SHP-2-Erk signaling regulates concanavalin A-dependent production of TIMP-2

To search for the signaling critical for the production of tissue inhibitor of metalloproteinase-2 (TIMP-2), we investigated the role of SHP-2 in TIMP-2 production with Concanavalin A (Con A)-treated cells. In wild-type fibroblasts, Con A-treatment dramatically activated TIMP-2 production. In contrast, production of TIMP-2 in response to Con A-treatment was severely impaired in cells expressing mutant SHP-2 whose 65 amino acids in the SH2-N domain were deleted. Con A-treatment activated dual signaling pathways, Erk and p38, in a SHP-2-dependent manner. Pretreatment of wild-type cells with U0126, a potent inhibitor of MEK1, significantly inhibited the production of TIMP-2, whereas SB203580, a specific inhibitor for p38, could not. Finally, expression of exogenous wild-type SHP-2 in SHP-2 mutant cells clearly rescued Erk activation and TIMP-2 production in response to Con A-treatment. Taken together, our results strongly suggest that SHP-2 plays a critical role as a positive modulator for the production of TIMP-2 via MEK1-Erk signaling in fibroblasts.     

Enhanced photosystem 2 thermostability during leaf growth of Elm (Ulmus pumila) seedlings

We examined photosynthetic activities and thermostability of photosystem 2 (PS2) in leaves of elm (Ulmus pumila) seedlings from initiation to full expansion. During leaf development, net photosynthetic rate (P _N) increased gradually and reached the maximum when leaves were fully developed. In parallel with the increase of P _N, chlorophyll (Chl) content was significantly elevated. Chl a fluorescence measurements showed that the maximum quantum yield of PS2 (ϕ_PS2), the efficiency a trapped exciton, moved an electron into the electron transport chain further than Q_A ⁻ (Ψ_o), and the quantum yield of electron transport beyond Q_A (ϕ_Eo) increased gradually. These results were independently confirmed by our low irradiance experiments. When subjected to progressive heat stress, the young leaves exhibited considerably lower ϕ_PS2 and higher minimal fluorescence (F₀) than the mature leaves, revealing the highly sensitive nature of PS2 under heat in the newly initiating leaves. Further analysis showed that PS2 structure in the newly initiating leaves was strongly altered under heat, as evidenced by the increased fluorescence signals at the position of the K step. We therefore demonstrated an inhibition in the oxygen-evolving complex (OEC) in the young leaves. This resulted in decrease in amount of the functional PS2 reaction centres and relative increase in the PS2 reaction centres with inhibited electron transport at the acceptor side under heat. We suggest that the enhanced thermostability of PS2 during leaf development is associated with improved OEC stability.     

Indomethacin inactivates gastric peroxidase to induce reactive-oxygen-mediated gastric mucosal injury and curcumin protects it by preventing peroxidase inactivation and scavenging reactive oxygen

We have investigated the mechanism of indomethacin-induced gastric ulcer caused by reactive oxygen species (ROS) and the gastroprotective effect of curcumin thereon. Curcumin dose-dependently blocks indomethacin-induced gastric lesions, showing 82% protection at 25 mg/kg. Indomethacin-induced oxidative damage by ROS as shown by increased lipid peroxidation and thiol depletion is almost completely blocked by curcumin. Indomethacin causes nearly fivefold increase in hydroxyl radical (()OH) and significant inactivation of gastric mucosal peroxidase to elevate endogenous H(2)O(2) and H(2)O(2)-derived ()OH, which is prevented by curcumin. In vitro studies indicate that indomethacin inactivates peroxidase irreversibly only in presence of H(2)O(2) by acting as a suicidal substrate. 5,5-Dimethyl-pyrroline-N-oxide (DMPO) protects the peroxidase, indicating involvement of indomethacin radical in the inactivation. Indomethacin radical was also detected in the peroxidase-indomethacin-H(2)O(2) system as DMPO adduct (a(N) = 15 G, a(beta)(H) = 16 G) by electron spin resonance spectroscopy. Curcumin protects the peroxidase in a concentration-dependent manner and consumes H(2)O(2) for its oxidation as a suitable substrate of the peroxidase, thereby blocking indomethacin oxidation. Curcumin can also scavenge ()OH in vitro. We suggest that curcumin protects gastric damage by efficient removal of H(2)O(2) and H(2)O(2) -derived ()OH by preventing peroxidase inactivation by indomethacin.     

DNA arms do the legwork to ensure the directionality of lambda site-specific recombination

The integrase protein of bacteriophage lambda (Int) catalyzes site-specific recombination between lambda phage and Escherichia coli genomes. Int is a tyrosine recombinase that binds to DNA core sites via a C-terminal catalytic domain and to a collection of arm DNA sites, distant from the site of recombination, via its N-terminal domain. The arm sites, in conjunction with accessory DNA-bending proteins, provide a means of regulating the efficiency and directionality of Int-catalyzed recombination. Recent crystal structures of lambda Int tetramers bound to synaptic and Holliday junction intermediates, together with new biochemical data, suggest a mechanism for the allosteric control of the recombination reaction through arm DNA binding interactions.     

Altered neuropeptide processing in prefrontal cortex of Cpe (fat/fat) mice: implications for neuropeptide discovery

The biosynthesis of most neuropeptides and peptide hormones requires a carboxypeptidase such as carboxypeptidase E, which is inactive in Cpe(fat/fat) mice due to a naturally occurring point mutation. To assess the role of carboxypeptidase E in the processing of peptides in the prefrontal cortex, we used a quantitative peptidomics approach to examine the relative levels of peptides in Cpe(fat/fat) versus wild-type mice. Peptides representing internal fragments of prohormones and other secretory pathway proteins were decreased two- to 10-fold in the Cpe(fat/fat) mouse prefrontal cortex compared with wild-type tissue. Degradation fragments of cytosolic proteins showed no major differences between Cpe(fat/fat) and wild-type mice. Based on this observation, a search strategy for neuropeptides was performed by screening for peptides that decreased in the Cpe(fat/fat) mouse. Altogether, 32 peptides were identified, of which seven have not been previously reported. The novel peptides include fragments of VGF, procholecystokinin and prohormone convertase 2. Interestingly, several of the peptides do not fit with the consensus sites for prohormone convertase 1 and 2, raising the possibility that another endopeptidase is involved with their biosynthesis. Taken together, these findings support the proposal that carboxypeptidase E is the major, but not the only, peptide-processing carboxypeptidase and also demonstrate the feasibility of searching for novel peptides based on their decrease in Cpe(fat/fat) mice.     

Role of protein kinase C-mediated protein phosphorylation in mitochondrial translocation of mouse CYP1A1, which contains a non-canonical targeting signal

A large number of mitochondrial proteins lack canonical mitochondrial-targeting signals. The bimodal transport of cytochromes P450 (CYPs) to endoplasmic reticulum and mitochondria (MT), reported previously by us, likely represents one mode of non-canonical protein targeting to MT. Herein, we have studied the mechanism of mouse MT-CYP1A1 targeting to gain insight into the regulatory features and evolutionary conservation of bimodal targeting mechanism. Mouse MT-CYP1A1 consists of two NH2-terminal-truncated molecular species, +91A1 and +331A1. Mutations Pro-2 --> Leu and Tyr-5 --> Leu, which increase the signal recognition particle (SRP) binding, diminished MT targeting of the protein in intact cells. By contrast, mutations Leu-7 --> Asn and Leu-17 --> Asn, which decreased SRP-binding affinity, enhanced MT targeting, thus suggesting that SRP binding is an important regulatory step that modulates bimodal targeting. Protein kinase C (PKC)-mediated phosphorylation of nascent chains at Thr-35 vastly decreased affinity for SRP binding suggesting an important regulatory step. In support of these results, COS cell transfection experiments show that phosphomimetic mutation Thr-35 --> Asp or induced cellular PKC caused increased CYP1A1 targeting to MT and correspondingly lower levels to the endoplasmic reticulum. Results suggest evolutionary conservation of chimeric signals and bimodal targeting of CYP1A1 in different species. The mouse MT-CYP1A1 is an extrinsic membrane protein, which exhibited high FDX1 plus FDXR-mediated N-demethylation of a number of tricyclic antidepressants, pain killers, anti-psychotics, and narcotics that are poor substrates for microsomal CYP1A1.     

Differential gene expression profiles and real-time measurements of growth parameters in Saccharomyces cerevisiae grown in microliter-scale bioreactors equipped with internal stirring

Combining real-time growth kinetics measurements with global gene expression analysis of microbial cultures is of significant value for high-throughput biological research. We have performed differential gene expression analysis in the eukaryotic model Saccharomyces cerevisiae grown in galactose and glucose media in 150 muL bioreactors equipped with sensors for in situ and real-time measurements of optical density (OD), pH, and dissolved oxygen (DO). The microbioreactors were fabricated from poly(dimethylsiloxane) (PDMS) and poly(methyl methacrylate) (PMMA) and equipped with internal magnetic ministirrers and evaporation compensation by water replacement. In galactose-grown cells, the core genes of the GAL operon GAL2, GAL1, GAL7, and GAL10 were upregulated at least 100-fold relative to glucose-grown cells. These differential gene expression levels were similar to those observed in large-scale culture vessels. The increasing rate at which complete genomic sequences of microorganisms are becoming available offers an unprecedented opportunity for comparative investigations of these organisms. Our results from S. cerevisiae cultures grown in instrumented microbioreactors show that it is possible to integrate high-throughput studies of growth physiology with global gene expression analysis of microorganisms.     

Establishment of the fungal entomopathogen Beauveria bassiana as a season long endophyte in jute (Corchorus olitorius) and its rapid detection using SCAR marker

An experiment was conducted to introduce the entomopathogen Beauveria bassiana (Balsamo) Vuillemin (Ascomycota: Hypocreales) as an endophyte in jute (Corchorus olitorius), a bast fibre crop through seed treatment. Colonization of root, leaf, stem, capsule, and seed were assessed through plating on selective medium and PCR based detection using B. bassiana specific SCAR markers. Endophytic colonization was detected in all the plants grown from treated seeds, but all the plant parts were not colonized. Colonization was detected in leaves, stems, and green capsules but not in roots and seeds. The endophytic colonization was influenced by both plant part and sampling period. Colonization was greater in leaves (55.87%) compared to stems (12.53%) and capsules (42.44%). The percent colonization was higher in case of 60?days old plants (43.34%) than in 30?days (23.89%) and 120?days (35.39%) old plants. As B. bassiana has already been reported to be pathogenic on jute pests, namely semilooper (Anomis sabulifera) and bihar hairy caterpillar (Spilosoma obliqua), its season long endophytic colonization within jute plant suggests a novel approach of biological control of these pests through seed treatment with the entomopathogen.     

An Extracelluar Protease,SepM, Generates Functional Competence-Stimulating Peptide in Streptococcus mutans UA159

Cell-cell communication in Gram-positive bacteria often depends on the production of extracellular peptides. The cariogenic bacterium Streptococcus mutans employs so-called competence-stimulating peptide (CSP) to stimulate mutacin (bacteriocin) production and competence development through the activation of the ComDE two-component pathway. In S. mutans, CSP is secreted as a 21-residue peptide; however, mass spectrometric analysis of culture supernatant indicates the presence of an 18-residue proteolytically cleaved species. In this study, using a transposon mutagenesis screening, we identified a cell surface protease that is involved in the processing of 21-residue CSP to generate the 18-residue CSP. We named this protease SepM for streptococcal extracellular protease required for mutacin production. We showed that the truncated 18-residue peptide is the biologically active form and that the specific postexport cleavage is a prerequisite to activate the ComDE two-component signal transduction pathway. We also showed that the CSP and the mutacins are exported outside the cell by the same ABC transporter, NlmTE. Our study further confirmed that the ComDE two-component system is absolutely necessary for mutacin production in S. mutans.