Detection and Genotyping of Leuconostoc spp. in a Sausage Processing Plant.

Some Leuconostoc spp. have the ability to produce slime and undesirable compounds in cooked sausage. The objectives of this research were to identify Leuconostoc sources in a Vienna-type sausage processing plant and to evaluate the genetic diversity of the isolated strains. Three hundred and two samples of sausage batter, sausages during processing, spoiled sausage, equipment surfaces, chilling brine, workers' gloves and aprons, and used casings were collected (March to November 2008 and February to April 2010) from a sausage processing plant. Lactic acid bacteria (LAB) were quantified, and Leuconostoc were detected using PCR. Strains were isolated and identified in Leuconostoc-positive samples. Leuconostoc strains were genotyped using randomly amplified polymorphic DNA and pulsed-field gel electrophoresis. LAB content of nonspoiled and spoiled sausage ranged from <0.8 to 4.4 log CFU/g and from 4.9 to 8.3 log CFU/g, respectively. LAB levels on equipment surfaces ranged from <1.3 to 4.8 log CFU/100 cm(2). Leuconostoc was detected in 35% of the samples, and 88 Leuconostoc spp. strains were isolated and genotyped. The main Leuconostoc spp. isolated were L. mesenteroides (37 genotypes), L. fallax (29 genotypes), and L. lactis (6 genotypes). Some strains of Leuconostoc isolated from equipment surfaces and sausages showed the same genotype. One L. lactis genotype included strains isolated from spoiled sausages analyzed in April 2008 and March to April 2010. Equipment and conveyor belts constitute Leuconostoc contamination sources. Leuconostoc persistence in the sausage processing environment and in the final product suggests the existence of microbial reservoirs, possibly on equipment surfaces.

Dietary supplementation of lutein reduces colon carcinogenesis in DMH-treated rats by modulating K-ras, PKB, and ß-catenin proteins.

In colon cancer, disturbances have been detected in genes coding for proteins involved in cellular proliferation, such as K-ras, ß-catenin, extracellular signal-regulated kinases (ERKs), and the protein kinase B (PKB). Although carotenoids such as lutein have an important role to prevent and treat some types of cancer, there are very few studies about the effect of lutein against colon cancer and its activity at the molecular level. Therefore, the aim of this study was to evaluate the chemoprotective activity of lutein against colon cancer induced by dimethylhydrazine (DMH). The results showed a significant increase in protein expression for K-ras and ß-catenin in tumors of DMH-treated rats. Simultaneously, we detected changes in the phosphorylation state of ERK1/2 and PKB in DMH-treated animals. Lutein given in the diet (0.002%), before (prevention) and after (treatment) DMH administration, diminished the number of tumors by 55% and 32%, respectively. Moreover, lutein significantly decreased in tumors the expression of K-ras (25%) and ß-catenin (28%) and the amount of pPKB (32%), during the prevention, and 39%, 26%, and 26% during the treatment stage, respectively. This study demonstrates the chemoprotective effect of lutein against colon cancer by modulating the proliferative activity of K-ras, PKB, and ß-catenin proteins.

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.

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.

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.

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.

Effect of cyclooxygenase-2 inhibition on renal function after renal ablation.

Kidney failure is the common end of hypertension and renal diseases. Several authors have suggested that vasodilatory prostaglandins participate in the hemodynamic mechanism responsible for the development of kidney failure. However, the mechanism by which prostaglandins are increased in renal disease is not clear. Recently, 2 isoforms of the enzyme responsible for prostaglandin synthesis, cyclooxygenase, have been described as cyclooxygenase-1 (COX-1), a constitutive isoform, and cyclooxygenase-2 (COX-2), an inducible isoform. In the present study, we investigated whether COX-2-dependent prostaglandins participate in the evolution of renal functional changes after renal ablation. We inhibited prostaglandin synthesis by COX-1 and COX-2 with indomethacin (3 mg/kg) and prostaglandin synthesis by COX-2 with NS-398 (3 mg/kg) and tested the effect of these inhibitors on the renal functional changes elicited by renal ablation. Renal ablation produced an increase in urinary volume, protein, and prostaglandin E(2), whereas urinary sodium and potassium were not affected and urinary osmolarity decreased; treatment with indomethacin or NS-398 partially prevented the renal functional changes elicited by renal ablation. Immunoblots for COX showed an increase in the expression of COX-2 protein 2 days after renal ablation. Furthermore, COX-2 mRNA expression was increased 1 day after renal ablation. These data suggest that COX-2-dependent prostaglandins participate in the renal mechanisms associated with the development of renal functional changes after renal ablation.

Entamoeba histolytica contains a gene encoding a homologue to the 54 kDa subunit of the signal recognition particle.

We have determined the nucleotide sequence and predicted amino acid sequence of the 54 kDa subunit of the signal recognition particle (SRP54) from the amitochondrial protist Entamoeba histolytica. The SRP54 gene was isolated from a genomic library using a polymerase chain reaction (PCR) probe. Nucleotide sequence analysis of a 2.3 kb fragment, derived from a 7 kb genomic clone, revealed an open reading frame encoding a protein of 487 amino acids (MW 53.8 kDa). The identities of the predicted amino acid sequence with its homologues from other species were between 24 and 47%. Functional domains previously defined for the SRP54-type proteins were present in the entamoebal sequence, such as the amino-terminal GTP binding domain (G domain) and the carboxy-terminal methionine rich domain (M domain). SRP54 mRNA contains an extra G residue at the 5' end, suggesting that capping of poly-A(+) transcripts is present in E. histolytica. Evolutionary analysis of the SRP54 based on phylogenetic inference placed the E. histolytica sequence as an early divergence of the eukaryotic tree. Although the function of the entamoebal homologue remains to be elucidated, the identification of the SRP54 gene constitutes the first evidence for SRP related proteins in protozoans.

Effect of bacterial association on the phenotype and genotype of an Entamoeba histolytica clonal population.

A several-times-cloned population of Entamoeba histolytica trophozoites (clone MAVIII) was cultured under axenic (MAVIIIax), monoxenic (MAVIIImx) and polyxenic (MAVIIIpx) conditions. Clones MAVIIIax and MAVIIImx presented similar virulence in vitro, but differed in their virulence in vivo, whereas MAVIIIpx trophozoites were neither virulent in vitro or in vivo. The MAVIII clones maintained their zymodeme and exhibited three unusual glucose phosphate isomerase bands, absent in other E. histolytica strains studied. Similar patterns were shown by the three MAVIII clones in the signature of a 482-bp DNA fragment from the M17 gene (which encodes for a variable immunodominant antigen), obtained by low stringency single specific primer PCR technique. However, MAVIII clones displayed genotypic variability in the patterns obtained by the random amplified polymorphic DNA technique using total DNA as template. Results suggest that monomorphism is kept in certain regions of the genome, mainly in those carrying protein encoding genes, but a high polymorphism is present in total DNA of cloned trophozoites cultured under different conditions, confirming the plasticity of the E. histolytica genome.

New Mutants of Phycomyces blakesleeanus for (beta)-Carotene Production.

The accumulation of (beta)-carotene by the zygomycete Phycomyces blakesleeanus is increased by mutations in the carS gene. The treatment of spores of carS mutants with N-methyl-N(prm1)-nitro-N-nitrosoguanidine led to the isolation, at very low frequencies, of mutants that produced higher levels of (beta)-carotene. Strain S556 produced about 9 mg of (beta)-carotene per g of dry mass when it was grown on minimal agar. Crosses involving strain S556 separated the original carS mutation from a new, unlinked mutation, carF. The carF segregants produced approximately as much carotene as did carS mutants, but they were unique in their ability to produce zygospores on mating and in their response to agents that increase carotenogenesis in the wild type. The carotene contents of carF segregants and carF carS double mutants were increased by sexual interaction and by dimethyl phthalate but were not increased by light or retinol. Mixed opposite-sex cultures of carF carS mutants contained up to 33 mg of (beta)-carotene per g of dry mass. Another strain, S444, produced more (beta)-carotene than did S556 but was marred by slow growth, defective morphology, and bizarre genetic behavior. In all the strains tested, the carotene concentration was minimal during the early growth phase and became higher and constant for several days in older mycelia.

Identification and analysis of the u6 small nuclear RNA gene from Entamoeba histolytica.

Among the small nuclear RNAs (snRNAs) involved in the spliceosomal processing of pre-mRNA, U6 is the most conserved. As a first evidence for the presence of the splicing machinery in the amitochondrial protozoan Entamoeba histolytica (Eh), we have cloned the u6 snRNA gene. We find that in this organism u6 is a single copy gene that is transcribed as a poly(A)- RNA molecule of approximately 105 nucleotides. We have mapped the 5' end of the U6 snRNA transcript, and identified typical elements of a putative polymerase III promoter. This is the first snRNA gene reported in Eh. Sequence analysis indicates that this gene contains all the conserved nucleotides known to be important for U6 snRNA function. These results, in conjunction with the earlier finding of genes that contain pre-mRNA introns, suggest that Eh has a functional spliceosomal complex.

Cloning of a ras-related gene from Hydra which responds to head-specific signals.

Members of the Ras family of proteins are important components of signal transduction pathways responding to external signals and leading to changes in cell behavior. Analysis of two ras-related genes in the phylogenetically old metazoan Hydra indicates that in normal animals both genes are expressed in all body regions of the polyp. Upon head removal, however, the transcript level of one of the two genes, ras2, decreases rapidly in the upper gastric region which is adjacent to the former head. The decrease is transient and specific for ras2, since no changes could be observed in the transcript level of the related ras1 gene or any other gene. The disappearance of the ras2 mRNA can be prevented completely by brief exposure of decapitated polyps to the protein kinase C activator TPA, which previously was shown to be capable of converting gastric tissue into head tissue [Müller, W.A. In: Othmer, H.G. (Ed.) Experimental and Theoretical Advances in Biological Pattern Formation. Plenum Press, New York, NY, 1993, pp. 237-253]. The finding that Hydra ras2 expression is strongly dependent on a signal from the head provides the first evidence for ras expression being regulated in pattern formation.

Ks1, an epithelial cell-specific gene, responds to early signals of head formation in Hydra.

As a molecular marker for head specification in Hydra, we have cloned an epithelial cell-specific gene which responds to early signals of head formation. The gene, designated ks1, encodes a 217-amino acid protein lacking significant sequence similarity to any known protein. KS1 contains a N-terminal signal sequence and is rich in charged residues which are clustered in several domains. ks1 is expressed in tentacle-specific epithelial cells (battery cells) as well as in a small fraction of ectodermal epithelial cells in the gastric region subjacent to the tentacles. Treatment with the protein kinase C activator 12-O-tetradecanoylphorbol-13-acetate (TPA) causes a rapid increase in the level of ks1 mRNA in head-specific epithelial cells and also induces ectopic ks1 expression in cells of the gastric region. Sequence elements in the 5'-flanking region of ks1 that are related to TPA-responsive elements may mediate the TPA inducibility of ks1 expression. The pattern of expression of ks1 suggests that a ligand-activated diacyglycerol second messenger system is involved in head-specific differentiation.

Hydra tropomyosin TROP1 is expressed in head-specific epithelial cells and is a major component of the cytoskeletal structure that anchors nematocytes.

A cDNA clone encoding a 253 amino acid tropomyosin was isolated from Hydra in a differential screen for head-specific genes. The Hydra tropomyosin gene, designated trop1, is a single copy gene, lacks introns and is strongly expressed in tentacle-specific epithelial cells. Analysis of protein synthesis in head and gastric tissue indicated a high rate of tropomyosin synthesis in head tissue. Immunolocalization of tropomyosin in tentacle tissue revealed a cushion-like tropomyosin-containing structure within battery cells at the base of nematocytes. The structure appears to form part of the cytoskeletal anchor for nematocytes. Tropomyosin cushions were also observed in epithelial cells along the body column, which contain mounted stenotele nematocytes.