Alcohol-conferred hemolysis in yeast is a consequence of increased respiratory burden

We have previously reported that growth on alcohol vapors confers hemolytic properties on certain yeast species and strains ('microbial alcohol conferred hemolysis'; MACH). Here, a Saccharomyces cerevisiae deletion library consisting of c. 4800 clones was screened for MACH mutants in the presence of n-butanol vapors; 136 mutants were MACH-negative, and 325 exhibited reduced hemolysis and/or growth. Of the MACH-negative mutants, 35.3% were affected in mitochondrial-related genes. The data suggest that intact mitochondrial and respiratory chain functions are critical for the observed MACH phenomenon. We propose that the uncontrolled cellular uptake of alcohol results in yeast 'hyper-respiration', leading to elaboration of hemolytic molecules such as hydrogen peroxide and hemolysis-causing lipids. To support this premise, we showed that: (1) exogenous catalase and glutathione reduce alcohol-conferred hemolysis in S. cerevisiae BY4741 and Candida tropicalis 59445; (2) C. tropicalis produces hydrogen peroxide following growth on ethanol and n-butanol, as shown using xylenol orange; and (3) a lysophospholipid-containing lipid extract from alcohol-grown C. tropicalis specifically causes hemolysis.     

Thioredoxin secreted upon ultraviolet A irradiation modulates activities of matrix metalloproteinase-2 and tissue inhibitor of metalloproteinase-2 in human dermal fibroblasts

Regulation of the balance of matrix metalloproteinase-2 (MMP-2) and its tissue inhibitor (TIMP-2) by thioredoxin (Trx) was investigated in human dermal fibroblasts. Expression and secretion of Trx and Trx reductase 1 (TR1) was increased after ultraviolet (UV) A irradiation. A significant increase in proMMP-2 activity and a decrease of TIMP-2 activity in supernatants of UVA-irradiated fibroblasts were observed in gelatin and reverse zymography compared to non-irradiated fibroblasts. Removal of Trx or TR1 by immunoprecipitation diminished these changes in proMMP-2 activity. Incubation with 5, 5'-dithio-bis-2-nitrobenzoic acid (DTNB) also suppressed these changes. Incubation with recombinant Trx or TR decreased TIMP-2 activity and increased MMP-2 activity. UVA-irradiated fibroblasts, transiently transfected with a dominant-negative mutant or wild-type Trx, showed down- or upregulation of proMMP-2 activities, respectively, without significant change of protein amount. In conclusion, thioredoxin secreted by UVA irradiation is involved in the regulation of MMP-2 and TIMP-2 activities through its redox activity in human dermal fibroblasts.     

Virus-induced dilated cardiomyopathy is characterized by increased levels of fibrotic extracellular matrix proteins and reduced amounts of energy-producing enzymes

The most relevant clinical phenotype resulting from chronic enteroviral myocarditis is dilated cardiomyopathy (DCM). Mice of the susceptible mouse strain A.BY/SnJ mimick well human DCM since they develop as a consequence of persistent infection and chronic inflammation a dilation of the heart ventricle several weeks after coxsackievirus B3 (CVB3) infection. Therefore, this model is well suited for the analysis of changes in the heart proteome associated with DCM. Here, we present a proteomic survey of the dilated hearts based on differential fluorescence gel electrophoresis and liquid chromatography-mass spectrometric centered methods in comparison to age-matched non-infected hearts. In total, 101 distinct proteins, which belong to categories immunity and defense, cell structure and associated proteins, energy metabolism and protein metabolism/modification differed in their levels in both groups. Levels of proteins involved in fatty acid metabolism and electron transport chain were found to be significantly reduced in infected mice suggesting a decrease in energy production in CVB3-induced DCM. Furthermore, proteins associated with muscle contraction (MLRV, MLRc2, MYH6, MyBPC3), were present in significantly altered amounts in infected mice. A significant increase in the level of extracellular matrix proteins in the dilated hearts indicates cardiac remodeling due to fibrosis.     

Emergent polyethism as a consequence of increased colony size in insect societies

A threshold reinforcement model in insect societies is explored over a range of colony sizes and levels of task demand to examine their effects upon worker polyethism. We find that increasing colony size while keeping the demand proportional to the colony size causes an increase in the differentiation among individuals in their activity levels, thus explaining the occurrence of elitism (individuals that do a disproportionately large proportion of work) in insect societies. Similar results were obtained when the overall work demand is increased while keeping the colony size constant. Our model can reproduce a whole suite of distributions of the activity levels among colony members that have been found in empirical studies. When there are two tasks, we demonstrate that increasing demand and colony size generates highly specialized individuals, but without invoking any strict assumptions about spatial organization of work or any inherent abilities of individuals to tackle different tasks. Importantly, such specialization only occurs above a critical colony size such that smaller colonies contain a set of undifferentiated equally inactive individuals while larger colonies contain both active specialists and inactive generalists, as has been found in empirical studies and is predicted from other theoretical considerations.     

Epigenetic variation as a consequence of homology-dependent gene interactions in transgenic plants

Unexpected results from gene transfer experiments in higher plants have revealed that unlinked duplicated genes can interact in trans in somatic cells, resulting in inactivation of one or both of the interacting pair. This inactivation is the result of epigenetic changes and not genetic mutations because it is reversible and is often accompanied by methylation of the affected genes. The fact that some duplications escape inactivation suggests that the interaction depends on the relative chromosomal positions of the duplicated genes. The similarities between homology-dependent trans-inactivation in plants and other 'homology-sensing' methylation phenomena, such as repeat-induced point mutation in fungi and mammals, are discussed.     

Intramolecular rearrangements as a consequence of the dephosphorylation of phosphoaspartate residues in proteins

Sedlin is an evolutionarily conserved protein encoded by the causative gene SEDL for spondyloepiphyseal dysplasia tarda. Nevertheless, how Sedlin mutations cause the disease remains unknown. Here, the intracellular chloride channel protein CLIC1 was shown to associate with Sedlin by yeast two-hybrid screening. Green fluorescence protein-CLIC1 readily co-immunoprecipitated with FLAG-Sedlin. In addition, both proteins colocalized extensively in cytoplasmic vesicular/reticular structures in COS-7 cells, suggesting their interaction at intracellular membranous organelles. Sedlin also associated with CLIC2 in yeast two-hybrid assays. The link between Sedlin and the intracellular chloride channels is the first step to understand their functional interplays.     

Macrophage migration inhibitory factor: a mediator of matrix metalloproteinase-2 production in rheumatoid arthritis

Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by destruction of bone and cartilage, which is mediated, in part, by synovial fibroblasts. Matrix metalloproteinases (MMPs) are a large family of proteolytic enzymes responsible for matrix degradation. Macrophage migration inhibitory factor (MIF) is a cytokine that induces the production of a large number of proinflammatory molecules and has an important role in the pathogenesis of RA by promoting inflammation and angiogenesis.     

Effect of a derivatized tetrapeptide from lactoferrin on nitric oxide mediated matrix metalloproteinase-2 production by synovial fibroblasts in collagen-induced arthritis in rats

Matrix metalloproteinases (MMPs) constitute a family of zinc-dependent proteolytic enzymes, which degrade several components of extracellular matrix, in arthritic synovial cells. In cultured synovial fibroblasts, both nitric oxide (NO) and reactive oxygen species (ROS) are potent inducers of MMPs production. PEP1261, a tetrapeptide derivative used in this study, corresponds to residues of 39-42 human lactoferrin. The parent protein lactoferrin is able to inhibit the production of free radicals in rheumatoid joints and it regulates many aspects of inflammation. This study is aimed to examine the effects of PEP1261 on MMP-2 production in the presence of nitric oxide donor in cultured synovial fibroblasts from collagen-induced arthritic rats. PEP1261 affects a significant reduction in nitrite levels as well as in MMP-2 production in SNAP stimulated synovial fibroblasts and this is validated by gelatin zymography and immunoblot analysis. Furthermore, RTPCR analysis has demonstrated that PEP1261 inhibits MMP-2 mRNA expression in SNAP treated synovial fibroblasts. The results of this study suggest that PEP1261 possesses antiarthritic activity by inhibiting nitrite levels as well as MMP-2 expression better than control peptides viz., KRDS and RGDS.     

Evidence that activation of platelet calpain is induced as a consequence of binding of adhesive ligand to the integrin, glycoprotein IIb-IIIa

Calpain (a Ca(2+)-dependent protease) is present in many cell types. Because it is present in the cytosol, the potential exists that it may regulate critical intracellular events by inducing crucial proteolytic cleavages. However, the concentrations of Ca2+ required to activate calpain are higher than those attained in the cytoplasm of most cells. Thus, the physiological importance of calpain and the mechanisms involved in its activation have remained elusive. In this study, we show that calpain rapidly moved to a peripheral location upon the addition of an agonist to suspensions of platelets, but it remained unactivated. We provide three lines of evidence that calpain was subsequently activated by a mechanism that required the binding of an adhesive ligand to the major platelet integrin, glycoprotein (GP) IIb- IIIa: calpain activation was prevented by RGDS, a tetrapeptide that inhibits the binding of adhesive ligand to GP IIb-IIIa; it was also prevented by monoclonal antibodies that inhibit adhesive ligand binding to GP IIb-IIIa; and its activation was markedly reduced in platelets from patients whose platelets have greatly reduced levels of functional GP IIb-IIIa. Thus, in platelets, binding of the extracellular domain of GP IIb-IIIa to its adhesive ligand can initiate a transmembrane signal that activates intracellular calpain. Because calpain is present in focal contacts of adherent cells, the interaction of integrins with adhesive ligands in the extracellular matrix may regulate activation of calpain in other cell types as well.     

Ultraviolet A-induced production of matrix metalloproteinase-1 is mediated by macrophage migration inhibitory factor (MIF) in human dermal fibroblasts

Matrix metalloproteinases (MMPs) are thought to be responsible for dermal photoaging in human skin. In the present study, we evaluated the involvement of macrophage migration inhibitory factor (MIF) in MMP-1 expression under ultraviolet A (UVA) irradiation in cultured human dermal fibroblasts. UVA (20 J/cm(2)) up-regulates MIF production, and UVA-induced MMP-1 mRNA production is inhibited by an anti-MIF antibody. MIF (100 ng/ml) was shown to induce MMP-1 in cultured human dermal fibroblasts. We found that MIF (100 ng/ml) enhanced MMP-1 activity in cultured fibroblasts assessed by zymography. Moreover, we observed that fibroblasts obtained from MIF-deficient mice were much less sensitive to UVA regarding MMP-13 expression than those from wild-type BALB/c mice. Furthermore, after UVA irradiation (10 J/cm(2)), dermal fibroblasts of MIF-deficient mice produced significantly decreased levels of MMP-13 compared with fibroblasts of wild-type mice. Next we investigated the signal transduction pathway of MIF. The up-regulation of MMP-1 mRNA by MIF stimulation was found to be inhibited by a PKC inhibitor (GF109203X), a Src-family tyrosine kinase inhibitor (herbimycin A), a tyrosine kinase inhibitor (genistein), a PKA inhibitor (H89), a MEK inhibitor (PD98089), and a JNK inhibitor (SP600125). In contrast, the p38 inhibitor (SB203580) was found to have little effect on expression of MMP-1 mRNA. We found that PKC-pan, PKC alpha/beta II, PKC delta (Thr505), PKC delta (Ser(643)), Raf, and MAPK were phosphorylated by MIF. Moreover, we demonstrated that phosphorylation of PKC alpha/beta II and MAPK in response to MIF was suppressed by genistein, and herbimycin A as well as by transfection of the plasmid of C-terminal Src kinase. The DNA binding activity of AP-1 was significantly up-regulated 2 h after MIF stimulation. Taken together, these results suggest that MIF is involved in the up-regulation of UVA-induced MMP-1 in dermal fibroblasts through PKC-, PKA-, Src family tyrosine kinase-, MAPK-, c-Jun-, and AP-1-dependent pathways.     

Starch synthesis in transgenic potato tubers with increased 3-phosphoglyceric acid content as a consequence of increased 6-phosphofructokinase activity

The aim of this work was to test the hypothesis that changes in cytosolic 3-phosphoglyceric acid (3-PGA) content can regulate the rate of starch synthesis in potato (Solanum tuberosum L.) tubers. The amount of 3-PGA was increased by expressing bacterial phosphofructokinase (PFK; EC 2.7.1.11) in transgenic potato tubers. The resultant 3-fold increase in PFK activity was accompanied by an increase in metabolites downstream of PFK, including a 3-fold increase in 3-PGA. There was also a decrease in metabolites upstream of PFK, most notably of glucose-6-phosphate. The increase in 3-PGA did not affect the amount of starch that accumulated in developing tubers, nor its rate of synthesis in tuber discs cut from developing tubers. This suggests that changes in cytosolic 3-PGA may not affect the rate of starch synthesis under all circumstances. We propose that in this case, a decrease in glucose-6-phosphate (which is transported into the amyloplast as a substrate for starch synthesis) may be sufficient to counteract the effect of increased 3-PGA.     

Carboxy derivatized glucosamine is a potent inhibitor of matrix metalloproteinase-9 in HT1080 cells

Experimental evidences have confirmed that matrix metalloproteinases (MMPs) play a fundamental role in a wide variety of pathologic conditions and recent advances in medicinal chemistry approach to the design of MMP inhibitors with desired structural and functional properties. Among MMPs, MMP-9 has demonstrated to play a major role in the establishment of metastases and it is substantially increased in the majority of malignant tumors. Inhibition of MMP-9 is thought to have a therapeutic benefit to cancer. Results of this study present a novel synthetic MMP-9 inhibitor that downregulates MMP-9 expression level in HT1080, human fibrosarcoma cells.     

Decreased diversity but increased substitution rate in host mtDNA as a consequence of Wolbachia endosymbiont infection

A substantial fraction of insects and other terrestrial arthropods are infected with parasitic, maternally transmitted endosymbiotic bacteria that manipulate host reproduction. In addition to imposing direct selection on the host to resist these effects, endosymbionts may also have indirect effects on the evolution of the mtDNA with which they are cotransmitted. Patterns of mtDNA diversity and evolution were examined in Drosophila recens, which is infected with the endosymbiont Wolbachia, and its uninfected sister species D. subquinaria. The level of mitochondrial, but not nuclear, DNA diversity is much lower in D. recens than in D. subquinaria, consistent with the hypothesized diversity-purging effects of an evolutionarily recent Wolbachia sweep. The d(N)/d(S) ratio in mtDNA is significantly greater in D. recens, suggesting that Muller's ratchet has brought about an increased rate of substitution of slightly deleterious mutations. The data also reveal elevated rates of synonymous substitutions in D. recens, suggesting that these sites may experience weak selection. These findings show that maternally transmitted endosymbionts can severely depress levels of mtDNA diversity within an infected host species, while accelerating the rate of divergence among mtDNA lineages in different species.