Characterization and virulence potential of phenotypically diverse Aeromonas veronii isolates recovered from moribund freshwater ornamental fishes of Kerala, India

In the present study, we investigated the involvement of Aeromonas spp. in eliciting disease outbreaks in freshwater ornamental fishes across the state of Kerala, India. We investigated three incidences of disease, in which the moribund fishes exhibited clinical signs such as haemorrhagic septicemia (in gouramy, Trichogaster sp.), dropsy (in Oscar, Astronotus ocellatus) and tail rot/fin rot (in gold fish, Carassius carassius). Pure cultures (n = 20 from each fish; 60 in total) of Aeromonas spp. were recovered from the abdominal fluid as well as from internal organs of affected fishes, although they could not be identified to species level because of the variations in their phenotypic characters. The molecular fingerprinting of the isolates using Enterobacterial Repetitive Intergenic Consensus PCR proved the genetic diversity of the isolates from the three sites. The phylogenetic trees constructed using concatenated sequences (using 16S rRNA, gyrA, gyrB and rpoD genes) indicated that they were related to Aeromonas veronii. They exhibited marked cytotoxic and haemolytic activity, which were responsible for the pathogenic potential of the isolates. The isolates possessed multiple virulence genes such as enterotoxins (act and alt), haemolytic toxins (aerA and hlyA), genes involved in type III secretion system (ascV, aexT and ascF–ascG), glycerophospholipid-cholesterol acyltransferase (gcat) and a type IV pilus (tapA) gene, as determined by PCR. Virulence of representative isolates to goldfish was also tested, and we found LD50 values of 104.07–105.35 cfu/fish. Furthermore, the organisms could be recovered as pure cultures from the lesions as well as from the internal organs.

Protein kinase C down-regulation, and not transient activation, correlates with melanocyte growth

The nontumorigenic, immortal line of murine melanocytes, Mel-ab, requires the continual presence of biologically active phorbol esters for growth (R. E. Wilson et al., Cancer Res., 49: 711–716, 1989). Comparable treatments of B16 murine melanoma cells result in partial inhibition of cell proliferation. The role of protein kinase C (PKC) in the modulation of growth of cells from these two melanocytic cell lines has been investigated. Significant levels of PKC were present in quiescent Mel-ab cells as determined by Western blotting, whereas no immunoreactive protein was detected in cell extracts from either proliferating Mel-ab or B16.F1 cells. Phosphorylation of a Mr 80,000 protein, which by one- and two-dimensional gel analysis comigrated with the known Mr 80,000 protein substrate of PKC in fibroblasts, was induced in 12-O-tetradecanoylphorbol-13-acetate-stimulated quiescent Mel-ab cells but not in proliferating Mel-ab cells or B16.F1 melanoma cells. Direct measurement of PKC activity in these cells demonstrated a 10-fold greater level of activity in quiescent Mel-ab cells (262 ± 50 pmol/min/mg SD) compared with growing cells (22.8 ± 11.8 pmol/min/mg SD). An intermediate level of activity was detected in proliferating B16.F1 melanoma cells (148.5 ± 20.4 pmol/min/mg SD). The subcellular distribution of PKC was dependent upon the growth state of the cells such that quiescent Mel-ab cells displayed a higher level of activity in the cytosol, whereas growing Melab cells displayed greater activity in the particulate fraction. Like many other transformed lines, B16.F1 melanoma cells constitutively expressed the majority of enzyme activity in the particulate fraction. Measurement of [3H]phorbol ester binding in intact cells paralleled the PKC activation data such that quiescent Mel-ab cells displayed binding of 1612 ± 147 cpm/106 cells, whereas proliferating Mel-ab and B16.F1 melanoma cells displayed binding of 652 ± 28 and 947 ± 81 cpm/106 cells, respectively. Membrane-permeant diacylglycerol analogues, which activated but did not down-regulate PKC, were devoid of growth-stimulating effects on melanocytes, even in the presence of the specific diacylglycerol kinase inhibitor, R59022. Together, these data show that PKC down-regulation, and not activation, correlates with the growth of melanocytes in culture.

Protein kinase D isoforms are expressed in rat and mouse primary sensory neurons and are activated by agonists of protease-activated receptor 2

Serine proteases generated during injury and inflammation cleave protease-activated receptor 2 (PAR(2)) on primary sensory neurons to induce neurogenic inflammation and hyperalgesia. Hyperalgesia requires sensitization of transient receptor potential vanilloid (TRPV) ion channels by mechanisms involving phospholipase C and protein kinase C (PKC). The protein kinase D (PKD) serine/threonine kinases are activated by diacylglycerol and PKCs and can phosphorylate TRPV1. Thus, PKDs may participate in novel signal transduction pathways triggered by serine proteases during inflammation and pain. However, it is not known whether PAR(2) activates PKD, and the expression of PKD isoforms by nociceptive neurons is poorly characterized. By using HEK293 cells transfected with PKDs, we found that PAR(2) stimulation promoted plasma membrane translocation and phosphorylation of PKD1, PKD2, and PKD3, indicating activation. This effect was partially dependent on PKCepsilon. By immunofluorescence and confocal microscopy, with antibodies against PKD1/PKD2 and PKD3 and neuronal markers, we found that PKDs were expressed in rat and mouse dorsal root ganglia (DRG) neurons, including nociceptive neurons that expressed TRPV1, PAR(2), and neuropeptides. PAR(2) agonist induced phosphorylation of PKD in cultured DRG neurons, indicating PKD activation. Intraplantar injection of PAR(2) agonist also caused phosphorylation of PKD in neurons of lumbar DRG, confirming activation in vivo. Thus, PKD1, PKD2, and PKD3 are expressed in primary sensory neurons that mediate neurogenic inflammation and pain transmission, and PAR(2) agonists activate PKDs in HEK293 cells and DRG neurons in culture and in intact animals. PKD may be a novel component of a signal transduction pathway for protease-induced activation of nociceptive neurons and an important new target for antiinflammatory and analgesic therapies.

CalDAG-GEFI is at the nexus of calcium-dependent platelet activation.

The importance of the second messengers calcium (Ca(2+)) and diacylglycerol (DAG) in platelet signal transduction was established more than 30 years ago. Whereas protein kinase C (PKC) family members were discovered as the targets of DAG, little is known about the molecular identity of the main Ca(2+) sensor(s). We here identify Ca(2+) and DAG-regulated guanine nucleotide exchange factor I (CalDAG-GEFI) as a critical molecule in Ca(2+)-dependent platelet activation. CalDAG-GEFI, through activation of the small GTPase Rap1, directly triggers integrin activation and extracellular signal-regulated kinase-dependent thromboxane A(2) (TxA(2)) release. CalDAG-GEFI-dependent TxA(2) generation provides crucial feedback for PKC activation and granule release, particularly at threshold agonist concentrations. PKC/P2Y12 signaling in turn mediates a second wave of Rap1 activation, necessary for sustained platelet activation and thrombus stabilization. Our results lead to a revised model for platelet activation that establishes one molecule, CalDAG-GEFI, at the nexus of Ca(2+)-induced integrin activation, TxA(2) generation, and granule release. The preferential activation of CalDAG-GEFI over PKC downstream of phospholipase C activation, and the different kinetics of CalDAG-GEFI- and PKC/P2Y12-mediated Rap1 activation demonstrate an unexpected complexity to the platelet activation process, and they challenge the current model that DAG/PKC-dependent signaling events are crucial for the initiation of platelet adhesion.

Regulation of phospholipases C and D in rat ventricular myocytes: stimulation by endothelin-1, bradykinin and phenylephrine.

The physiological activator of protein kinase C (PKC), diacylglycerol, is formed by hydrolysis of phosphoinositides (PI) by phospholipase C (PLC) or phosphatidylcholine by phospholipase D (PLD). We have measured activation of these phospholipases by endothelin-1 (ET-1), bradykinin (BK), or phenylephrine (PE) in ventricular myocytes cultured from neonatal rat. The stimulation of PI hydrolysis after 10 min by 0.1 microM ET-1 (about 12-fold) was much greater than for BK or PE (each about four-fold), and did not correlate with translocation of nPKC delta or nPKC epsilon (Clerk A. Bogoyevitch MA. Andersson MB. Sugden PH, 1994. J Biol Chem 269: 32848-32857: Clerk A, Gillespie-Brown J, Fuller SJ, Sugden PH, 1996. Biochem J 317: 109-118). However, ET-1 and BK stimulated a similar rapid increase in [3H]InsP, formation (< 30 s), which was much greater than that seen with PE. This early phase correlated with PKC translocation. Acute or chronic exposure to 12-O-tetradecanoylphorbol-13-acetate (TPA) or treatment with Ro-31-8220 showed that the stimulation of PI hydrolysis by PE, but not ET-1 or BK, was inhibited by activation of PKC. Furthermore, ET-1 and BK heterologously desensitized the stimulation of PI hydrolysis by PE, ET-1 or BK homologously uncoupled their own receptors from [3H]InsP3 formation, but there was no evidence of heterologous desensitization with these two agonists. Anomalously, chronic exposure to TPA increased the stimulation of PI hydrolysis by BK, but this probably resulted from an increase in BK receptor density. PLD was also rapidly activated by TPA. ET-1, BK or PE. Experiments with Ro-31-8220 showed that the stimulation of PLD by ET-1 and BK was mediated through activation of PKC. We discuss the characteristics of the activation of PI hydrolysis and PLD by ET-1, BK, and PE with respect to the translocation of PKC.

Endothelin signalling in the cardiac myocyte and its pathophysiological relevance

Endothelin A (ET(A)) transmembrane receptors predominate in rat cardiac myocytes. These are G protein-coupled receptors whose actions are mediated by the G(q) heterotrimeric G proteins. Through these, ET-1 binding to ET(A)-receptors stimulates the hydrolysis of membrane phosphatidylinositol 4,5-bisphosphate to diacylglycerol and inositol 1,4,5-trisphosphate. Diacylglycerol remains in the membrane whereas inositol 1,4,5-trisphosphate is soluble (though its importance in the cardiac myocyte is still debated). Isoforms of the phospholipid-dependent protein kinase, protein kinase C (PKC), are intracellular receptors for diacylglycerol. Cytoplasmic nPKCdelta and nPKCepsilon detect increases in membrane diacylglycerols and translocate to the membrane. This brings about PKC activation, though modifications additional to binding to phospholipids and diacylglycerol are involved. The next event (probably associated with PKC activation) is the activation of the membrane-bound small G protein Ras by exchange of GTP for GDP. Ras.GTP loading translocates Raf family mitogen-activated protein kinase (MAPK) kinase kinases to the membrane, initiates the activation of Raf, and thus activates the extracellular signal-regulated kinase 1/2 (ERK1/2) cascade. Over longer times, two analogous protein kinase cascades, the c-Jun N-terminal kinase and p38-mitogen-activated protein kinase cascades, become activated. As the signals originating from the ET(A) receptor are transmitted through these protein kinase pathways, other signalling molecules become phosphorylated, thus changing their biological activities. For example, ET-1 increases the expression of the c-jun transcription factor gene, and increases abundance and phosphorylation of c-Jun protein. These changes in c-Jun expression and phosphorylation are likely to be important in the regulation of gene transcription.

Short communication: Variable number of tandem repeat polymorphisms in DGAT1 gene of buffaloes (Bubalus bubalis) is associated with milk constituents

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Drosophila melanogaster lipins are tissue-regulated and developmentally regulated and present specific subcellular distributions

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Studio della trasduzione del Segnale Nucleare Inositide-Dipendente: identificazione di eEF1A2 come nuovo Fosfosubstrato di PKC βI

Introduction Phospholipase Cb1 (PLC-β1) is a key player in the regulation of nuclear inositol lipid signaling and of a wide range of cellular functions, such as proliferation and differentiation (1,2,3). PLCb1 signaling depends on the cleavage of phosphatidylinositol 4,5-bisphosphate and the formation of the second messengers diacylglycerol and Inositol tris-phosphate which activate canonical protein kinase C (cPKC) isoforms. Here we describe a proteomic approach to find out a potential effector of nuclear PLC-b1 dependent signaling during insulin stimulated myogenic differentiation. Methods Nuclear lysates obtained from insulin induced C2C12 myoblasts were immunoprecipitated with anti-phospho-substrate cPKC antibody. Proteins, stained with Comassie blue, were excised, digested and subsequently analysed in LC-MS/MS. For peptide sequence searching, the mass spectra were processed and analyzed using the Mascot MS/MS ion search program with the NCBI database. Western blotting, GST-pull down and co-immunoprecipitation were performed to study the interaction between eEF1A2 and cPKCs. Site direct mutagenesis was performed to confirm the phosphorylated motif recognized by the antibody. Immunofluorescence analysis, GFP-tagged eEF1A2 vector and subcellular fractionation were performed to study nuclear localization and relative distribution of eEF1A2. Results We have previously shown that PLC-β1 is greatly increased at the nuclear level during insulin-induced myoblasts differentiation and that this nuclear localization is essential for induction of differentiation. Thus, nuclear proteins of insulin stimulated C2C12 myoblasts, were immunoprecipitated with an anti-phospho-substrate cPKC antibody. After Electrophoretic gel separation of proteins immunoprecipitated, several molecules were identified by LC-MS/MS. Among these most relevant and unexpected was eukaryotic elongation factor 1 alpha 2 (eEF1A2). We found that eEF1A2 is phosphorylated by PKCb1 and that these two molecules coimmunolocalized at the nucleolar level. eEF1A2 could be phosphorylated in many sites among which both threonine and serine residues. By site direct mutagenesis we demonstrated that it is the serine residue of the motif recognized by the antibody that is specifically phosphorylated by PKCb1. The silencing of PLCb1 gives rise to a reduction of expression and phosphorylation levels of eEF1A2 indicating this molecule as a target of nuclear PLCb1 regulatory network during myoblasts differentiation.

New DAG-dependent mechanisms modulate cell cycle progression

Through the years, several studies reported the involvement of nuclear lipid signalling as highly connected with cell cycle progression. Indeed, nuclear Phosphatidylinositol-4,5-Biphosphate (PIP2) hydrolisis mediated by Phospholipases C (PLC), which leads to production of the second messengers Diacylglycerol (DAG) and Inositol-1,4,5-Triphosphate (IP3), is a fundamental event for both G1/S and G2/M checkpoints. In particular, we found that nuclear DAG production was mediated by PLCbeta1, enzyme mainly localized in the nucleus of K562 human erythroleukemia cells. This event triggered the activation and nuclear translocation of PKCalpha, which, in turn, resulted able to affect cell cycle via modulation of Cyclin D3 and Cyclin B1, two important enzymes for G1/S transition and G2/M progression respectively.

Modulation of signal transduction by vitamin E

The ability of vitamin E to modulate signal transduction and gene expression has been observed in numerous studies; however, the detailed molecular mechanisms involved are often not clear. The eight natural vitamin E analogues and synthetic derivatives affect signal transduction with different potency, possibly reflecting their different ability to interact with specific proteins. Vitamin E modulates the activity of several enzymes involved in signal transduction, such as protein kinase C, protein kinase B, protein tyrosine kinases, 5-, 12-, and 15-lipoxygenases, cyclooxygenase-2, phospholipase A2, protein phosphatase 2A, protein tyrosine phosphatase, and diacylglycerol kinase. Activation of some these enzymes after stimulation of cell surface receptors with growth factors or cytokines can be normalized by vitamin E. At the molecular level, the translocation of several of these enzymes to the plasma membrane is affected by vitamin E, suggesting that the modulation of protein-membrane interactions may be a common theme for vitamin E action. In this review the main effects of vitamin E on enzymes involved in signal transduction are summarized and the possible mechanisms leading to enzyme modulation evaluated. The elucidation of the molecular and cellular events affected by vitamin E could reveal novel strategies and molecular targets for developing similarly acting compounds.

Inflammation induces hemorrhage in thrombocytopenia

The role of platelets in hemostasis is to produce a plug to arrest bleeding. During thrombocytopenia, spontaneous bleeding is seen in some patients but not in others; the reason for this is unknown. Here, we subjected thrombocytopenic mice to models of dermatitis, stroke, and lung inflammation. The mice showed massive hemorrhage that was limited to the area of inflammation and was not observed in uninflamed thrombocytopenic mice. Endotoxin-induced lung inflammation during thrombocytopenia triggered substantial intra-alveolar hemorrhage leading to profound anemia and respiratory distress. By imaging the cutaneous Arthus reaction through a skin window, we observed in real time the loss of vascular integrity and the kinetics of skin hemorrhage in thrombocytopenic mice. Bleeding-observed mostly from venules-occurred as early as 20 minutes after challenge, pointing to a continuous need for platelets to maintain vascular integrity in inflamed microcirculation. Inflammatory hemorrhage was not seen in genetically engineered mice lacking major platelet adhesion receptors or their activators (alphaIIbbeta3, glycoprotein Ibalpha [GPIbalpha], GPVI, and calcium and diacylglycerol-regulated guanine nucleotide exchange factor I [CalDAG-GEFI]), thus indicating that firm platelet adhesion was not necessary for their supporting role. While platelets were previously shown to promote endothelial activation and recruitment of inflammatory cells, they also appear indispensable to maintain vascular integrity in inflamed tissue. Based on our observations, we propose that inflammation may cause life-threatening hemorrhage during thrombocytopenia.

Liver fat content and lipid metabolism in dairy cows during early lactation and during a mid-lactation feed restriction.

During the transition period, the lipid metabolism of dairy cows is markedly affected by energy status. Fatty liver is one of the main health disorders after parturition. The aim of this study was to evaluate the effects of a negative energy balance (NEB) at 2 stages in lactation [NEB at the onset of lactation postpartum (p.p.) and a deliberately induced NEB by feed restriction near 100 d in milk] on liver triglyceride content and parameters of lipid metabolism in plasma and liver based on mRNA abundance of associated genes. Fifty multiparous dairy cows were studied from wk 3 antepartum to approximately wk 17 p.p. in 2 periods. According to their energy balance in period 1 (parturition to wk 12 p.p.), cows were allocated to a control (CON; n=25) or a restriction group (RES; 70% of energy requirements; n=25) for 3 wk in mid lactation starting at around 100 d in milk (period 2). Liver triglyceride (TG) content, plasma nonesterified fatty acids (NEFA), and β-hydroxybutyrate were highest in wk 1 p.p. and decreased thereafter. During period 2, feed restriction did not affect liver TG and β-hydroxybutyrate concentration, whereas NEFA concentration was increased in RES cows as compared with CON cows. Hepatic mRNA abundances of tumor necrosis factor α, ATP citrate lyase, mitochondrial glycerol-3-phosphate acyltransferase, and glycerol-3-phosphate dehydrogenase 2 were not altered by lactational and energy status during both experimental periods. The expression of fatty acid synthase was higher in period 2 compared with period 1, but did not differ between RES and CON groups. The mRNA abundance of acetyl-coenzyme A-carboxylase showed a tendency toward higher expression during period 2 compared with period 1. The solute carrier family 27 (fatty acid transporter), member 1 (SLC27A1) was upregulated in wk 1 p.p. and also during feed restriction in RES cows. In conclusion, the present study shows that a NEB has different effects on hepatic lipid metabolism and TG concentration in the liver of dairy cows at early and later lactation. Therefore, the homeorhetic adaptations during the periparturient period trigger excessive responses in metabolism, whereas during the homeostatic control of endocrine and metabolic systems after established lactation, as during the period of feed restriction in the present study, organs are well adapted to metabolic and environmental changes.

Western diet changes cardiac acyl-CoA composition in obese rats: a potential role for hepatic lipogenesis.

The "lipotoxic footprint" of cardiac maladaptation in diet-induced obesity is poorly defined. We investigated how manipulation of dietary lipid and carbohydrate influenced potential lipotoxic species in the failing heart. In Wistar rats, contractile dysfunction develops at 48 weeks on a high-fat/high-carbohydrate "Western" diet, but not on low-fat/high-carbohydrate or high-fat diets. Cardiac content of the lipotoxic candidates--diacylglycerol, ceramide, lipid peroxide, and long-chain acyl-CoA species--was measured at different time points by high-performance liquid chromatography and biochemical assays, as was lipogenic capacity in the heart and liver by qRT-PCR and radiometric assays. Changes in membranes fluidity were also monitored using fluorescence polarization. We report that Western feeding induced a 40% decrease in myocardial palmitoleoyl-CoA content and a similar decrease in the unsaturated-to-saturated fatty acid ratio. These changes were associated with impaired cardiac mitochondrial membrane fluidity. At the same time, hepatic lipogenic capacity was increased in animals fed Western diet (+270% fatty acid elongase activity compared with high-fat diet), while fatty acid desaturase activity decreased over time. Our findings suggest that dysregulation of lipogenesis is a significant component of heart failure in diet-induced obesity.

Identification and characterization of genes encoding phospholipid biosynthetic enzymes of {\it Saccharomyces cerevisiae\/}

Phospholipids are the major component of cellular membranes. In addition to its structural role, phospholipids play an active and diverse role in cellular processes. The goal of this study is to identify the genes involved in phospholipid biosynthesis in a model eukaryotic system, Saccharomyces cerevisiae. We have focused on the biosynthetic steps localized in the inner mitochondrial membrane; hence, the identification of the genes encoding phosphatidylserine decarboxylase (PSD1), cardiolipin synthase (CLS1), and phosphatidylglycerophosphate synthase (PGS1).^ The PSD1 gene encoding a phosphatidylserine decarboxylase was cloned by complementation of a conditional lethal mutation in the homologous gene in Escherichia coli strain EH150. Overexpression of the PSD1 gene in wild type yeast resulted in 20-fold amplification of phosphatidylserine decarboxylase activity. Disruption of the PSD1 gene resulted in 20-fold reduction of decarboxylase activity, but the PSD1 null mutant exhibited essentially normal phenotype. These results suggest that yeast has a second phosphatidylserine decarboxylation activity.^ Cardiolipin is the major anionic phospholipid of the inner mitochondrial membrane. It is thought to be an essential component of many biochemical functions. In eukaryotic cells, cardiolipin synthase catalyzes the final step in the synthesis of cardiolipin from phosphatidylglycerol and CDP-diacylglycerol. We have cloned the gene CLS1. Overexpression of the CLS1 gene product resulted in significantly elevated cardiolipin synthase activity, and disruption of the CLS1 gene, confirmed by PCR and Southern blot analysis, resulted in a null mutant that was viable and showed no petite phenotype. However, phospholipid analysis showed undetectable cardiolipin level and an accumulation of phosphatidylglycerol. These results support the conclusion that CLS1 encodes the cardiolipin synthase of yeast and that normal levels of cardiolipin are not absolutely essential for survival of the cell.^ Phosphatidylglycerophosphate (PGP) synthase catalyzes the synthesis of PGP from CDP-diacylglycerol and glycerol-3-phosphate and functions as the committal and rate limiting step in the biosynthesis of cardiolipin. We have identified the PGS1 gene as encoding the PGP synthase. Overexpression of the PGS1 gene product resulted in over 15-fold increase in in vitro PGP synthase activity. Disruption of the PGS1 gene in a haploid strain of yeast, confirmed by Southern blot analysis, resulted in a null mutant strain that was viable but had significantly altered phenotypes, i.e. inability to grow on glycerol and at $37\sp\circ$C. These cells showed over a 10-fold decrease in PGP synthase activity and a decrease in both phosphatidylglycerol and cardiolipin levels. These results support the conclusion that PGS1 encodes the PGP synthase of yeast and that neither phosphatidylglycerol nor cardiolipin are absolutely essential for survival of the cell. ^