Real-time PCR-based mismatch amplification mutation assay for specific detection of CS6-expressing allelic variants of enterotoxigenic Escherichia coli and its application in assessing diarrheal cases and asymptomatic controls.

Enterotoxigenic Escherichia coli (ETEC) expressing the colonization factor CS6 is widespread in many developing countries, including India. The different allelic variants of CS6, caused by point mutations in its structural genes, cssA and cssB, are designated AIBI, AIIBII, AIIIBI, AIBII, and AIIIBII. A simple, reliable, and specific mismatch amplification mutation assay based on real-time quantitative PCR (MAMA-qPCR) was developed for the first time for the detection of CS6-expressing ETEC, along with the identification of allelic variations. The assay was based on mismatched nucleotide incorporation at the penultimate base at the 3' ends of the reverse primers specific for cssA and cssB and was validated using 38 CS6-expressing ETEC isolates. This strategy was effective in detecting all the alleles containing single-nucleotide polymorphisms. Using MAMA-qPCR, we also tested CS6 allelic variants in 145 ETEC isolates from children with acute diarrhea and asymptomatic infections, with the latter serving as controls. We observed that the AIBI and AIIIBI allelic variants were mostly associated with cases rather than controls, whereas the AIIBII variants were detected mostly in controls. In addition, the AIBI and AIIIBI alleles were frequently associated with ETEC harboring the heat-stable toxin gene (est) alone or with the heat-labile toxin gene (elt), whereas the AIIBII allele was predominant in ETEC isolates harboring the elt gene. This study may help in understanding the association of allelic variants in CS6-expressing ETEC with the clinical features of diarrhea, as well as in ETEC vaccine studies.

Cholera toxin production by the El Tor variant of Vibrio cholerae O1 compared to prototype El Tor and classical biotypes.

Vibrio cholerae O1 El Tor variant strains produced much more cholera toxin than did prototype El Tor strains. The amount of cholera toxin produced by El Tor variant strains both in vitro and in vivo was more or less equivalent to that produced by classical strains.

Characterization and studies of the cellular interaction of native colonization factor CS6 purified from a clinical isolate of enterotoxigenic Escherichia coli.

CS6 is a widely expressed colonization factor of enterotoxigenic Escherichia coli (ETEC). To date, CS6 has not been well characterized in its native state. Here, we purified CS6 for the first time from an ETEC clinical isolate. Purified CS6 was composed of two structural subunits, CssA and CssB, which were present in equal amounts and tightly linked through noncovalent, detergent-stable association. The CssA subunit was poorly immunogenic, whereas CssB was highly immunogenic. Although the predicted molecular mass of CssA is 15 kDa, the purified CssA has an effective molecular mass of 18.5 kDa due to fatty acid modification. When purified CS6 was screened for its ability to bind with different extracellular matrix proteins, fibronectin (Fn) was found to interact with CS6 as well as CssA in a dose-dependent and saturable manner. This interaction was inhibited both by a synthetic peptide corresponding to the C-terminal hydrophilic, surface-exposed region of CssA (positions 112 to 126) and by the antibody derived against this region. Enzyme-linked immunosorbent assay results showed that CssA interacted with the 70-kDa N-terminal domain of Fn. The modifications on CssA probably do not play a role in Fn binding. Preincubation of INT 407 cells with CssA, but not CssB, inhibited ETEC binding to these cells. The results suggested that CS6-expressing ETEC binds to Fn of INT 407 cells through the C-terminal region of CssA. Purified CS6 was found to colocalize with Fn along the junctions of INT 407 cells. Based on the results obtained, we propose that CS6-expressing ETEC binds to the intestinal cells through Fn for colonization.

Genetic characteristics of Matlab variants of Vibrio cholerae O1 that are hybrids between classical and El Tor biotypes.

The Matlab variants of Vibrio cholerae O1, defined as hybrids between the classical and El Tor biotypes, were first isolated from hospitalized patients with acute secretory diarrhoea in Matlab, a rural area of Bangladesh. These variants could not be categorized as classical or El Tor biotypes by phenotypic and genotypic tests, and had representative traits of both the biotypes. A number of virulence-associated genes and/or gene clusters were screened by PCR and DNA sequencing. El Tor-specific gene clusters, Vibrio seventh-pandemic islands (VSP)-I and -II and repeat toxin (RTX) were present in the genome of these variants, indicating their El Tor lineage, whereas the nucleotide-sequence-derived CtxB amino acid sequence of these strains grouped them under the classical biotype. Matlab variants possessed all the necessary genes to initiate pandemics. The genetic relatedness of Matlab variants to the V. cholerae strains recently isolated in Mozambique is another important observation of this study, which underscores the epidemiological significance of Matlab variants.

CD146 is a novel marker for highly tumorigenic cells and a potential therapeutic target in malignant rhabdoid tumor.

Malignant rhabdoid tumor (MRT) is a rare, highly aggressive pediatric malignancy that primarily develops during infancy and early childhood. Despite the existing standard of intensive multimodal therapy, the prognosis of patients with MRT is dismal; therefore, a greater understanding of the biology of this disease is required to establish novel therapies. In this study, we identified a highly tumorigenic sub-population in MRT, based on the expression of CD146 (also known as melanoma cell adhesion molecule), a cell adhesion molecule expressed by neural crest cells and various derivatives. CD146+ cells isolated from four MRT cell lines by cell sorting exhibited enhanced self-renewal and invasive potential in vitro. In a xenograft model using immunodeficient NOD/Shi-scid IL-2Rγ-null mice, purified CD146+ cells obtained from MRT cell lines or a primary tumor exhibited the exclusive ability to form tumors in vivo. Blocking of CD146-related mechanisms, either by short hairpin RNA knockdown or treatment with a polyclonal antibody against CD146, effectively suppressed tumor growth of MRT cells both in vitro and in vivo via induction of apoptosis by inactivating Akt. Furthermore, CD146 positivity in immunohistological analysis of 11 MRT patient samples was associated with poor patient outcomes. These results suggest that CD146 defines a distinct sub-population in MRT with high tumorigenic capacity and that this marker represents a promising therapeutic target.

Retrotransposon-induced ectopic expression of cut causes the Om(1A) mutant in Drosophila ananassae.

Optic morphology (Om) mutations in Drosophila ananassae map to at least 22 loci scattered throughout the genome. They are semidominant, neomorphic, nonpleiotropic, and are associated with the insertion of a retrotransposon, tom. The Om(1A) gene, which is cytogenetically linked to the cut locus, was cloned using a DNA fragment of the cut locus of Drosophila melanogaster as a probe. Three of the eight alleles of Om(1A) examined have insertion of the tom element within a putative cut region. The gamma-ray-induced revertants of Om(1A) are accompanied with cut lethal mutations and rearrangements within the cut coding region. In the eye imaginal discs of the Om(1A) mutants, differentiation of photoreceptor clusters is suppressed, abnormal cell death occurs in the center and the cut protein is expressed ectopically. D. melanogaster flies transformed with a chimeric cut gene under the control of a heat-inducible promoter show excessive cell death in the region anterior to the morphogenetic furrow, suppressed differentiation to photoreceptor clusters and defect in the imaginal eye morphology when subjected to temperature elevation. These findings suggest that the tom element inserted within the Om(1A) region induces ectopic cut expression in the eye imaginal discs, thus resulting in the Om(1A) mutant phenotype.

Inhibition of in vitro growth of Shiga toxin-producing Escherichia coli O157:H7 by probiotic Lactobacillus strains due to production of lactic acid.

The inhibiting characteristics of lactic acid bacteria on Shiga toxin-producing Escherichia coli (STEC) O157:H7 (three strains, clinically isolated) was investigated by using a batch fermentation system. The species such as Lactobacillus casei strain Shirota or L. acidophilus YIT 0070 exert growth inhibitory and bactericidal activities on STEC. The pH value and undissociated lactic acid (U-LA) concentration of the culture medium of STEC cocultured with L. casei or L. acidophilus dramatically lowered or increased, respectively [corrected], when compared with those of the control culture. The cytotoxic properties of U-LA on STEC strain 89020087 analyzed in vitro was divided into two phases, i.e., the bacteriostatic phase (between 3.2 to 62 mM) and the bactericidal phase (over 62 mM). These data suggest that the bactericidal effect of Lactobacillus on STEC depends on its lactic acid production and pH reductive effect.

Allelic variation in colonization factor CS6 of enterotoxigenic Escherichia coli isolated from patients with acute diarrhoea and controls.

Colonization factor antigens (CFAs) are important virulence factors in enterotoxigenic Escherichia coli (ETEC). Using a multiplex PCR and RT-PCR, this study tested the presence of common colonization factor-encoding genes and their expression in 50 ETEC strains isolated from stool specimens. The samples were from patients (children) with acute diarrhoea (cases) admitted to the Infectious Disease Hospital (Kolkata, India) and from normal children (controls) under 5 years of age from the community. The results indicated that coli surface antigen 6 (CS6) was the most prevalent CFA (78 %) expressed by these ETEC strains. Sequence analysis of both of the CS6 structural genes, i.e. cssA and cssB, in different ETEC isolates revealed the presence of point mutations in a systematic fashion. Based on the analysis of these variations, it was found that CssA had three alleles and CssB had two. Based on the allelic variations, subtyping of CS6 into AIBI, AIIBII, AIIIBI, AIBII and AIIIBII is proposed. The point mutations in the different alleles were reflected in a partial alteration in the secondary structure of both subunits, as determined by computational analysis. The functional significance of these changes was confirmed with cellular binding studies in Caco-2 cells with representative ETEC isolates. CS6 with AI or AIII allelic subtypes showed a higher binding capacity than AII, whereas BI showed stronger binding than BII. The AII and BII alleles were mostly detected in controls rather than in cases. The antibody specificity of BI and BII also varied due to alteration of the amino acids. Thus, CS6 variants are formed as a result of different allelic combinations of CssA and CssB, and these changes at the functional level might be important in the development of an effective ETEC vaccine.

Transcriptional activation of the Drosophila melanogaster glucose-6-phosphate dehydrogenase gene by insertion of defective P elements.

Tandem insertions of defective P elements (1.15 kb KP and 0.6 kb core P) accelerate the transcription rate of the glucose-6-phosphate dehydrogenase (G6PD) gene in Drosophila melanogaster. In this report, we have analyzed the activation mechanism of the G6PD promoter by in vitro transcription and gel retardation assays. Results showed that one cis-acting region in the core P and two such regions in the KP are associated with activation of the G6PD promoter, and that putative transcriptional regulatory protein(s) which specifically bind to each of the cis-acting regions are present in nuclear extracts of Canton S embryos. On the other hand, the P elements do not activate the normal actin 5C promoter, but activate the promoter when the 20 bp sequence around the G6PD transcription start site is placed in front of the promoter. It appears that the GC-rich region in this 20 bp sequence is required for the activation.

Purification, characterization, and localization of follipsin, a novel serine proteinase from the fluid of porcine ovarian follicles.

Follipsin, an enzyme that accumulates in the follicular fluid of porcine ovaries during follicular maturation, was purified to apparent homogeneity. The purified enzyme consists of two different polypeptide chains having M(r) = 45,000 and 32,000 each, associated covalently. The enzyme activity was strongly inhibited by diisopropyl fluorophosphate, benzamidine, leupeptin, and antipain, indicating that follipsin is a serine proteinase. Using synthetic peptide substrates containing 4-methylcoumaryl-7-amide, follipsin was shown to preferentially hydrolyze Arg-X bonds but not Lys-X bonds. The NH2-terminal amino acid sequences of the 45- and 32-kDa polypeptides were highly homologous with those of the heavy and light chains, respectively, of human plasma kallikrein and human factor XIa. Immunological analyses and substrate specificity studies, together with other existing evidence, indicated that follipsin is distinct from kallikrein and factor XIa, thus being a novel type of serine proteinase. Follipsin is immunohistochemically localized in follicular fluid as well as in stroma cells of porcine ovaries. The results strongly suggest that follipsin originates from interstitial cells of the ovarian stroma.

Cleavage specificity of porcine follipsin.

Follipsin purified from the follicular fluid of porcine ovaries was studied for its specificity against various synthetic and peptide substrates. The enzyme cleaved only by an endopeptidase activity at the amide and peptide bonds of Arg-X, indicating strict specificity of the S1 pocket for arginine. The specificity for pocket S2 appears to favor either hydrophobic or basic side chains. A 10-residue peptide containing a portion of the activation site of human tissue plasminogen activator was synthesized and tested with the enzyme. The peptide was cleaved by follipsin at the Arg-Ile bond, as expected from the specificity deduced above. Furthermore, the enzyme successfully converted single-chain precursor tissue plasminogen activator (sctPA) to its active, two-chain form by cleaving the corresponding peptide bond. Comparison of the rates of single-chain precursor tissue plasminogen activator activation and tissue plasminogen activator peptide hydrolysis revealed that the former is a more efficient substrate than the latter.

Localization of prolyl endopeptidase mRNA in small growing follicles of porcine ovary.

Prolyl endopeptidase (EC3.4.21.26) has been considered a unique intracellular enzyme catalyzing internal peptide bond hydrolysis of Pro-X. In this study, the distribution of prolyl endopeptidase activity and its mRNA was investigated in the follicles of porcine ovary. Both follicular fluid and granulosa cell fractions from small follicles showed higher activity than those from large follicles. Molecular cloning and Northern blot analysis suggested that only one species of prolyl endopeptidase gene was expressed in the ovary. In addition, in situ hybridization study revealed that the prolyl endopeptidase mRNA expression was more noticeable in the granulosa cell layers of small ovarian follicles than in those of large follicles, suggesting its importance in the early stage of follicular development.

Identification of porcine follipsin as plasma kallikrein, and its possible involvement in the production of bradykinin within the follicles of porcine ovaries.

To determine the identity of porcine follipsin, a plasma kallikrein cDNA clone was isolated from a porcine liver cDNA library. The clone encoded a protein of 643 amino acids, exhibiting identities 79.7, 72. 9, and 74.4% homologous to human, rat, and mouse plasma prekallikrein, respectively. The amino acid sequences of four internal peptides isolated from the tryptic digest of follipsin were all found in the deduced sequence. Authentic plasma kallikrein was purified from porcine plasma and compared directly with follipsin. Actions on synthetic substrates and behaviors with proteinase inhibitors were indistinguishable between these two enzymes. The cDNA was expressed in COS-7 cells and the recombinant protein was prepared from the culture medium of these cells. No active enzyme could be obtained, but the expressed protein was reacted with anti-porcine plasma kallikrein antibody. The mRNA was detected only in the liver in northern blot analysis. RT-PCR analysis of RNAs revealed that porcine testis, in addition to the liver, expressed the corresponding mRNA. In the ovary, plasma kallikrein was detected as a main band of the active form (Mr = 85,000) and the band of the minor inactive precursor form (Mr = 80,000), respectively. In contrast, the liver extract contained only the precursor form. Incubation of high molecular weight kininogen with follicular fluid plasma kallikrein resulted in an increased production of bradykinin. Further, the fresh fluid of large-sized follicles of porcine ovaries was found to contain this peptide hormone at a detectable level. These results indicate that porcine follipsin is plasma kallikrein, and that the enzyme may be involved in the production of bradykinin within ovarian follicles.

Protective effect of Lactobacillus casei strain Shirota on Shiga toxin-producing Escherichia coli O157:H7 infection in infant rabbits.

We examined colonization patterns of Shiga toxin-producing Escherichia coli (STEC), concentrations of Shiga toxins (Stxs) and specific immunoglobulin A (lgA) against Stxs and STEC bacterial cell surface antigen in various portions of the gastrointestinal tract in an infant rabbit infection model. After inoculation of 3-day-old infant rabbits with STEC strain 89020087 at low doses (approximately 10(3) CFU/body), numbers of colonizing STEC bacteria and concentrations of Stxs in the intestine increased dramatically and the animals developed diarrhea within a couple of days after infection. Daily administration of Lactobacillus casei from the day of birth dramatically decreased the severity of diarrhea and lowered STEC colonization levels in the gastrointestinal tract 100-fold day 7 after infection. Both Stx1 and Stx2 concentrations in the intestines and histological damage to the intestinal mucus induced by STEC infection were decreased by the administration of L. casei. Examination of the concentrations of volatile fatty acids and pH of the intestinal contents revealed that the protective effect of L. casei administration against STEC infection was not due to fermented products such as lactic acid in the gastrointestinal tract. Administration of L. casei increased levels of lgAs against Stx1, Stx2, and formalin-killed STEC cells in the colon approximately two-, four-, and threefold, respectively, compared with those of the untreated controls by day 7 after infection. These results suggest that administration of L. casei strain Shirota enhances the local immune responses to STEC cells and Stxs and leads to elimination of STEC and thus decreases Stx concentrations in the intestines.

Clonal diversity among recently emerged strains of Vibrio parahaemolyticus O3:K6 associated with pandemic spread.

The genomes of the O3:K6 strains of Vibrio parahaemolyticus which abruptly emerged in Calcutta, India, in February 1996 and which demonstrated an unusual potential to spread and an enhanced propensity to cause infections were examined by different molecular techniques to determine clonality. No restriction fragment length polymorphism (RFLP) in the gene encoding the thermostable direct hemolysin was observed among the O3:K6 isolates of V. parahaemolyticus. Clonal diversity among the O3:K6 strains became evident by examining the RFLPs of the rrn operons and by the use of pulsed-field gel electrophoresis. Five ribotypes were distinguished among the O3:K6 strains examined, with ribotype R4 constituting the major type. Strains of O3:K6 isolated between June and August 1996 showed different pulsotypes compared to the pulsotypes of strains isolated before and after this period, indicating genetic reassortment among these strains, but those isolated between August 1996 and March 1998 showed identical or nearly similar pulsotypes. It is clear that there is a certain degree of genomic reassortment among the O3:K6 clones but that these strains are predominantly one clone.

Diverse CTX phages among toxigenic Vibrio cholerae O1 and O139 strains isolated between 1994 and 2002 in an area where cholera is endemic in Bangladesh.

PCR surveillance of the rstR genes of CTX phages in Vibrio cholerae O1 and O139 showed no relationship between the incidence of disease and changes in the rstR but showed variations in their presence in O1 and O139 strains and the occurrence of multiple types in a few strains.