Antibacterial activity of bovine lactoferrin and its peptides against enterohaemorrhagic Escherichia coli O157:H7

OBJECTIVES: To assess the long term effects of small increases in dietary folic acid on the concentration of plasma homocysteine, an independent risk factor for occlusive vascular disease, in a general population. DESIGN: A randomized double-blind placebo-controlled intervention study. SUBJECTS: One hundred and nineteen healthy volunteers, whose intake of fortified or supplemental folic acid was low, were recruited by letter from the patient register of a large inner-city group general practice. METHODS: Volunteers were randomized to receive unfortified cereals, or cereals fortified with 200 microg of folic acid per portion, with or without other vitamins. Blood samples were taken presupplement and at 4, 8 and 24 weeks on treatment and analysed for plasma homocysteine, cysteine and vitamin B12 and serum and red cell folate. Ninety-four subjects completed the study providing blood samples on all four occasions. RESULTS: There were no significant changes in any measured parameter in those eating unfortified cereals. Overall, folic acid fortification of cereals led to significant increases (P < 0.001) in serum folate (66%), and red cell folate (24%), and a decrease in plasma homocysteine (10%; P < 0.001). There were no changes in vitamin B12 or cysteine. The homocysteine decrease persisted until the end of the study and was primarily seen in those who initially had the highest plasma homocysteine or the lowest serum folate. CONCLUSIONS: If homocysteine is found to be a causative risk factor in occlusive vascular disease, food fortification with physiological levels of folic acid should have a significant impact on the prevalence of the disease in the general population.     

The C-terminal region of the Escherichia coli UvrC protein, which is homologous to the C-terminal region of the human ERCC1 protein, is involved in DNA binding and 5''-incision

STUDY OBJECTIVES: To examine the incidence and consequences of atrial arrhythmias in surgical ICU patients following major noncardiac, nonthoracic surgery. DESIGN: Prospective observational study. SETTING: University hospital surgical ICU. PATIENTS: Four hundred sixty-two consecutive patients after noncardiothoracic surgery. INTERVENTIONS: None. MEASUREMENTS AND RESULTS: Patients were assigned to one of three groups: group 1-new-onset atrial arrhythmias (n=47); group 2-history of atrial arrhythmias (n=58); and group 3-no atrial arrhythmias (n=357). New arrhythmias occurred in 10.2% of patients. Most began within the first 2 postoperative days. These patients had a higher mortality rate (23.4%), longer ICU stay (8.5+/-17.4 [SD] days), and extended hospital stay (23.3+/-23.6 days) than patients without atrial arrhythmias (mortality, 4.3%; ICU stay, 2.0+/-4.5 days; hospital stay; 13.3+/-17.7 days; p<0.02). Thirteen percent of patients had a history of atrial arrhythmias. They had a higher mortality rate (8.6%) and longer ICU stays (2.9+/-4.9 days; p<0.02) than patients without arrhythmias. Most deaths in the two arrhythmia groups were not due to cardiac problems, but to sepsis or cancer. CONCLUSIONS: Patients admitted to a surgical ICU after noncardiothoracic surgery with a history of or who developed new atrial arrhythmias had greater mortality and longer ICU stays than patients without arrhythmias. The incidence of new-onset arrhythmias was lower than reported after cardiac and thoracic surgery, but higher than in the general population. Atrial arrhythmias were not the cause of death and appear to be markers of increased mortality and morbidity.     

Adhesion of Helicobacter pylori and Escherichia coli to human and bovine surface mucus cells in vitro

Helicobacter pylori shows in vivo a specific affinity for epithelial surface mucus cells (SMC) of the human stomach. We studied the in vitro adhesion of five different H. pylori strains and one non-pathogenic Escherichia coli-strain to (a) human antral SMC, obtained during gastroscopy; (b) human tumour SMC, from a carcinoma cell line (CRL 1739 AGS); and (c) bovine SMC, obtained from the abomasum. SMC of different origin were characterized by means of electron microscopy and immunohistochemistry, and showed similar main features: all cells showed intra-cellular structures like zymogens and PAS-positive mucin granules. HSMC were antibody-positive against epithelial cell markers. All five H. pylori strains adhered to human SMC (HSMC) and tumour SMC (TSMC). Only one strain additionally adhered to bovine SMC (BSMC). No adhesion to any of these cells was observed with E. coli. Adhesion in vitro is characterized by a close membrane-to-membrane association between H. pylori and the target cells. This phenomenon suggests a specific receptor-ligand interaction.     

Membrane interaction of synthetic peptides related to the putative fusogenic region of PH-30 alpha, a protein in sperm-egg fusion

In order to investigate the relationship between structure and function of a putative fusogenic region of PH-30a, a protein active in sperm-egg fusion, two peptides, SFP22 and SFP23, whose sequences correspond to the residues 90-111 and 89-111 of PH-30 alpha, respectively, were chemically synthesized. An analog of SFP23, SFP23AA, which has an Ala-Ala sequence instead of the Pro-Pro sequence in SFP23, was also prepared. The CD study indicated that SFP22 and SFP23 mainly took a beta-structure in the presence of DPPC and DPPC/DPPG (3/1) vesicles, while SFP23AA showed an alpha-helical pattern though the alpha-helical content calculated was low (25-30%). alpha-Helical CD curve was observed for these peptides in trifluoroethanol. The membrane-perturbing activity of SFP22 and SFP23 was weaker than that of SFP23AA. On the other hand, the membrane-fusogenic activity of SFP22 and SFP23 to acidic phospholipid bilayers was much stronger than that of SFP23AA. All the peptides caused very weak cell lysis. These results are consistent with the reported speculation [Blobel, C. P. et al. (1992), Nature (London) 356, 248-252] that residues 90-111 of PH-30 alpha may be the fusogenic region and suggest that the Pro-Pro sequence is one of the important factors for holding the active secondary structure of the fusogenic region of PH-30 alpha in membranes.     

EspP, a novel extracellular serine protease of enterohaemorrhagic Escherichia coli O157:H7 cleaves human coagulation factor V

In this study, we identified and characterized a novel secreted protein, the extracellular serine protease EspP, which is encoded by the large plasmid of enterohaemorrhagic Escherichia coli (EHEC) O157:H7. The corresponding espP gene consists of a 3900 bp open reading frame that is able to encode a 1300-amino-acid protein. EspP is synthesized as a large precursor which is then processed at the N- and C-termini during secretion. It can be grouped into the autotransporter protein family. The deduced amino acid sequence of EspP showed homology to several secreted or surface-exposed proteins of pathogenic bacteria, in particular EspC of enteropathogenic E. coli and IgA1 proteases from Neisseria spp. and Haemophilus influenzae. Hybridization experiments and immunoblot analysis of clinical EHEC isolates showed that EspP is widespread among EHEC of the serogroup O157 and that it also exists in serogroup 026. A specific immune response against EspP was detected in sera from patients suffering from EHEC infections. Functional analysis showed that EspP is a protease capable of cleaving pepsin A and human coagulation factor V. Degradation of factor V could contribute to the mucosal haemorrhage observed in patients with haemorrhagic colitis.     

Detection, distribution and probable fate of Escherichia coli O157 from asymptomatic cattle on a dairy farm

We conducted this study to address concerns regarding a perceived increase in bronchitis, skin rashes, and diarrhea and long term health effects in workers at two Refuse Derived Fuel (RDF) processing plants. We abstracted medical records to assess symptom occurrence, and to identify spirometry and lab abnormalities. Overall FEV1 and FVC showed no decrease over time for workers with more than five years of exposure. The primary self reported symptoms were low back pain and headaches followed by skin rashes, colds, 'flu', hay fever and ear problems. Conclusions from this analysis are limited by lack of standardized protocols or a control group. However, results from this analysis pointed out potential occupational health problems among RDF workers for future epidemiological and environmental studies.     

Isolation of a periplasmic molecular chaperone-like protein of Rhodobacter sphaeroides f. sp. denitrificans that is homologous to the dipeptide transport protein DppA of Escherichia coli

A periplasmic protein has been found to prevent aggregation of the acid-unfolded dimethyl sulfoxide reductase (DMSOR), the periplasmic terminal reductase of dimethyl sulfoxide respiration in the phototroph Rhodobacter sphaeroides f. sp. denitrificans, in a manner similar to that of the Escherichia coli chaperonin GroEL (Matsuzaki et al., Plant Cell Physiol. 37:333-339, 1996). The protein was isolated from the periplasm of the phototroph. It had a molecular mass of 58 kDa and had no subunits. The sequence of 14 amino-terminal residues of the protein was completely identical to that of the periplasmic dipeptide transport protein (DppA) of E. coli. The 58-kDa protein prevented aggregation to a degree comparable to that of GroEL on the basis of monomer protein. The 58-kDa protein also decreased aggregation of guanidine hydrochloride-denatured rhodanese, a mitochondrial matrix protein, during its refolding upon dilution. The 58-kDa protein is a kind of molecular chaperone and could be involved in maintaining unfolded DMSOR, after secretion of the latter into the periplasm, in a competent form for its correct folding.     

Characterization of EspC, a 110-kilodalton protein secreted by enteropathogenic Escherichia coli which is homologous to members of the immunoglobulin A protease-like family of secreted proteins

Enteropathogenic Escherichia coli (EPEC) secretes at least five proteins. Two of these proteins, EspA and EspB (previously called EaeB), activate signal transduction pathways in host epithelial cells. While the role of the other three proteins (39, 40, and 110 kDa) remains undetermined, secretion of all five proteins is under the control of perA, a known positive regulator of several EPEC virulence factors. On the basis of amino-terminal protein sequence data, we cloned and sequenced the gene which encodes the 110-kDa secreted protein and examined its possible role in EPEC signaling and interaction with epithelial cells. In accordance with the terminology used for espA and espB, we called this gene espC, for EPEC-secreted protein C. We found significant homology between the predicted EspC protein sequence and a family of immunoglobulin A (IgA) protease-like proteins which are widespread among pathogenic bacteria. Members of this protein family are found in avian pathogenic Escherichia coli (Tsh), Haemophilus influenzae (Hap), and Shigella flexneri (SepA). Although these proteins and EspC do not encode IgA protease activity, they have considerable homology with IgA protease from Neisseria gonorrhoeae and H. influenzae and appear to use a export system for secretion. We found that genes homologous to espC also exist in other pathogenic bacteria which cause attaching and effacing lesions, including Hafnia alvei biotype 19982, Citrobacter freundii biotype 4280, and rabbit diarrheagenic E. coli (RDEC-1). Although these strains secrete various proteins similar in molecular size to the proteins secreted by EPEC, we did not detect secretion of a 110-kDa protein by these strains. To examine the possible role of EspC in EPEC interactions with epithelial cells, we constructed a deletion mutant in espC by allelic exchange and characterized the mutant by standard tissue culture assays. We found that EspC is not necessary for mediating EPEC-induced signal transduction in HeLa epithelial cells and does not play a role in adherence or invasion of tissue culture cells.     

Expression of thioredoxin random peptide libraries on the Escherichia coli cell surface as functional fusions to flagellin: a system designed for exploring protein-protein interactions

The administration of high dose chemotherapy and or radiotherapy with autologous hematopoietic rescue has become a treatment modality with increasing number of indications in a variety of malignant conditions. Improvements in the conditioning regimens and supportive measures used, as well as a more refined patient selection based on prognostic factors, have resulted in progressively better results. The availability of precursor cells from peripheral blood has allowed a faster restoration of hematopoiesis, decreasing the period and intensity of myelosuppression. The following revision gives an updated image of the accumulated experience with this mode of support in malignant lymphomas.     

Immunoreactivity and in-vitro effect on human sperm-egg binding of antibodies against peptides corresponding to bonnet monkey zona pellucida-3 glycoprotein

The following synthetic peptides were made as immunogens for development of a zona-based contraceptive vaccine: P1, KQPFWLLQGGASRAETSVQPVLVE [amino acids (aa) 23-45 with an additional K at the N-terminus]; P2, FSEEKLVFSLRLMEENC (aa 164-179 with an additional C at the C-terminus and T170 replaced by V); and P3, CSFSKSSNSWFPVEGPADICQCC (aa 300-322). The aa are numbered on the basis of bonnet monkey ZP3 precursor protein. Antibodies against an additional peptide P4, KGDCGTPSHSRRQPHVVSQWSRSA (aa 324-347), significantly inhibits human sperm-oocyte binding. In addition, antibodies against cocktail of peptide-diphtheria toxoid conjugates also significantly inhibit the binding of spermatozoa to zona pellucida in a hemizona assay. These results will further help in the design of an immunocontraceptive vaccine based upon synthetic peptides corresponding to ZP3.     

Human cathepsin O. Molecular cloning from a breast carcinoma, production of the active enzyme in Escherichia coli, and expression analysis in human tissues

A cDNA encoding a novel member of the cysteine proteinase family of proteins has been cloned from a human breast carcinoma cDNA library, by using a polymerase chain reaction-based cloning strategy. The isolated cDNA contains an open reading frame coding for a polypeptide of 321 amino acids that has been tentatively called cathepsin O. This protein presents all the structural features characteristic of the different cysteine proteinases identified to date, including the active site cysteine residue that is involved in covalent intermediate formation during peptide hydrolysis. The cathepsin O cDNA was expressed in Escherichia coli, and after purification and refolding, the recombinant protein was able to degrade the synthetic peptides benzyloxycarbonyl-Phe-Arg-7-amido-4- methylcoumarin and benzyloxycarbonyl-Arg-Arg-7-amido-4-methylcoumarin widely used as substrates for cysteine proteinases. Cathepsin O proteolytic activity was abolished by trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane (E-64), an inhibitor of this subclass of proteolytic enzymes, thus providing additional evidence that the isolated cDNA codes for an authentic cysteine proteinase. Northern blot analysis of poly(A)+ RNAs isolated from a variety of human tissues demonstrated that cathepsin O is expressed in all examined tissues, which is consistent with a putative role of this protein as a proteolytic enzyme involved in normal cellular protein degradation and turnover.     

Isolation of cDNA for a novel human protein KNP-I that is homologous to the E. coli SCRP-27A protein from the autoimmune polyglandular disease type I (APECED) region of chromosome 21q22.3

We have isolated cDNA clones for a novel human protein KNP-I from fetal brain and bone marrow cDNA libraries. Northern blot analysis indicated that the KNP-I gene is ubiquitously expressed in various human tissues. Significant homology of the KNP-I protein with Escherichia coli anti-sigma cross-reacting protein (SCRP-27A) (44% identity) and zebrafish (Brachydanio rerio) esl protein (49% identity) suggested that the KNP-I protein may be involved in a basic cellular function. Genomic sequencing revealed that the KNP-I gene consists of seven exons spanning 12 kb. Exon 5 was involved in alternative splicing. The KNP-I gene was mapped between D21S1460 and D21S25 on human chromosome 21q22.3, 26 kb distal to a Not 1 site of D21S1460. Thus, this novel KNP-I gene could be a candidate gene for autoimmune polyglandular disease type I (APECED) and other disorders mapped to this region.     

Antibacterial actin of vinegar against food-borne pathogenic bacteria including Escherichia coli O157:H7 (Part 2). Effect of sodium chloride and temperature on bactericidal activity

Bactericidal effects of various kinds of AWASEZU (processed vinegar, 2.5% acidity) on food-borne pathogenic bacteria including Escherichia coli O157:H7 and other bacteria were examined. the order of bactericidal activities was NIHAIZU (3.5% NaCl was added) > SANBA-IZU (3.5% NaCl and 10% sucrose were added) > plain vinegar (spirit vinegar) > AMAZU (10% sucrose was added). This indicates that their activities were enhanced by the addition of sodium chloride and suppressed by the addition of sugar. On the other hand, when soy sauce was used instead of sodium chloride, the order of bactericidal activities was plain vinegar > AMAZU > NIHAIZU > SANBAIZU. This is mainly because their activities were suppressed by the increase in the pH value. The effect of sodium chloride (0.01-15%) and temperature (10-50 degrees C) on bactericidal activities against E. coli O157:H7 in spirit vinegar (0.5-2.5% acidity) was further examined. When vinegar was used in combination with sodium chloride, predominant synergism on the bactericidal activity was observed. Their activities were markedly enhanced by the addition of sodium chloride in proportion to the concentration. In addition to this, at higher temperatures spirit vinegar killed bacteria much more rapidly. It should be noted that the bactericidal activity of spirit vinegar was extremely enhanced by the combined use of the addition of sodium chloride and the rise of temperature. For example, in 2.5% acidity vinegar, the time required for 3 log decrease in viable cell numbers at 20 degrees C was shortened to 1/140-fold by the addition of 5% sodium chloride, shortened to 1/51-fold by the rise of the reaction temperature at 40 degrees C, and shortened to 1/830-fold; 0.89 minutes by both the addition of 5% sodium chloride and the rise of temperature at 40 degrees C. In order to propose the methods to prevent food poisoning by bacterial infection, bactericidal activities of vinegar solution containing sodium chloride on cooking tools and raw vegetables were examined. Vinegar solution (1-2% acidity, 3-7% NaCl) produced more than 3 log decrease in viable cell numbers of E. coli O157:H7 on the surface of cutting board, and cabbage and cucumber at 20-50 degrees C. These results suggested that the treatment with vinegar solution containing sodium chloride may be one of the useful methods to prevent food poisoning.     

Localization of a domain in the FimH adhesin of Escherichia coli type 1 fimbriae capable of receptor recognition and use of a domain-specific antibody to confer protection against experimental urinary tract infection

The FimH subunit of type 1-fimbriated Escherichia coli has been implicated as an important determinant of bacterial adherence and colonization of the urinary tract. Here, we sought to localize the functionally important domain(s) within the FimH molecule and to determine if antibodies against this domain would block adherence of type 1-fimbriated E. coli to the bladder mucosa in situ and in vivo in an established mouse model of cystitis. We generated translational fusion proteins of disparate regions of the FimH molecule with an affinity tag MalE, and tested each of the fusion products in vitro for functional activity. The minimum region responsible for binding mouse bladder epithelial cells and a soluble mannoprotein, horseradish peroxidase, was contained within residues 1-100 of the FimH molecule. We validated and extended these findings by demonstrating that antibodies directed at the putative binding region of FimH or at synthetic peptides corresponding to epitopes within the binding domain could specifically block type 1 fimbriae-mediated bacterial adherence to bladder epithelial cells in situ and yeast cells in vitro. Next, we compared the ability of mice passively immunized intraperitoneally with antisera raised against residues 1-25 and 253-264 of FimH or 1-13 of FimA to resist bladder colonization in vivo after intravesicular challenge with type 1-fimbriated E. coli. Only the antibody directed at the putative binding region of FimH (anti- s-FimH1-25) significantly reduced E. coli bladder infections in the experimental mouse model of urinary tract infections. Similar results were obtained when the mice were actively immunized with synthetic peptides corresponding to residues 1-25 and 253-264 of FimH or 1-13 of FimA. The mechanism of protection was attributed, at least in part, to inhibition of bacterial adherence to the bladder surface by s-FimH1-25-specific antibody molecules that had filtered through the kidneys into the urine. The level of FimH antibodies entering the bladder from the circulatory system of the immunized mice was found to be markedly enhanced upon bacterial challenge. The potential broad spectrum activity of the protective FimH antibody was indicated from its serologic cross-reactivity with various urinary tract bacterial isolates bearing type 1 fimbriae. These findings could be relevant in the design of an efficacious and broadly reactive FimH vaccine against urinary tract infections.     

Influence of synthetic peptides, corresponding to fragments of the human alpha-2 interferon molecule, on the proliferation of lymphoblastoid cells in vitro. Growth inhibition and receptor binding

Influence of nine synthetic peptides from the C-terminal part of human alpha-2 interferon (IFN) molecule on growth of a human T-lymphoblastoid cell line MT-4 was investigated. It was shown that some peptides inhibited the proliferation of MT-4 cells. It was also found that MT-4 cells expressed specific receptors on the outer surface of the plasma membrane. All studied peptides competed for binding to a common binding site. Antiproliferative activity of the peptides correlated with their length and affinity to receptors on the outer membrane of MT-4 cells.