against NaOCl. The crp mutation reduced the transcription levels of group 1 CPS operon on a per cell basis. Glucose addition in the absence of Crp stimulated V. vulnificus growth, changed translucent colonies to opaque colonies, and increased the transcription levels of group 1 CPS operon. These results indicate that cAMP or Crp is indirectly involved in optimal CPS production by positively affecting metabolism or V. vulnificus growth rather than by directly controlling the expression of group 1 CPS operon.
Adenylyl Cyclases ; Complement System Proteins ; Cyclic AMP Receptor Protein ; Cyclic AMP* ; Glucose ; Metabolism ; Operon ; Vibrio vulnificus

The cyclic AMP receptor protein (CRP) complexed with cyclic AMP (CRP.cAMP) regulates expression of many genes by binding to sites at or near many promoters of Escherichia coli. The regulatory effect of CRP.cAMP was studied by in vitro transcription assay with lacUV5 promoter derivatives that have the CRP binding site at different locations (-56 to -69 from the transcription start site of lacUV5 promoter) upstream of the promoter. The CRP binding site itself influenced differently on the promoter activity depending on the distances from the promoter. Depending on the helix phasing of the CRP.cAMP relative to RNA polymerase CRP.cAMP activated, repressed or had no effect on the promoter. These results imply that a regulator is not a dedicated protein for repression or activation but that any regulator may have a potential of dual functionalities when it is under appropriate condition.
Binding Sites ; Cyclic AMP ; Cyclic AMP Receptor Protein ; DNA-Directed RNA Polymerases ; Escherichia coli* ; Escherichia* ; Repression, Psychology ; Transcription Initiation Site

Cyclic amp receptor protein (CRP) is a global transcriptional factor in many prokaryotes, capable of governing nearly half of the total genes in Escherichia coli. Through the method of error-prone PCR or DNA shuffling, we can first obtain CRP mutant library and then get the expected cell phenotype with enhanced resistance. In this article, we reviewed the following desired phenotype: enhanced tolerance towards oxidative stress, improved osmotolerance, enhanced organic solvent (toluene) tolerance, improved acetate tolerance of E. coli fermentation and improved ethanol tolerance during bio-ethanol production. We then concluded that CRP can also be applied in other host cells to get desired phenotypes. Last, we predicted potential applications of mutant CRP transcriptional factor.
Cyclic AMP Receptor Protein ; biosynthesis ; DNA Shuffling ; Escherichia coli ; metabolism ; Fermentation ; Metabolic Engineering

OBJECTIVEThe complex of the cyclic AMP receptor protein (CRP) and cAMP is an important transcriptional regulator of numerous genes in prokaryotes. The transport of mannitol through the phosphotransferase systems (PTS) is regulated by the CRP-cAMP complex. The aim of the study is to investigate how the CRP-cAMP complex acting on the mannitol PTS operon mtl of the Vibrio cholerae El Tor biotype.

METHODSThe crp mutant strain was generated by homologous recombination to assess the need of CRP to activate the mannitol PTS operon of V. cholerae El Tor. Electrophoretic mobility shift assays (EMSA) and the reporter plasmid pBBRlux were used to confirm the role that the CRP-cAMP complex playing on the mannitol PTS operon mtl.

RESULTSIn this study, we confirmed that CRP is strictly needed for the activation of the mtl operon. We further experimentally identified five CRP binding sites within the promoter region upstream of the mannitol PTS operon mtl of the Vibrio cholerae El Tor biotype and found that these sites display different affinities for CRP and provide different contributions to the activation of the operon.

CONCLUSIONThe five binding sites collectively confer the strong activation of mannitol transfer by CRP in V. cholerae, indicating an elaborate and subtle CRP activation mechanism.

Bacterial Proteins ; genetics ; Base Sequence ; Cyclic AMP ; metabolism ; Cyclic AMP Receptor Protein ; Gene Expression Regulation, Bacterial ; Mannitol ; Molecular Sequence Data ; Operon ; Phosphotransferases ; Vibrio cholerae

Roundabout (Robo) is the transmembrane receptor for slit, the neuronal guidance molecule. In this study, the tyrosine phosphorylation of Robo was observed in Robo-transfected human embryonic kidney cells and developing rat brains, and found to be increased by the treatment with protein kinase A activator, forskolin. In contrast, protein kinase C activation by phorbol-12-myristate-13-acetate decreased the phosphorylation of Robo. Intracellular calcium was required for the tyrosine phosphorylation. Furthermore, the transfection of an Eph receptor tyrosine kinase dramatically enhanced the tyrosine phosphorylation. These findings indicate that the tyrosine phosphorylation of Robo is regulated by multiple mechanisms, and that Eph receptor kinases may play a role in the regulation of tyrosine phosphorylation of Robo in the rat brain.
Animals ; Brain ; Calcium ; Colforsin ; Cyclic AMP-Dependent Protein Kinases ; Humans ; Kidney ; Neurons ; Phosphorylation* ; Phosphotransferases ; Protein Kinase C ; Protein Kinases ; Rats ; Receptor, EphA1 ; Receptors, Eph Family ; Transfection ; Tyrosine

In the intestinal mucosal surface, microfold cells (M cells) are the representative gateway for the uptake of luminal antigens. At the same time, M cells are the primary infection site for pathogens invading mucosal surface for their infection. Although it is well recognized that many mucosal pathogens exploit the M cells for their infection, the mechanism to infect M cells utilized by pathogens is not clearly understood yet. In this study, we found that M cells expressing complement 5a (C5a) receptor (C5aR) also express Toll-like receptor (TLR) 1/2 and TLR4. Infection of Yersinia enterocolitica, an M cell-invading pathogen, synergistically regulated cyclic adenosine monophosphate-dependent protein kinase A (cAMP-PKA) signaling which are involved in signal crosstalk between C5aR and TLRs. In addition, Y. enterocolitica infection into M cells was enhanced by C5a treatment and this enhancement was abrogated by C5a antagonist treatment. Finally, Y. enterocolitica infection into M cells was unsuccessful in C5aR knock-out mice. Collectively, we suggest that exploit the crosstalk between C5aR and TLR signaling is one of infection mechanisms utilized by mucosal pathogens to infect M cells.
Adenosine ; Animals ; Complement C5a* ; Complement System Proteins* ; Cyclic AMP-Dependent Protein Kinases ; Mice ; Mice, Knockout ; Phenobarbital ; Receptor, Anaphylatoxin C5a* ; Toll-Like Receptors* ; Yersinia enterocolitica* ; Yersinia*

To study the correlation between the spatial cognitive impairment and different subtypes of estrogen receptor α (ERα) of hippocampus in diabetic mice, we used alloxan (intraperitoneal injection) to induce type 1 diabetes in male Kunming mice and compared the spatial cognitive ability of the model mice with that of control mice through Morris water maze test. Meanwhile, using Western blot, we detected the protein expressions of ER-α36, ER-α66, caveolin-1, PKCα, cAMP-response element binding protein 2 (CREB2), and synaptophysin (Syn) in the hippocampus of the mice. The results showed that on the 3rd and 5th days of training, the ability of spatial learning and memory in the diabetic mice was significantly inferior to that of the control mice (P < 0.05). In the diabetic mice, the protein expressions of caveolin-1 and PKCα were decreased (P < 0.05), but ER-α66 expression was unaffected, while ER-α36 and CREB2 expressions were significantly increased (P < 0.05) compared with those of the control mice. The results suggest that abnormal expression of ER-α36 and related signal molecules may be important factors for diabetes-induced spatial cognitive impairment.
Animals ; Caveolin 1 ; metabolism ; Cognitive Dysfunction ; Cyclic AMP Response Element-Binding Protein ; metabolism ; Diabetes Mellitus, Experimental ; physiopathology ; Estrogen Receptor alpha ; metabolism ; Hippocampus ; metabolism ; physiopathology ; Male ; Maze Learning ; Memory ; Mice ; Protein Kinase C-alpha ; metabolism ; Synaptophysin ; metabolism

Salmonella choleraesuis C500 strain was an attenuated vaccine strain to prevent piglet paratyphoid, attenuated by chemical method. Although the vaccine has good immunogenicity, it remains some residual virulence. In order to develop a safer vaccine strain and exploit C500 as a live vaccine vector for mucosal immunization, delta crp delta asd double deletion mutant was constructed. Firstly, the recombination suicide vector with 320 bp-deleted crp (cAMP receptor protein) gene and sacB (sucrose-sensitive gene) gene was constructed and conjugated with C500. The unmarked crp deleted strain without resistance was selected by two-step method and crp deletion on the genome was determined by PCR. Then the asd (beta-aspartic semialdehyde dehydrogenase) gene was further deleted in the delta crp strain by the same method. Foreign DAP (diaminopimelic acid) must be supplied for delta crp delta asd mutant to grow. The phenotype, growth properties and virulence in mice of delta crp mutant were further characterized. In conclusion, the delta crp delta asd double-deletion mutant was successfully constructed. The delta crp delta asd mutant can be used as a live vector to express foreign genes and to develop potential oral multivalent vaccines.
Amino Acid Oxidoreductases ; genetics ; Animals ; Antibodies, Bacterial ; biosynthesis ; Bacterial Proteins ; genetics ; Cyclic AMP Receptor Protein ; genetics ; Gene Deletion ; Mice ; Mutation ; Salmonella ; genetics ; immunology ; pathogenicity ; Salmonella Vaccines ; genetics ; immunology ; Swine ; Transduction, Genetic ; Vaccines, Attenuated ; genetics ; immunology ; Virulence

OBJECTIVETo study the effect of MNNG on inducement of non-targeted mutation and activation of several cellular signal transduction pathways, and to determine whether the activation of these signaling pathways was dependent on the DNA-damage.

METHODSVero cells were enucleated by discontinuous density centrifugation. The PKA activities were measured by enzyme-linked immunosorbent assay. The status of cell membrane receptors was studied with immunofluorescent staining and confocal microscopy.

RESULTIn enucleated cytoplasts, MNNG-treatment increased PKA activity for about 2.3-fold in accordance with the 2.7-fold up-regulation of PKA activity in whole vero cells exposed to MNNG. The clustering of cell surface receptors of epidermal growth factor and tumor necrosis factor alpha was also observed in cells exposed to MNNG; this phenomenon was also found in enucleated cells.

CONCLUSIONThe results indicate that the initiation of signal cascades induced by low concentration of MNNG might be associated with its interaction with cell surface receptors and/or direct activation of related signal proteins but not its DNA damage.

Animals ; Cell Nucleus ; physiology ; Cercopithecus aethiops ; Cyclic AMP-Dependent Protein Kinases ; metabolism ; DNA Damage ; Enzyme Activation ; drug effects ; Methylnitronitrosoguanidine ; toxicity ; Receptor, Epidermal Growth Factor ; drug effects ; metabolism ; Receptors, Tumor Necrosis Factor ; drug effects ; metabolism ; Signal Transduction ; drug effects ; Vero Cells

An important property of the intestine is the ability to secrete fluid. The intestinal secretion is regulated by a number of substances including vasoactive intestinal peptide (VIP), ATP and different inflammatory mediators. One of the most important secretagogues is adenosine during inflammation. However, the controversy concerning the underlying mechanism of adenosine-stimulated Cl- secretion in intestinal epithelial cells still continues. To investigate the effect of adenosine on Cl- secretion and its underlying mechanism in the rabbit colon mucosa, we measured short circuit current (ISC) under automatic voltage clamp with DVC-1000 in a modified Ussing chamber. Adenosine, when added to the basolateral side of the mucosa, increased ISC in a dose-dependent manner. The adenosine-stimulated ISC response was abolished when Cl- in the bath solution was replaced completely with gluconate. In addition, the ISC response was inhibited by a basolateral Na-K-Cl cotransporter blocker, bumetanide, and by apical Clchannel blockers, dephenylamine-2-carboxylate (DPC), 5-nitro-2-(3-phenyl-propylamino)-benzoate (NPPB), glibenclamide. Amiloride, an epithelial Na+ channel blocker, and 4,4-diisothiocyanato-stilbene-2,2-disulphonate (DIDS), a Ca2+-activated Cl- channel blocker, had no effect. In the mucosa pre-stimulated with forskolin, adenosine did not show any additive effect, whereas carbachol resulted in a synergistic potentiation of the ISC response. The adenosine response was inhibited by 10 micrometer H-89, an inhibitor of protein kinase A. These results suggest that the adenosine-stimulated ISC response is mediated by basolateral to apical Cl- secretion through a cAMP-dependent Cl- channel. The rank order of potencies of adenosine receptor agonists was 5'-(N-ethylcarboxamino)adenosine(NECA) > N6-(R-phenylisopropyl)adenosine(R-PIA)>2-(p-(2-carbonylethyl)-phenyl-et hylamino)-5'-N-ethylcarboxaminoadenosine(CGS21680). From the above results, it can be concluded that adenosine interacts with the A2b adenosine receptor in the rabbit colon mucosa and a cAMP-dependent signalling mechanism underlies the stimulation of Cl- secretion.
Adenosine ; Adenosine Triphosphate ; Amiloride ; Baths ; Bumetanide ; Carbachol ; Colforsin ; Colon* ; Cyclic AMP-Dependent Protein Kinases ; Epithelial Cells ; Glyburide ; Inflammation ; Intestinal Secretions ; Intestines ; Mucous Membrane* ; Purinergic P1 Receptor Agonists ; Receptors, Purinergic P1 ; Vasoactive Intestinal Peptide