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PubMed2003

Quorum sensing and expression of virulence in Escherichia coli O157:H7.

Enterohaemorrhagic Escherichia coli O157:H7 causes a characteristic histopathology in intestinal cells known as attaching and effacing lesion. The genes for the lesion are encoded by the Locus of Enterocyte Effacement (LEE) pathogenicity island, that encodes a type III secretion system, the intimin intestinal colonization factor, and the translocated intimin receptor protein that is translocated from the bacterium to the host epithelial cells. Expression of the operons encoded within LEE is complex, but recent studies have demonstrated that quorum sensing influences transcription from four of the LEE operon promoters. A transcriptional regulator (LuxR homologue), signal synthase (LuxI homologue), and autoinducer (acylhomoserine lactone) are indispensable for this system in most gram-negative bacteria. Vibrio harveyi, a gram-negative bioluminescent marine bacterium, regulates light production in response to two autoinducers (AI-1 and AI-2). AI-1 is a homoserine lactone produced by most gram-negative bacteria. The structure of AI-2 is not known, but many species of gram positive and gram-negative bacteria, including E. coli and more specifically O157:H7, have been shown to produce AI-2 depending on the function encoded by the luxS gene. The LuxS acts as an AI-2 synthase and the AI-2 is produced from S-adenosylmethionine in three enzymatic steps. The substrate for LuxS is S-ribosylhomocysteine, which is cleaved to form two products, one of which is homocysteine, and the other is AI-2. The biosynthetic pathways and the biochemical intermediates in AI-2 biosynthesis have been observed to be identical in several gram-negative bacteria, such as E. coli, Salmonella typhimurium, V. harveyi, Vibrio cholerae, and Enterococcus faecalis. Thus, unlike quorum sensing via the family of related homoserine autoinducers, AI-2 is a universal signal, which may be used by a variety of bacteria for communication among and between species and may be responsible for regulation of virulence genes in E. coli O157:H7.

Escherichia coli O157↗
PubMed2002

The effect of vaccination with a Salmonella enteritidis aroA mutant on early cellular responses in caecal lamina propria of newly-hatched chickens.

When newly hatched chicks are inoculated with a Salmonella strain, they induce a rapid onset of resistance to intestinal colonization by other Salmonella strains. The exact mechanism of this early colonization-inhibition is not known. To study host-related contributions to this phenomenon, the kinetics of immune cell infiltration in the caecal wall was analyzed during the first 10 days after vaccination of newly hatched chickens with a Salmonella enterica serovar Enteritidis aroA mutant, and infection 1 day later with a virulent S. enterica serovar Enteritidis strain. These data were correlated with bacterial colonization and clearance of the Salmonella Enteritidis challenge strain. Bacteriological data showed that vaccinated animals had a much lower number of challenge bacteria in their organs and caecal contents the first days post-challenge, relative to unvaccinated animals. Immunohistochemical analysis of the caecal lamina propria revealed that heterophils started infiltrating the caecal lamina propria from 12 h post-vaccination. Macrophages and T-lymphocytes started infiltrating from 20 h and B-lymphocytes from 24 h post-vaccination. These data imply that immune cells already colonized the caecal wall at the time of challenge in vaccinated animals. The presence and activity of these cells in the caecal wall shortly after administration of a Salmonella Enteritidis aroA mutant might contribute to the inhibition of colonization of a virulent Salmonella strain, subsequently administered.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗
PubMed1988

Safe, live Vibrio cholerae vaccines?

Mutants of Vibrio cholerae defective in intestinal colonization have been constructed. Characterization of these mutants has led to the identification of a gene cluster involved in the assembly of a pilus colonization factor called TCP. The tcp operon has been cloned and strains of V. cholerae have been constructed that overproduce this pilus and the B subunit of cholera toxin. Together these studies may contribute to the eventual construction of efficient live and killed, oral cholera vaccines.

Administration, Oral↗
PubMed1998

Short- and intermediate-term carcinogenicity testing--a review. Part 2: available experimental models.

Numerous experimental protocols for short- and intermediate-term carcinogenicity assays have been available for many years. This paper surveys various of these test systems in rodents, fish species, non-vertebrates and avian embryos in ovo. The mouse skin tumour assay and the rat liver foci assay were used to introduce the basic concepts of short- and intermediate-term carcinogenicity testing in the previous part of the review. The focus of this second part of the review is on rodent assays for carcinogenicity testing in the lung, kidney, urinary bladder, pancreas, stomach, oral cavity, small intestine, colon, and on the possibility to combine several target organs in multi-organ models. The potential use of various fish species, non-vertebrates and hatching eggs for carcinogenicity testing is outlined and the advantages and limitations are discussed. This review also presents the problem of validation of any carcinogenicity test system and proposes a strategy for contemporary safety assessment of chemicals with regard to the detection and evaluation of carcinogenicity.

Animals↗
PubMed1998

Formation of DNA adducts of the food-derived mutagen 2-amino-9H-pyrido-[2,3-b]indole (A(alpha)C) and bioassay of mammary gland carcinogenicity in Sprague-Dawley rats.

2-amino-9H-pyrido[2,3-b]indole (AalphaC) is a heterocyclic amine found at relatively high concentrations in barbecued or grilled meats. In the current study, the mammary gland carcinogenicity of AalphaC was examined in female Sprague-Dawley rats given 10 doses of AalphaC (75 mg/kg, orally, once per day starting at 43 days of age) and placed on a defined high-fat diet (23.5% corn oil), a strong promotional factor for rat mammary gland carcinogenesis. Within 1 year, one out of 20 rats dosed with AalphaC developed a tubulopapillary carcinoma, indicating that the bioassay was largely negative. As DNA adduct formation is considered to play a role in carcinogenesis, AalphaC-DNA adduct levels were measured in the mammary gland and other tissues by the 32P-postlabelling method. Under intensification conditions, one major adduct and up to three minor adducts were detected in isolated mammary gland epithelial cells and other tissues (liver, stomach, small intestine, colon and kidney) of AalphaC-treated rats; the adduct patterns were similar in all tissues examined. The major adduct, comprising 60-100% of total DNA adduct levels in tissues, was chromatographically identical to the principal adduct found in 3'-dGp-AalphaC (synthesized by reacting 3'-phospho-2'-deoxyguanosine (3'-dGp) with N-acetoxy-AalphaC). Of the tissues examined, the highest AalphaC-DNA adduct levels were found in the liver. In male rats given a single dose of AalphaC (75 mg/kg, orally, 3 hr prior to necropsy), no AalphaC-DNA adducts were detected in extrahepatic tissues. In female rats given a single dose or 12 daily doses of AalphaC, hepatic DNA adduct levels were at least 12-13-fold higher than those in any other tissue. Mean total AalphaC-DNA adduct levels in mammary gland epithelial cells and liver from female rats given multiple doses of AalphaC were 3.5 and 50.7 (RAL x 10(7)), respectively. Although factors in addition to DNA adduct formation are likely to play a role in mammary gland carcinogenesis, the results suggest that the weak mammary gland carcinogenicity of AalphaC may in part be associated with low AalphaC-DNA adduct levels in the mammary gland epithelium.

Adenocarcinoma↗
PubMed2003

Chemoprevention of lung carcinogenesis by cacao liquor proanthocyanidins in a male rat multi-organ carcinogenesis model.

The effects of cacao liquor proanthocyanidins (CLPr) on tumorigenesis were investigated using a multi-organ carcinogenesis model in male F344 rats receiving combined treatment with a single i.p. injection of diethylnitrosamine (100 mg/kg body wt), four i.p. injections of N-methylnitrosourea (20 mg/kg body wt), four s.c. injections of dimethylhydrazine (40 mg/kg body wt), along with 0.05% N-butyl-N-(4-hydroxybutyl)nitrosamine and then 0.1% 2,2'-dihydroxy-di-n-propylnitrosamine, both in the drinking water, for 2 weeks each, during the initial 4-week period (DMBDD treatment). Starting 1 week thereafter, rats were administered CLPr at a dose of 0.025% or 0.25% and the experiment was terminated at week 36. The final survival rate for the DMBDD+0.25% CLPr group was significantly greater than for the DMBDD alone group. In the lung, significant reduction in the incidence and multiplicity of carcinomas was also observed, and in the thyroid, quantitative values for adenomas also tended to decrease in a CLPr dose-dependent manner. No significant modification in the small intestine, colon or kidney was evident. These results indicate that CLPr exerts chemopreventive effects in the lung without any promoting influence in other major organs.

1,2-Dimethylhydrazine↗
PubMed1999

Guinea pig alpha 1-microglobulin/bikunin: cDNA sequencing, tissue expression and expression during acute phase.

cDNA encoding alpha 1-microglobulin/bikunin (AMBP) was amplified from guinea pig (Cavia porcellus) liver mRNA by reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends methods, cloned and sequenced. The deduced amino acid sequence was found to be homologous to the sequence of AMBP of other mammals (69-76% amino acid identity). It has two Kunitz-type trypsin inhibitor domains in the bikunin part as reactive sites, one in the N-terminal region and another in the C-terminal region. The N-terminal inhibitor domain sequence is well-conserved, but the P1 residue of the C-terminal inhibitor domain sequence was found to be Gln rather than Arg, a residue highly conserved in the AMBP of seven other mammals examined to date. By RT-PCR and nested PCR, AMBP mRNA was detected not only in liver tissue, previously known to be a site of its synthesis, but also in pancreas, stomach, small intestine, colon, lung, spleen, kidney, testis, skeletal muscle, and leukocytes, but not in brain or heart. We examined the AMBP mRNA levels in guinea pig liver by RT-PCR, comparing normal levels and those in a state of inflammation. The mRNA levels, however, did not significantly change.

Amino Acid Sequence↗
PubMed1994

Gastrointestinal amyloid deposits in old age. Report on 110 consecutive autopsical patients and 98 retrospective bioptic specimens.

The prevalence of gastrointestinal amyloid was determined in 110 consecutive autopsy patients aged 85 years and older. Paraffin sections from the esophagus, stomach, small intestine, colon and rectum were stained with Congo red and inspected in polarized light. Amyloid was found in 38 patients (36%). Four patients had generalized amyloidosis and the remaining 34 revealed more localized varieties of amyloid. Immunohistochemical classification with a panel of antisera directed against five major amyloid fibril proteins displayed at least four different types of amyloid. Twenty cases exhibited amyloid of transthyretin origin, five cases were of lambda light chain origin and one case was of amyloid A-type. Thus far, unclassifiable amyloid deposits were found in 18 cases, 16 of which were strictly localized in subserosal veins. This consecutive autopsy series was compared with 98 randomly-selected bioptical gastrointestinal tissues with amyloid according to distribution pattern, sex, frequency of the various types of amyloid and associated diseases.

Aged↗
PubMed2004

Role of exopolysaccharide, the rugose phenotype and VpsR in the pathogenesis of epidemic Vibrio cholerae.

Vibrio cholerae, the causative agent of cholera can produce an exopolysaccharide (EPS). Some strains can also phenotypically switch from a smooth to a 'rugose' phenotype characterized by small wrinkled colonies, overproduction of EPS, increased biofilm formation in vitro and increased resistance to various stressful conditions. High frequency switching to the rugose phenotype is more common in epidemic strains than in non-pathogenic strains, suggesting EPS production and the rugose phenotype are important in cholera epidemiology. VpsR up-regulates Vibrio polysaccharide (VPS) genes and the synthesis of extracellular EPS (VPS). However, the function of VPS, the rugose phenotype and VpsR in pathogenesis is not well understood. We report that rugose strains of both classical and El Tor biotypes of epidemic V. cholerae are defective in the in vitro production of extracellular collagenase activity. In vivo studies in rabbit ileal loops suggest that VpsR mutants are attenuated in reactogenicity. Intestinal colonization studies in infant mice suggest that VPS production, the rugose phenotype and VpsR have a role in pathogenesis. Our results indicate that regulated VPS production is important for promoting in vivo biofilm formation and pathogenesis. Additionally, VpsR might regulate genes with roles in virulence. Rugose strains appear to be a subpopulation of cells that might act as a 'helper' phenotype promoting the pathogenesis of certain strains. Our studies provide new insight into the potential role of VPS, the rugose phenotype and VpsR in the pathogenesis of epidemic V. cholerae.

Animals↗
PubMed2003

A novel myosin heavy chain gene in human chromosome 19q13.3.

A human myosin heavy chain gene was identified in chromosome 19q13 by computational sequence analysis, RT-PCR and DNA sequencing of the cDNA. The complete cDNA has a length of 6786 bp and comprises 41 exons (40 coding) included in 108 kb of genomic sequence. Alternative splicing variants were also identified. The gene is expressed in a multitude of tissues, but mainly in small intestine, colon and skeletal muscle. The putative protein (228 kDa) carries the common myosin domains and presents high homology with the non-muscle myosin heavy chains (MYH9 and MYH10) as well as the smooth muscle myosin heavy chain MYH11. Nevertheless, phylogenetic analysis indicated that these homologous proteins are more related among themselves than to MYH14, suggesting that possibly this myosin heavy chain should be classified in a new myosin-subfamily.

Blotting, Northern↗
PubMed1998

Molecular cloning and characterization of FHL2, a novel LIM domain protein preferentially expressed in human heart.

A full-length cDNA clone encoding a novel LIM-only protein was isolated and sequenced from a human fetal heart cDNA library. This full-length clone consists of 1416 base pairs and has a predicted open reading frame (ORF) encoding 279 amino acids. The ORF of this polypeptide codes for the human heart-specific four and a half LIM-only protein 2 (FHL2). It possesses an extra zinc finger that is a half LIM domain and four repeats of LIM domain. When the human FHL2 cDNA probe was used to hybridize with poly-A RNA of various human tissues, a very strong signal could be seen in heart tissues, and only moderately low signals could be detected in placenta, skeletal muscle and ovary. Virtually no signal could be detected in brain, lung, liver, kidney, pancreas, spleen, thymus, prostate, testis, small intestine, colon or peripheral blood leukocyte. FHL2 was mapped to chromosome 2q12-q13 by fluorescent in-situ hybridization (FISH).

Amino Acid Sequence↗
PubMed1998

In-frame elimination of exon 10 in Cftrtm1Unc CF mice.

Cystic fibrosis (CF) is a severe autosomal-linked inherited disease in humans. Transgenic CF animals play a crucial role in the study of molecular mechanisms underlying disease pathology. In the present study, CFTR mRNA expression was examined in different tissues from one CF mouse model that contains a disruption in exon 10 sequence of the CF transmembrane conductance regulator (CFTR) gene. Multiple tissue samples were collected from new-born normal (+/+), homozygous (-/-), and heterozygous (+/-) mice and compared for their CFTR mRNA expression. Total RNA samples were prepared from eight different tissues (nasal mucosa, trachea, lung, colon, intestine, pancreas, liver, gonads, and brain) and then analyzed by reverse transcription polymerase chain reaction (RT-PCR) amplification. A 329-bp fragment comprising exon 9 through exon 11 of the CFTR gene was amplified from all tissues of the normal mouse. In contrast, a 137-bp fragment was observed in tissue samples from homozygous CF mice. Both the 329-bp and 137-bp fragments were detected in samples from heterozygous mice. Direct sequencing analysis of the amplified fragments showed an exon 9-exon 11 splice junction, indicating that the entire exon 10 sequence was eliminated from homozygous CF mice. RT-PCR analysis of the 3' end of CFTR mRNA showed the presence of a 682-bp, exon 20-24 fragment in -/- and +/- mice. These results demonstrate that an alternately spliced CFTR mRNA is produced in this CF 'knock-out' mouse. A semi-quantitative comparison of the wild-type and exon 10 minus CFTR (CFTR-E10) mRNA in heterozygote animals indicated that less (but a detectable amount) mutant CFTR mRNA was present in all organs tested. There was, however, a significant reduction of CFTR-E10 mRNA in the liver and the pancreas. Since the deletion of exon 10 is in-frame, the significance of the CFTR-E10 mRNA in terms of CFTR protein and function requires further analysis.

Animals↗
PubMed1998

Chromosomal mapping, tissue distribution and cDNA sequence of four-and-a-half LIM domain protein 1 (FHL1).

We have isolated and sequenced a human heart cDNA clone encoding a novel LIM-only protein. This full-length cDNA clone has a predicted open reading frame (ORF) encoding 280 amino acids. The ORF of this cDNA codes for a LIM-only protein that possesses four repeats of LIM domain and an extra zinc finger and this putative protein is named four-and-a-half LIM domain protein 1 (FHL1). FHL1 is unique when compared with other LIM-only proteins because it possesses an odd number of zinc fingers. When the FHL1 cDNA probe was used to hybridize with poly-(A) RNA of various human tissues, a very strong signal was detected in skeletal muscle, a moderate one in the heart; only weak signals were associated with the placenta, ovary, prostate, testis, small intestine, colon and spleen, and virtually no signal could be detected in brain, lung, liver, kidney, pancreas, thymus and peripheral blood leukocytes. The FHL1 gene was located to human chromosome at Xq27.2 by somatic cell hybrid mapping, fluorescent in situ hybridization (FISH) and radiation hybrid mapping.

Amino Acid Sequence↗
PubMed2002

Localization and expression of usherin: a novel basement membrane protein defective in people with Usher's syndrome type IIa.

People with Usher's syndrome type IIa have mutations in a novel gene encoding a protein with domains commonly found in many types of extracellular matrix and cell surface receptor proteins. Here we report that this protein, which we refer to as usherin, is a new basement membrane protein. In the mouse, usherin has a broad, but not ubiquitous, tissue distribution. Usherin is found in all of the capillary and structural basement membranes of the human and mouse retina and in the murine inner ear at both post-natal day 0 and in the adult. High levels of usherin are also observed in tissues not affected in Usher's syndrome type IIa, including spleen, testis, oviduct, epididymis, submaxillary gland, and large and small intestines. Many organs are completely devoid of usherin, including the brain, skin, kidney, lung, liver, and skeletal muscle. Expression was observed in the smooth muscle of the small intestine, colon, and oviduct, however, usherin is not present in cardiac smooth muscle. Usherin is critical for normal development and tissue homeostasis in the inner ear and retina, illustrating yet another example of the importance of basement membranes in the development and function of tissues.

Animals↗
PubMed2000

Addition of tyrosyl-arginine to parenteral nutrition is anabolic in unstressed rats.

To determine whether the addition of tyrosine and arginine (Arg), as tyrosyl-arginine (TyrArg), to parenteral nutrition (PN) can promote anabolism, rats were assigned to: 1) PN (1.20 MJ. kg body weight [BW](-1) x d(-1) and 1.22 gN x kgBW(-1) x d(-1); PN control group, n = 5), 2) PN plus TyrArg (2.6 mmol. kgBW(-1) x d(-1); TyrArg group, n = 6), or 3) PN plus Arg (2.6 mmol. kgBW(-1). d(-1); Arg group, n = 5). Results from these three groups were compared with an unoperated chow-fed reference group (chow control group, n = 5). The BW gain during PN and the proportion of lipid in the total body after 14 d of PN was greater for the TyrArg group than for the PN group (P<0.01). Although the differences in weight gain, body water, lipid, and protein between the TyrArg and Arg groups were not significant, the mean weight gain throughout PN was greater in the TyrArg group than in the Arg group. The proportion of protein in the small intestine, colon, and gastrocnemius muscle was greater in the TyrArg and Arg groups than in the PN group (P<0.01). A distinct requirement for tyrosine has not been demonstrated in this model, and additional studies in stressed animals are required. In contrast, arginine had tissue-specific anabolic activity.

Animals↗
PubMedDate not supplied

Immunonutrition: role of biosurfactants, fiber, and probiotic bacteria.

Phospholipids constitute an important part of cellular membranes, and membrane fluidity and permeability are dependent on the fatty acid composition of the phospholipid. The composition, which changes with aging and disease is, to a large degree, influenced by nutrient supply. Phospholipids have been effective in protecting cellular membranes such as those of the gastrointestinal tract to an extent not much different from that observed with external supply of established mucosa-protective drugs such as misoprostol and sucralfate. Polar lipids have also been shown to be effective in preventing microbial translocation. The effect is further potentiated by an external supply of probiotic fibers such as pectin, guar gum, and oat gum. These and many other fibers also have documented strong mucosa preventive effects. Prebiotic bacteria such as Lactobacillus plantarum have demonstrated a strong ability to preserve food and prevent spoilage. In addition, L. plantarum seems to not only preserve key nutrients such as omega-3 fatty acids, but also increases its content during storage conditions. L. plantarum alone or in combination with various fibers has demonstrated a strong ability to reduce and eliminate potentially pathogenic microorganisms both in vitro and in vivo. It has recently been shown that L. plantarum possesses the ability to adhere to and colonize intestinal mucosa. It seems unique among the lactobacilli for L. plantarum to use mannose-specific adhesins, uncommon among gram-positive, but common among gram-negative bacteria, which makes it possible that L. plantarum competes with gram-negative other potential pathogens for receptor sites at the mucosal cell surfaces. Additionally, L. plantarum seems to be effective in eliminating nitrate and producing nitric oxide. These functions of L. plantarum are among the reasons why it has been used in combination with various fibers and polar lipids to recondition the gastrointestinal mucosa. For the purpose of a L. plantarum-containing formula being produced and tried, a treatment policy is regarded as an extension of the immunonutrition program and called ecoimmunonutrition.

Bacteria↗
PubMed2002

[Human milk: a perpetual (re)appreciation].

Based upon recent studies, it appears more and more evident that exclusive breast-feeding can be said as the best functional food, the last being defined as: "a food that has components or ingredients bringing a specific medical or physiological benefit to the host, other than purely nutritional effects". In particular, human breast milk does favour optimal bacterial intestinal colonization with bifidobacteria and lactic acid bacteria in the neonates and contains a large number of components with antimicrobial activity.

Adjuvants, Immunologic↗
PubMed1999

Enteropathogenic bacteria in faecal swabs of young children fed on lactic acid-fermented cereal gruels.

The influence of consumption of a lactic acid-fermented cereal gruel togwa with pH < or = 4 on the presence of faecal enteric bacteria such as campylobacter, enterohaemorrhagic Escherichia coli (EHEC:O157), enterotoxigenic Escherichia coli (ETEC), salmonella and shigella was evaluated. Under 5 years old healthy children listed in an ascending order of age were alternatively assigned and given either a lactic-acid fermented cereal gruel togwa (test diet) or an unfermented cereal gruel uji (control diet) once a day for 13 consecutive days. The presence of the enteropathogens was examined in rectal swabs collected from the children at baseline (before feeding session started), on days 7 and 13, and additionally 14 days (follow-up day) after the feeding session had stopped. The swabs were cultured on to different optimal media for respective enteropathogen and confirmed by standard microbiological and serological methods. Campylobacter spp. dominated among the enteropathogens (62% out of total) followed by Salmonella spp., ETEC and Shigella spp. Children with isolated enteropathogens in the togwa group was significantly reduced (P < 0.001) from 27.6% at baseline to 7.8, 8.2 and 12.7% on days 7, 13 and follow-up day, respectively. The effect was more pronounced in those children taking togwa > 6 times during the study period. In the control group, there was a slight decrease from 16.7% at baseline to 11.4% on day 7 and 8.1% on day 13. On the follow-up day, enteropathogens were found in 22.6% of the children, which was significantly higher than in those children taking togwa > 6 times. We conclude, that regular consumption of togwa with pH < or = 4, once a day, three times a week may help to control intestinal colonization with potential diarrhoea-causing pathogens in young children.

Beverages↗