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PubMed1994

Contraction uncoupling with butanedione monoxime versus low calcium or high potassium solutions on flow and contractile function of isolated hearts after prolonged hypothermic perfusion.

BACKGROUND: Normal ionic perfusate containing butanedione monoxime (BDM), a reversible myofilament inhibitor, could be better than either a high potassium (KCl) or a low calcium (CaCl2) perfusate for long-term cardiac preservation. This hypothesis was tested in 70 isolated guinea pig hearts. METHODS AND RESULTS: Three groups--time control (8 hours, 37 degrees C), cold control (22 hours, 3.8 degrees C), and cold+BDM (22 hours)--were perfused with typical Krebs-Ringer solution (2.5 mmol/L CaCl2 and 4.5 mmol/L KCl). Two other groups were cold perfused for 22 hours either with 2.5 mmol/L CaCl2 + 20 mmol/L KCl (high) or with 0.5 mmol/L CaCl2 (low) + 4.5 mmol/L KCl. These changes were maintained from 20 minutes before cold perfusion until 30 minutes after rewarming to 37 degrees C. Coronary vasodilator reserve was tested before cold perfusion and 2 hours after warm reperfusion with adenosine (Ade), acetylcholine (Ach, endothelium dependent), and nitroprusside (NP, endothelium independent). Each treatment decreased left ventricular pressure (LVP) by more than 80% before cold perfusion. During warm reperfusion, LVP was lower in cold control (-72 +/- 5%), high KCl (-76 +/- 4%), and low CaCl2 (-80 +/- 4%) groups than in BDM (-38 +/- 3%) or time control (-18 +/- 4%) groups; coronary flow (CF) was lower in high KCl (-67 +/- 4%) and low CaCl2 (-54 +/- 7%) groups than in cold control (-37 +/- 6%), BDM (-30 +/- 5%), or time control (+2 +/- 3%) groups; and percent oxygen extraction (controls, 62 +/- 4%) was higher in the high KCl group (83 +/- 6%) than in cold control (72 +/- 3%), BDM (73 +/- 3%), low CaCl2 (72 +/- 5%), or time control (63 +/- 3%) groups. CF responses to Ade, Ach, and NP (+103 +/- 7%, +24 +/- 5%, and +34 +/- 5% before cold) were attenuated (+76 +/- 6%, +18 +/- 5%, and +23 +/- 4%) in the time control group (5 hours later), were reduced but present in the BDM group (+10 +/- 5%, -5 +/- 5%, and -5 +/- 5%), and were absent in both low CaCl2 and high KCl groups after 2 hours of reperfusion. CONCLUSIONS: Normal ionic BDM solution better preserves cardiac function and basal CF after prolonged cold perfusion than do cold control, high KCl, and low CaCl2 solutions. Vasodilatory capacity is markedly diminished after perfusion with either the high KCl or the low CaCl2 solution.

Animals↗

PubMed2001

Attempts to improve treatment outcomes in acute myeloid leukemia (AML) in older patients: the results of the United Kingdom Medical Research Council AML11 trial.

In an attempt to improve induction chemotherapy for older patients with acute myeloid leukemia (AML),1314 patients were randomized to 1 of 3 induction treatments for 2 courses of DAT (daunorubicin, cytarabine, and thioguanine) 3 + 10, ADE (daunorubicin, cytarabine, and etoposide) 10 + 3 + 5, or MAC (mitoxantrone-cytarabine). The remission rate in the DAT arm was significantly better than ADE (62% vs 50%; P =.002) or MAC (62% vs 55%; P =.04). This benefit was seen in patients younger and older than 70 years. There were no differences between the induction schedules with respect to overall survival at 5 years (12% vs 8% vs 10%). A total of 226 patients were randomized to receive granulocyte colony-stimulating factor (G-CSF) or placebo as supportive care from day 8 after the end of treatment course 1. The remission rate or survival were not improved by G-CSF, although the median number of days to recover neutrophils to 1.0 x 10(9)/L was reduced by 5 days. Patients who entered remission (n = 371) were randomized to stop after a third course (DAT 2 + 7) or after 6 courses, ie, a subsequent COAP (cyclophosphamide, vincristine, cytarabine, and prednisolone), DAT 2 + 5, and COAP. The relapse risk (81% vs 73%), disease-free survival (16% vs 23%), and overall survival at 5 years (23% vs 22%) did not differ between the 3-course or 6-course arms. In addition to a treatment duration randomization, 362 patients were randomized to receive 12-month maintenance treatment with low-dose interferon, but no benefit was seen with respect to relapse risk, disease-free survival, or overall survival.

Age Factors↗

PubMedDate not supplied

Improving patient safety by identifying side effects from introducing bar coding in medication administration.

OBJECTIVE: In addition to providing new capabilities, the introduction of technology in complex, sociotechnical systems, such as health care and aviation, can have unanticipated side effects on technical, social, and organizational dimensions. To identify potential accidents in the making, the authors looked for side effects from a natural experiment, the implementation of bar code medication administration (BCMA), a technology designed to reduce adverse drug events (ADEs). DESIGN: Cross-sectional observational study of medication passes before (21 hours of observation of 7 nurses at 1 hospital) and after (60 hours of observation of 26 nurses at 3 hospitals) BCMA implementation. MEASUREMENTS: Detailed, handwritten field notes of targeted ethnographic observations of in situ nurse-BCMA interactions were iteratively analyzed using process tracing and five conceptual frameworks. RESULTS: Ethnographic observations distilled into 67 nurse-BCMA interactions were classified into 12 categories. We identified five negative side effects after BCMA implementation: (1) nurses confused by automated removal of medications by BCMA, (2) degraded coordination between nurses and physicians, (3) nurses dropping activities to reduce workload during busy periods, (4) increased prioritization of monitored activities during goal conflicts, and (5) decreased ability to deviate from routine sequences. CONCLUSION: These side effects might create new paths to ADEs. We recommend design revisions, modification of organizational policies, and "best practices" training that could potentially minimize or eliminate these side effects before they contribute to adverse outcomes.

Clinical Pharmacy Information Systems↗

PubMed2003

The zinc-finger protein CNBP is required for forebrain formation in the mouse.

Mouse mutants have allowed us to gain significant insight into axis development. However, much remains to be learned about the cellular and molecular basis of early forebrain patterning. We describe a lethal mutation mouse strain generated using promoter-trap mutagenesis. The mutants exhibit severe forebrain truncation in homozygous mouse embryos and various craniofacial defects in heterozygotes. We show that the defects are caused by disruption of the gene encoding cellular nucleic acid binding protein (CNBP); Cnbp transgenic mice were able to rescue fully the mutant phenotype. Cnbp is first expressed in the anterior visceral endoderm (AVE) and, subsequently, in the anterior definitive endoderm (ADE), anterior neuroectoderm (ANE), anterior mesendoderm (AME), headfolds and forebrain. In Cnbp(-/-) embryos, the visceral endoderm remains in the distal tip of the conceptus and the ADE fails to form, whereas the node and notochord form normally. A substantial reduction in cell proliferation was observed in the anterior regions of Cnbp(-/-) embryos at gastrulation and neural-fold stages. In these regions, Myc expression was absent, indicating CNBP targets Myc in rostral head formation. Our findings demonstrate that Cnbp is essential for the forebrain induction and specification.

Animals↗

PubMed2003

Anterior identity is established in chick epiblast by hypoblast and anterior definitive endoderm.

Previous studies of head induction in the chick have failed to demonstrate a clear role for the hypoblast and anterior definitive endoderm (ADE) in patterning the overlying ectoderm, whereas data from both mouse and rabbit suggest patterning roles for anterior visceral endoderm (AVE) and ADE. Based on similarity of gene expression patterns, fate and a dual role in 'protecting' the prospective forebrain from caudalising influences of the organiser, the chick hypoblast has been suggested to be the homologue of the mouse anterior visceral endoderm. In support of this, when transplanted to chick embryos, the rabbit AVE induces anterior markers in the chick epiblast. To reevaluate the role of the hypoblast/ADE (lower layer) in patterning the chick ectoderm, we used rostral blastoderm isolates (RBIs) as an assay, that is, rostral regions of blastoderms transected at levels rostral to the node. RBIs are, therefore, free from the influences of Hensen's node and ingressing axial mesoderm - tissues that are able to induce Ganf, the earliest specific marker of anterior neural plate. We demonstrate, using such RBIs (or RBIs dissected to remove the lower layer with or without tissue replacement), that the hypoblast/ADE (lower layer) is required and sufficient for patterning anterior positional identity in the overlying ectoderm, leading to expression of Ganf in neuroectoderm. Our results suggest that patterning of anterior positional identity and specification of neural identity are separable events operating to pattern the rostral end of the early chick embryo. Based on this new evidence we propose a revised model for establishing anteroposterior polarity, neural specification and head patterning in the early chick that is consonant with that occurring in other vertebrates.

Animals↗

PubMed1985

Direct alkylation of calf thymus DNA by acrylonitrile. Isolation of cyanoethyl adducts of guanine and thymine and carboxyethyl adducts of adenine and cytosine.

Reaction of the rodent carcinogen acrylonitrile (AN) at pH 7.0 for 10 and/or 40 days with 2'-deoxyadenosine (dAdo), 2'-deoxycytidine (dCyd), 2'-deoxyguanosine (dGuo), 2'-deoxyinosine (dIno), and thymidine (dThd) resulted in the formation of cyanoethyl and carboxyethyl adducts. The adducts isolated were 1-(2-carboxyethyl)-dAdo (1-CE-dAdo), N6-CE-dAdo, 3-CE-dCyd, 7-(2-cyanoethyl)-Gua (7-CNE-Gua), 7,9-bis-CNE-Gua, imidazole ring-opened 7,9-bis-CNE-Gua, 1-CNE-dIno, and 3-CNE-dThd. Structures were assigned on the basis of UV spectra and electron impact (EI), desorption chemical ionization (DCI) and Californium-252 fission fragment ionization mass spectra. The carboxyethyl adducts resulted from initial cyanoethylation at a ring nitrogen adjacent to an exocyclic nitrogen followed by rapid hydrolysis of the nitrile moiety to a carboxylic acid. It was postulated that the facile hydrolysis is the result of an intramolecular-catalyzed reaction resulting from the formation of a transient cyclic intermediate between nitrile carbon and exocyclic nitrogen. AN was reacted with calf thymus DNA (pH 7.0, 37 degrees C, 40 days) and the relative amounts of adducts isolated was 1-CE-Ade (25.8%), N6-CE-Ade (7.6%), 3-CE-Cyt (1.3%), 7-CNE-Gua (25.8%), 7,9-bis-CNE-Gua (4.3%), imidazole ring-opened 7,9-bis-CNE-Gua (18.9%) and 3-CNE-Thy (16.3%). Thus a carcinogen once adducted to a base in DNA was shown to be subsequently modified resulting in a mixed pattern of cyanoethylated and carboxyethylated AN-DNA adducts.(ABSTRACT TRUNCATED AT 250 WORDS)

Acrylonitrile↗

PubMed2004

The heritability of breast cancer: a Bayesian correlated frailty model applied to Swedish twins data.

The aim of this study was to investigate the role of genes and environment in susceptibility to breast cancer and to give an estimate of heritability in the propensity to develop the disease. To do this we applied an interdisciplinary approach, merging models developed in the field of demography and survival analysis - so-called frailty models - and models coming from quantitative genetics and epidemiology, namely genetic models. In our study, the inferential problem was solved in a Bayesian framework and the numerical work was carried out using MCMC methods. We used the special information coming from twin data, particularly breast cancer data, from the Swedish Twin Register. The application of a correlated log-normal frailty model leads to a very large estimate of the population heterogeneity (sigma = 6.7), and relatively small correlations between co-twins' frailties - around 0.3 for monozygotic and 0.1 for dizygotic twins. Comparing three different genetic models (an ACE, an AE and an ADE model), we furthermore concluded that genetic effects would explain globally almost 30% of the total variability of propensity to breast cancer. Environmental effects would be predominant in determining breast cancer susceptibility and these effects would be primarily individual-specific, that is, non-shared effects. Finally, a model which includes dominance genetic effects (ADE model) is preferred for genetic and statistical reasons.

Bayes Theorem↗

PubMed2000

Characterization of ligands for galectins, natural galactoside-binding immunoglobulin G subfractions and sarcolectin and also of the expression of calcyclin in thyroid lesions.

The purpose of this study was to characterize ligands for galectins, natural galactoside-binding immunoglobulin G subfractions and sarcolectin and also the expression of calcyclin in various benign and malignant thyroid lesions. The extent of the binding of eight glycochemical probes was quantitatively assessed using computer-assisted microscopy on 76 thyroid lesions including 10 not-otherwise-specified multinodular goiters (S_MNG), 11 multinodular goiters with adenomatous hyperplasia (AH_MNG), 8 normomacrovesicular (NM_ADE) and 12 microvesicular (MIC_ADE) adenomas, and 9 papillary (P_CAR), 10 follicular variants of papillary (FvarP_CAR), 7 follicular (F_CAR) and 9 anaplastic (A_CAR) carcinomas. The 8 histochemical probes included 5 animal lectins (including galectins and sarcolectin), 1 polyclonal antibody (raised against calcyclin) and 2 immunoglobulin G subfractions from human serum with selectivity to alpha- and beta-galactosyl residues. The results show that multinodular goiters with adenomatous hyperplasia exhibited histochemical characteristics intermediate to those of normal multinodular goiters and microvesicular adenomas. Normomacrovesicular adenomas behaved very distinctly from microvesicular ones. Microvesicular adenomas were more closely related to differentiated thyroid carcinomas than any other type of benign thyroid lesions of epithelial origin. Papillary and follicular carcinomas seemed to represent the two extremes of the same biological entity with the follicular variant of the papillary carcinoma serving as a biological link between these two extremes. Anaplastic carcinomas behaved in a significantly different manner when compared to the differentiated forms of thyroid carcinomas. The results suggest that the patterns of expression of the glycoconjugates investigated in the present study may constitute useful tools for characterizing lesions in the human thyroid.

Adenocarcinoma↗

PubMed1987

Felodipine versus Moduretic. A double-blind parallel-group multicentre study.

In a double-blind parallel-group study 133 patients with mild to moderate essential hypertension were randomised to felodipine 5mg twice daily or Moduretic mite every morning after a run-in placebo period of 1 to 2 weeks. All previous antihypertensive therapy was withdrawn at the start of the run-in period. After 4 weeks the dose of felodipine was increased to 10mg twice daily, and Moduretic mite was replaced by Moduretic in patients with a diastolic blood pressure of greater than 80mm Hg. On the low dose, the supine blood pressure on felodipine was reduced by 24/14mm Hg from 174/105mm Hg, and on Moduretic mite by 19/11mm Hg from 171/103mm Hg. After the increase in dosage blood pressure was lowered further. There was no statistically significant difference in blood pressure reduction between the 2 groups. Severe adverse drug experiences (ADEs) were few and equally distributed. However, more felodipine patients were withdrawn because of ADEs. Potassium levels decreased and uric acid levels increased significantly in the Moduretic group.

Adult↗

PubMed2006

Contrasting genome-wide distribution of 8-hydroxyguanine and acrolein-modified adenine during oxidative stress-induced renal carcinogenesis.

Oxidative stress is a persistent threat to the genome and is associated with major causes of human mortality, including cancer, atherosclerosis, and aging. Here we established a method to generate libraries of genomic DNA fragments containing oxidatively modified bases by using specific monoclonal antibodies to immunoprecipitate enzyme-digested genome DNA. We applied this technique to two different base modifications, 8-hydroxyguanine and 1,N6-propanoadenine (acrotein-Ade), in a ferric nitrilotriacetate-induced murine renal carcinogenesis model. Renal cortical genomic DNA derived from 10- to 12-week-old male C57BL/6 mice, of untreated control or 6 hours after intraperitoneal injection of 3 mg iron/kg ferric nitrilotriacetate, was enzyme digested, immunoprecipitated, cloned, and mapped to each chromosome. The results revealed that distribution of the two modified bases was not random but differed in terms of chromosomes, gene size, and expression, which could be partially explained by chromosomal territory. In the wild-type mice, low GC content areas were more likely to harbor the two modified bases. Knockout of OGG1, a repair enzyme for genomic 8-hydroxyguanine, increased the amounts of acrolein-Ade as determined by quantitative polymerase chain reaction analyses. This versatile technique would introduce a novel research area as a high-throughput screening method for critical genomic loci under oxidative stress.

Acrolein↗

PubMed1989

Norfloxacin versus trimethoprim-sulfamethoxazole. A study in patients with known bacteriuria undergoing transurethral resection of the prostate.

Three hundred and forty-three patients with suspected bacteriuria undergoing transurethral resection of the prostate (TUR-P) were randomized to treatment with either trimethoprim-sulfamethoxazole (TMP-SMX) or norfloxacin (NF) for 5 1/2 days beginning the evening prior to operation. It was possible to analyse 165 patients for efficacy. Elimination of bacteria on days 10 to 20 was achieved in 78.1% and 78.3% in the TMP-SMX and NF group, respectively. The accumulated elimination rates for the follow up period (days 10-42) were 68.5% for the TMP-SMX group and 76.2% for the NF group. The differences were not statistically significant. No patient had any clinical signs of upper urinary tract infection or septicemia. Three hundred and twelve patients were analysed for safety. Twenty-six patients reported 32 adverse drug events (ADEs). Four reactions in the TMP-SMX group were considered severe while in the NF group all the ADEs were of mild or moderate intensity. In this study NF seems to be at least as effective and safe as TMP-SMX.

Aged↗

PubMed2004

Cytochrome C oxidase III interacts with hepatitis B virus X protein in vivo by yeast two-hybrid system.

AIM: To screen and identify the proteins which interact with hepatitis B virus (HBV) X protein in hepatocytes by yeast two-hybrid system and to explore the effects of X protein in the development of hepatocellular carcinoma (HCC). METHODS: With HBV X gene amplified by polymerase chain reaction (PCR), HBV X bait plasmid, named pAS2-1-X, was constructed by yeast-two hybridization system3 and verified by auto-sequencing assay. pAS2-1-X was transformed into the yeast AH109, and X-BD fusion protein expressed in the yeast cells was detected by Western blotting. The yeast cells cotransformed with pAS2-1-X and normal human liver cDNA library were grown in selective SC/-trp-leu-his-ade medium. The second screen was performed with beta-gal activity detection, and false positive clones were eliminated by segregation analysis, true positive clones were amplified, sequenced and analyzed with bioinformatics. Mating experiment was performed to confirm the binding of putative proteins to X protein in the yeast cells. RESULTS: Bait plasmid pAS2-1-X was successfully constructed and pAS2-1-X correctly expressed BD-X fusion protein in yeast AH109. One hundred and three clones grew in the selective SC/-trp-leu-his-ade medium, and only one clone passed through beta-gal activity detection and segregation analysis. The inserted cDNA fragment showed high homology with Homo sapiens cytochrome C oxidase III (COXIII). Furthermore, mating experiment identified that the binding of COXIII to X protein was specific. CONCLUSION: COXIII protein is a novel protein that can interact with X protein in vivo by yeast two-hybrid system, and may contribute to the development of HCC through the interaction with X protein.

Carcinoma, Hepatocellular↗

PubMed2005

Cloning and characterization of a novel hepatitis B virus core binding protein C12.

AIM: To elucidate the biological function of HBV core antigen (HBcAg) on pathogenesis of hepatitis B, a novel gene C12 coding for protein with unknown function interacting with HBcAg in hepatocytes was identified and characterized. METHODS: HBcAg bait plasmid pGBKT7-HBcAg was constructed and transformed into yeast AH109, then the transformed yeast was mated with yeast Y187 containing liver complementary DNA (cDNA) library plasmid in 2XYPDA medium. Diploid yeast was plated on synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) and synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) containing X-alpha-gal for screening twice. After extracting and sequencing of plasmid from blue colonies, we isolated a cDNA clone encoding a novel protein designated as C12 that directly interacted with HBcAg. The interaction between HBcAg and C12 was verified again by re-mating. pEGFP-N1-C12 fluorescent protein fusion gene was transfected in 293 and L02 cell, and observed by fluorescent microscope. MTT reduction assay was used to study the action of C12 protein effect on metabolism of mammal cell. Yeast two-hybrid and cDNA microarray were performed to search binding protein and differential expression genes regulated by C12 protein. RESULTS: C12 gene was screened and identified by yeast two-hybrid system 3. The interaction between HBcAg and the novel protein coded by the new gene C12 was further confirmed by re-mating. After 48 h, fluorescence of fusion protein could be observed steadily in the 293 and L02 cell plasma. Under MTT assay, we found that the expression of C12 did not influence the growth of liver cells. Seventeen differential expression genes in HepG2 cells transfected with C12 protein expression plasmid by cDNA microarray, of which 16 genes were upregulated and 1 gene was downregulated by C12 protein. Twenty-one colonies containing 16 different genes coding for C12 protein binding proteins were isolated by yeast two-hybrid, there were 2 new genes with unknown function. CONCLUSION: The novel protein C12 is located in cell plasma, and its overexpression has no significant effect on the metabolism of liver cell. It interacts with many proteins in hepatocytes and may be involved in many processes of gene expression.

Amino Acid Sequence↗

PubMed2006

Screening and identification of interacting proteins with hepatitis B virus core protein in leukocytes and cloning of new gene C1.

AIM: To investigate the biological function of HBcAg in pathogenesis of HBV replication in peripheral blood mononuclear cells (PBMCs). METHODS: HBcAg region was amplified by polymerase chain reaction (PCR) and HBV HBcAg bait plasmid pGBKT7-HBcAg was constructed by routine molecular biological methods. Then the recombinant plasmid DNA was transformed into yeast AH109. After the HBV core protein was expressed in AH109 yeast strains (Western blot analysis), yeast-two hybrid screening was performed by mating AH109 with Y187 containing leukocyte cDNA library plasmid. Diploid yeast cells were plated on synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) (QDO) and synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) (TDO). The second screening was performed with the LacZ report gene ( yeast cells were grown in QDO medium containing X-alpha-gal). The interaction between HBV core protein and the protein obtained from positive colonies was further confirmed by repeating yeast-two hybrid. After plasmid DNA was extracted from blue colonies and sequenced, the results were analyzed by bioinformatic methods. RESULTS: Eighteen colonies were obtained and sequenced, including hypermethylated in cancer 2 (3 colones), eukaryotic translation elongation factor 2 (2 colones), acetyl-coenzyme A synthetase 3 (1 colone), DNA polymerase gamma (1 colone), putative translation initiation factor (1 colone), chemokine (C-C motif) receptor 5 (1 colone), mitochondrial ribosomal protein L41 (1 colone), kyot binding protein genes (1 colone), RanBPM (1 colone), HBeAg-binding protein 3 (1 colone), programmed cell death 2 (1 colone). Four new genes with unknown function were identified. CONCLUSION: Successful cloning of genes of HBV core protein interacting proteins in leukocytes may provide some new clues for studying the biological functions of HBV core protein.

Amino Acid Sequence↗

PubMed2002

Seek protein which can interact with hepatitis B virus X protein from human liver cDNA library by yeast two-hybrid system.

AIM: To seek the X associated protein (XAP) with the constructed bait vector pAS2-1X from normal human liver cDNA library. METHODS: The X region of the HBV gene was amplied by PCR and cloned into the eukaryotic expression vector pAS2-1. The reconstituted plasmid pAS2-1X was transformed into the yeast cells and the expression of X protein (pX) was confirmed by Western blot analysis. Yeast cells were cotransformed with pAS2-1X and the normal human liver cDNA library and were grown in selective SC/-trp-leu-his-ade medium, the second screen was performed with the LacZ report gene. Furthermore, segregation analysis and mating experiment were performed to eliminate the false positive and the true positive clones were selected for PCR and sequencing. RESULTS: Reconstituted plasmid pAS2-1X including the anticipated fragment of X gene was proved by auto-sequencing assay. Western blot analysis showed that reconstituted plasmid pAS2-1X expressed BD:X fusion protein in yeast cells. Of 5 x 10(6) transformed colonies screened,65 grew in the selective SC/-trp-leu-his-ade medium, 5 scored positive for beta-gal activity, and only 2 remaining clones passed through the segregation analysis and mating experiment. Sequence analysis identified that two clones contained similar cDNA fragment:GAACTTGCG. CONCLUSION: The short peptide(glutacid-leucine-alanine)is a possible required site for XAP binding to pX. Normal human liver cDNA library has difficulties in expressing the integrated XAP on yeast cells.

Base Sequence↗

PubMed2003

Identification of RanBMP interacting with Shigella flexneri IpaC invasin by two-hybrid system of yeast.

AIM: Bacillary dysentery caused by Shigella flexneri is still a threat to human health. Of four invasion plasmid antigen proteins (IpaA,B,C and D), IpaC plays an important role in the pathogenicity of this pathogen. The purpose of this study was to investigate the proteins interacting with IpaC in the host cell during the pathogenic process of this disease. METHODS: By applying two-hybrid system, the bait plasmid containing ipaC gene was constructed and designated pGBKT-ipaC. The bait plasmid was transformed AH109, and proved to express IpaC and then HeLa cDNA library plasmids were introduced into the above transformed AH109. The transformation mixture was plated on medium lacking Trp, Leu, and His in the initial screen, then restreaked on medium lacking Trp, Leu, His and Ade. Colonies growing on the selection medium were further assayed for beta-galactosidase activity. BLAST was carried out in the database after sequencing the inserted cDNA of the positive library plasmid. RESULTS: Among the 2X10(6) transformants, 64 positive clones were obtained as determined by activation of His, Ade and LacZ reporter genes. Sequence analysis revealed that cDNA inserts of two colonies were highly homologous to a known human protein, RanBPM. CONCLUSION: These results provide evidence that IpaC may be involved in the invasion process of S. flexneri by interacting with RanBPM, and RanBPM is most likely to be the downstream target of IpaC in the cascade events of S. flexneri infection.

Adaptor Proteins, Signal Transducing↗

PubMed1977

Production of vitamin B 12 analogues in patients with small-bowel bacterial overgrowth.

We investigated the presence of vitamin B 12 analogues (cobamides) and the bacterial conversion of 57Co-B12 (vitamin B12 cyanocobalamin, [57Co]-CN-Cbl) into cobamides in the intestinal contents of four patients with bacterial overgrowth. The (57Co)-CN-Cbl bound to intrinsic factor was given orally. Jejunal contents were aspirated for 24 h and cultured aerobically and anaerobically. The CN-Cbl and cobamides were separated by electrophoresis and chromatography and identified by bioautography. Radioactivity of cobamide zones from duplicate chromatograms showed bacterial conversion of (57Co)-cn-cbl into cobamides. Cobamides ([Ade]CNCBA, [2-Me Ade] CNCba, [CN]2Cbi and factor E) were found in the intestinal contents in three of the four patients, and in two of three patients cobamides represented more than 25% of the administered CN-Cbl. Thus bacterial production of cobamides, both de novo and from ingested CN-Cbl bound to intrinsic factor, occurs in humans with bacterial overgrowth states and results in a significant loss of vitamin B12 to the host.

Anemia, Megaloblastic↗

PubMed1978

[UV-light induction of "true" revertants to adenine independence in Bacillus subtilis cells].

The UV-irradiation of Bacillus subtilis Mu5u8u16 (met5 leu8 purA) induces with relatively high frequency the revertants to adenine independence (Ade+) which form the rapidly growing morphologically uniform colonies on the solid selective medium. The genetic analysis of a portion of UV-induced Ade+ revertants (crosses in transformation system) has cleared up that their DNA does not contain the original mutation ade16. This means that they arise as the result of "true" reversions. This reversion in purA gene can serve as a good model for the study of UV-induced mutagenesis in a proximal structural locus of Bac. subtilis chromosome.

Adenine↗