Luis M. Salgado

High levels of palmitic acid lead to insulin resistance due to changes in the level of phosphorylation of the insulin receptor and insulin receptor substrate-1

Insulin resistance is defined as the decrease in the glucose disposal in response to insulin by the target tissues. High concentrations of nonesterified fatty acids (NEFA) in plasma have been implicated with many insulin resistance states. We evaluated several aspects of the insulin resistance induced by palmitic acid in rats and found that after treatment with 0.09 g/kg of palmitic acid there is a delay in the curve of tolerance to glucose. We measured the changes in protein phosphorylation in samples from abdominus rectus muscle and there was a decrease of 64 and 75% in the levels of phosphorylation in tyrosine of the insulin receptor and insulin receptor substrate-1, respectively. This diminution in the tyrosine phosphorylation is consistent with a decrease in the main pathway known to be activated after insulin treatment, the mitogen activated protein kinases (MAPKs). If the animals were treated with inhibitors of PKC, like sphingosine, there was a prevention of the effect of palmitic acid determined at the level of tyrosine phosphorylation. According with this result, we found an increase in the phosphorylations in serine of the insulin receptor after the treatment with palmitate. These results suggest that PKC has a role as negative regulator (by phosphorylation in serine) of the insulin receptors activation in the insulin resistance induced by palmitic acid.

Differential expression of cellulases and xylanases by Cellulomonas flavigena grown on different carbon sources

The diversity of cellulases and xylanases secreted by Cellulomonas flavigena cultured on sugar cane bagasse, Solka-floc, xylan, or glucose was explored by two-dimensional gel electrophoresis. C. flavigena produced the largest variety of cellulases and xylanases on sugar cane bagasse. Multiple extracellular proteins were expressed with these growth substrates, and a limited set of them coincided in all substrates. Thirteen proteins with carboxymethyl cellulase or xylanase activity were liquid chromatography/mass spectrometry sequenced. Proteins SP4 and SP18 were identified as products of celA and celB genes, respectively, while SP20 and SP33 were isoforms of the bifunctional cellulase/xylanase Cxo recently sequenced and characterized in C. flavigena. The rest of the detected proteins were unknown enzymes with either carboxymethyl cellulase or xylanase activities. All proteins aligned with glycosyl hydrolases listed in National Center for Biotechnology Information database, mainly with cellulase and xylanase enzymes. One of these unknown enzymes, protein SP6, was cross-induced by sugar cane bagasse, Solka-floc, and xylan. The differences in the expression maps of the presently induced cultures revealed that C. flavigena produces and secretes multiple enzymes to use a wide range of lignocellulosic substrates as carbon sources. The expression of these proteins depends on the nature of the cellulosic substrate.

Selective protein kinase inhibitors block head-specific differentiation in hydra

Several studies have suggested that morphogenesis and patterning in hydra are regulated through pathways involving protein kinase C (PKC). Nevertheless, the complete signal system for regeneration in hydra is still not completely understood. Using inhibitors of different signalling pathways we are dissecting this system. We found that sphingosine (2 microM), staurosporine (0.1 microM), PP1/AGL1872 (1 microM) and H7 (25 microM) were able to inhibit head but not foot regeneration. The inhibition was reversible. When the inhibitor was replaced with hydra medium the animals continue their regeneration in a normal way. The exception was PP1/AGL1872, in this case the animals regenerated only one or two tentacles. These results imply that head and foot regeneration are independent processes and they are not directly related as has been proposed. Sphingosine and PP1/AGL1872 inhibit the transcription of ks1, an early regeneration gene, at 24 and 48 h of treatment. Sphingosine 2 microM arrested the cells on the G1 phase of the cell cycle, but 1 microM of PP1/AGL1872 did not. The regeneration was not affected if the animals were exposed to inhibitors of human growth factor receptors. We propose that head regeneration in hydra may be regulated at least by two pathways, one going through PKC and the other through Src. The first pathway could be related to cellular proliferation and the second one to cellular differentiation.

PI3K and ERK 1‐2 regulate early stages during head regeneration in hydra

Different signaling systems coordinate and regulate the development of a multicellular organism. In hydra, the canonical Wnt pathway and the signal transduction pathways mediated by PKC and Src regulate early stages of head formation. In this paper, we present evidence for the participation of a third pathway, the PI3K‐PKB pathway, involved in this process. The data presented here are consistent with the participation of ERK 1‐2 as a point of convergence for the transduction pathways mediated by PKC, Src and PI3K for the regulation of the regeneration of the head in hydra. The specific developmental point regulated by them appears to be the commitment of tissue at the apical end of the regenerate to form the head organizer.

Selective inhibition of protein kinases blocks the formation of a new axis, the beginning of budding, in Hydra

In Hydra, head regeneration and bud formation appear to be very similar processes. The fact that there are genes whose expression is specific for one of the two processes suggests that they do not have identical molecular bases. We analyzed the signal transduction pathways regulating bud development using inhibitors of protein kinase C, Src, PI3K and ERK. The four inhibitors reversibly blocked bud formation in Hydra when applied before stage 1. Once the bud reached stage 3, three of them had no effect and the bud developed normally. The inhibitors blocked the expression of Budhead, an early head marker, and of CnOtx which are specific for bud formation. The results are in agreement with the central role of a signaling pathway mediated by Src on bud development.

Characterization of a β-Glucosidase Produced by a High-Specific Growth-Rate Mutant of Cellulomonas flavigena

The mutant strain PN-120 of Cellulomonas flavigena produces a ß-glucosidase that is 10-fold more active than the corresponding enzyme isolated from the parental strain. These enzymes were partially purified through Q Sepharose and Bio-Gel filtration. A single protein band was detected on polyacrylamide–gel electrophoresis/zymogram using 4-methylumbelliferyl-β-D-glucoside. On sodium dodecyl sulfate–PAGE, the enzyme displayed three protein bands, suggesting that in C. flavigena the enzyme is oligomeric with a molecular mass of 210 kDa. On purification, the specific activity of ß-glucosidase isolated from PN-120 was increased 16-fold and showed three times more affinity for cellobiose than the enzyme of the parental strain; nevertheless, the optimum pH and temperature were similar for both enzymes. The kinetic parameters suggested that the increase in the activity of the enzyme, from the mutant strain, was caused by a mutation that affects the catalytic site of the enzyme. The partial amino-acid sequence of the isolated enzyme confirmed that it is a β-glucosidase because of its homology with other β-glucosidases produced by cellulolytic bacteria and fungi.

Expression and Characterization of the celcflB Gene from Cellulomonas flavigena Encoding an Endo-β-1,4-Glucanase

An endoglucanase gene called celcflB was isolated from a genomic library of C. flavigena. Its nucleotide sequence showed an ORF of 1725 bp with a GTG start codon. A promoter sequence was found inside the adjacent gene upstream from the start point of translation of celcflB gene. The gene celcflB was induced by sugarcane bagasse and repressed by glucose when C. flavigena was grown on these carbon sources, suggesting that celcflB gene is regulated at transcriptional level. The predicted amino acid sequence of the CelcflB protein presented a catalytic domain with a high homology to family 5 of glycosil hydrolases, and a cellulose binding domain identical to cellulose binding domains type II. The coding region of the celcflB gene was cloned into the expression vector pQE30 and the recombinant protein of 58 kDa presented endoglucanase activity towards carboxymethyl cellulose (CMC).

Isolation and analysis of a new developmentally regulated gene from amastigotes of Leishmania mexicana mexicana

Leishmania differentiates from the promastigote to the amastigote stage during its digenetic life cycle. Characterization of the developmentally regulated genes during that process would help to elucidate the mechanisms of gene regulation. In this study, specific fragments of mRNAs from the amastigote stage of L. mexicana mexicana were discriminated from those of the promastigote and metacyclic stages by differential display. This technique combined with spliced-leader polymerase chain reaction allowed isolation of the complete gene VG7A5. The sequence of this gene did not align with any published L. mexicana sequence. More than one copy of this gene was identified in the genome by Southern-blot analysis and was transcribed exclusively in the amastigote stage. At 20 bp upstream from the splice AG site it has a trans-splicing polypyrimidine tract. The gene encodes the subcellular localization motifs 5′-GGACT and AAGCT-3′ in the 3′ untranslated region of the mRNA. The open reading frame of the gene VG7A5 predicts a polypeptide of 587 amino acid residues that has a KGRR amidation motif near its carboxyl terminus, suggesting that in the mammalian host this protein may be involved in the process of acute inflammation.

An IQGAP-related gene is activated during tentacle formation in the simple metazoan Hydra

Abstract Differentiation of body column epithelial cells into tentacle epithelial cells in Hydra is accompanied by changes in both cell shape and cell-cell contact. The molecular mechanism by which epithelial cells acquire tentacle cell characteristics is unknown. Here we report that expression of a Hydra homologue of the mammalian IQGAP1 protein is strongly upregulated during tentacle formation. Like mammalian IQGAP, Hydra IQGAP1 contains an N-terminal calponin-homology domain, IQ repeats and a conserved C terminus. In adult polyps a high level of Hydra IQGAP1 mRNA is detected at the basis of tentacles. Consistent with a role in tentacle formation, IQGAP1 expression is activated during head regeneration and budding at a time when tentacles are emerging. The observations support the previous hypothesis that IQGAP proteins are involved in cytoskeletal as well as cell-cell contact rearrangements.