Characterization of the thermal stress response of Campylobacter jejuni

Campylobacter jejuni, a microaerophilic, gram-negative bacterium, is a common cause of gastrointestinal disease in humans. Heat shock proteins are a group of highly conserved, coregulated proteins that play important roles in enabling organisms to cope with physiological stresses. The primary aim of this study was to characterize the heat shock response of C. jejuni. Twenty-four proteins were preferentially synthesized by C. jejuni immediately following heat shock. Upon immunoscreening of Escherichia coli transformants harboring a Campylobacter genomic DNA library, one recombinant plasmid that encoded a heat shock protein was isolated. The recombinant plasmid, designated pMEK20, contained an open reading frame of 1,119 bp that was capable of encoding a protein of 372 amino acids with a calculated molecular mass of 41,436 Da. The deduced amino acid sequence of the open reading frame shared similarity with that of DnaJ, which belongs to the Hsp-40 family of molecular chaperones, from a number of bacteria. An E. coli dnaJ mutant was successfully complemented with the pMEK20 recombinant plasmid, as judged by the ability of bacteriophage lambda to form plaques, indicating that the C. jejuni gene encoding the 41-kDa protein is a functional homolog of the dnaJ gene from E. coli. The ability of each of two C. jejuni dnaJ mutants to form colonies at 46 degreesC was severely retarded, indicating that DnaJ plays an important role in C. jejuni thermotolerance. Experiments revealed that a C. jejuni DnaJ mutant was unable to colonize newly hatched Leghorn chickens, suggesting that heat shock proteins play a role in vivo.     

Analysis of distribution of Campylobacter jejuni and Campylobacter coli in broilers by using restriction fragment length polymorphism of flagellin gene

The incidence of Campylobacter jejuni and Campylobacter coli in broiler farms was 33.9% (19/56). C. jejuni-positive flocks accounted for 20.0% (17/85) and C. coli-positive ones was 4.7% (4/85). There were 14 patterns (fla type) of restriction fragment length polymorphism (RFLP) of flagellin A gene among these 22 strains of C. jejuni and C. coli including the standard strain C. jejuni ATCC 33560. Different fla types of Campylobacter were isolated from broilers in different growing cycles on the same farms. Four strains of C. jejuni were isolated from four breeder farms and four fla types of C. jejuni were detected from their progenies reared on growing farms. Three fla types of C. jejuni detected from the progenies were different from those of each breeder. Also, the other three fla types of C. jejuni were detected from different progenies of each growing farm during the next growing cycle. These findings indicate that the RFLP analysis may contribute to epidemiological studies of C. jejuni and C. coli contamination of broilers and suggest the risk of contamination with different types of Campylobacter in every growing cycle of broilers on the farm even on the same farm. They also supported that there was little likeliness of the vertical transmission of C. jejuni and C. coli from breeders to broilers.     

Antimicrobial susceptibilities of Campylobacter jejuni and coli by using E-test in Taiwan

Cytogenetic abnormalities in human malignancies frequently involve chromosome 7. The existence of several tumor suppressor genes on the long arm of chromosome 7 has been suggested in both epithelial and hematologic malignancies. From the Danish Cytogenetic Register, we identified 183 persons with constitutional abnormalities involving chromosome 7, including 16 patients with Williams syndrome. By linkage to the Danish Cancer Registry, we found five persons with cancer, including one thyroid carcinoma, three carcinomas of the digestive tract, and one malignant melanoma. There were no cases of leukemia. The overall risk of developing cancer was not increased.     

Specific identification of the enteropathogens Campylobacter jejuni and Campylobacter coli by using a PCR test based on the ceuE gene encoding a putative virulence determinant

A PCR method for the rapid identification and discrimination of thermophilic Campylobacter jejuni and Campylobacter coli was developed by using a gene encoding a protein involved in siderophore transport (ceuE). A nucleotide sequence divergence of approximately 13% in the ceuE genes of C. jejuni and C. coli facilitated the design of two species-specific PCR primer sets. The specificity of the PCR amplification reactions was confirmed by using two nonradioactively labelled species-specific internal oligonucleotide hybridization probes for each of these species.     

PCR detection, identification to species level, and fingerprinting of Campylobacter jejuni and Campylobacter coli direct from diarrheic samples

Three sets of primers were designed for PCR detection and differentiation of Campylobacter jejuni and Campylobacter coli. The first PCR assay was designed to coidentify C. jejuni and C. coli based on their 16S rRNA gene sequences. The second PCR assay, based on the hippuricase gene sequence, identified all tested reference strains of C. jejuni and also strains of that species which lack detectable hippuricase activity. The third PCR assay, based on the sequence of a cloned (putative) aspartokinase gene and the downstream open reading frame, identified all tested reference strains of C. coli. The assays will find immediate application in the rapid identification to species level of isolates. The assays combine with a protocol for purification of total DNA from fecal samples to allow reproducible PCR identification of campylobacters directly from stools. Of 20 clinical samples from which campylobacters had been cultured, we detected C. jejuni in 17, C. coli in 2, and coinfection of C. jejuni and Campylobacter hyointestinalis in 1. These results were concordant with culture and phenotypic identification to species level. Strain typing by PCR-restriction fragment length polymorphism of the flagellin (flaA) gene detected identical flaA types in fecal DNA and the corresponding campylobacter isolate. Twenty-five Campylobacter-negative stool samples gave no reaction with the PCR assays. These PCR assays can rapidly define the occurrence, species incidence, and flaA genotypes of enteropathogenic campylobacters.     

Enzymatic activities of Campylobacter jejuni, C. coli, and C. lari

Many immunohistochemical studies have investigated the relationship between immunohistochemical characteristics and histopathological findings in colorectal tumors. One of the most extensively studied markers has been tissue CEA, although the prognostic significance of this and other antigens is still uncertain. The authors report results relative to three tumoral antigens (carcinoembryonic antigen, CEA; tissue polypeptide antigen. TPA, and carbohydrate antigen 19-9, CA 19-9) determined by immunohistochemical methods in tissue samples of 52 colorectal carcinomas. The relationship between the immunohistochemical characteristics of the neoplasms and the clinicopathologic parameters, as well as their influence on the prognosis of the patients, were examined. Positive CEA reaction has a significant relationship with grade of differentiation of the tumor while diffuse cellular expression of this antigen often indicates neoplasms extending beyond the intestinal wall and invading the lymph vessels. The number of tissue antigens expressed is significantly related to the extent of tumor spread through the intestinal wall. A greater incidence of recurrence and shorter disease-free interval and survival were observed in neoplasms that expressed tissue TPA antigen or more than one tissue antigens. In the present study the latter parameter has demonstrated to have independent prognostic significance for the disease-free interval. Immunohistochemical evaluation of antigens in colorectal carcinoma tissue shows a possible independent prognostic value of the antigenic heterogeneity of tumors, which could be related to their different biological behavior.     

Identification of the enteropathogens Campylobacter jejuni and Campylobacter coli based on the cadF virulence gene and its product

Campylobacter jejuni and Campylobacter coli are common causes of gastroenteritis in humans. Infection with C. jejuni or C. coli is commonly acquired by eating undercooked chicken. The goal of this study was to develop specific detection assays for C. jejuni and C. coli isolates based on the cadF virulence gene and its product. The cadF gene from C. jejuni and C. coli encodes a 37-kDa outer membrane protein that promotes the binding of these pathogens to intestinal epithelial cells. A fragment of approximately 400 bp was amplified from 38 of 40 (95%) C. jejuni isolates and 5 of 6 (83.3%) C. coli isolates with primers designed to amplify an internal fragment of the cadF gene. PCR was then used to amplify Campylobacter DNA from store-bought chickens. A 400-bp band was amplified from 26 of the 27 chicken carcasses tested by the PCR-based assay. The CadF protein was detected in every C. jejuni and C. coli isolate tested, as judged by immunoblot analysis with a rabbit anti-C. jejuni 37-kDa serum. In addition, methanol-fixed samples of whole-cell C. jejuni and C. coli were detected with the rabbit anti-37-kDa serum by using an indirect-immunofluorescence microscopy assay. These findings indicate that the cadF gene and its product are conserved among C. jejuni and C. coli isolates and that a PCR assay based on the cadF gene may be useful for the detection of Campylobacter organisms in food products.     

The relationship of Campylobacter jejuni subsp. jejuni enterotoxigenicity and the increase of cAMP and electrolyte changes in the rat intestine

BACKGROUND: Small intestine alterations produced by the enterotoxigenic capacity of Campylobacter jejuni subsp. jejuni are similar to the hydric, electrolytic and pathological changes caused by choleraic and thermolabile Escherichia coli toxins. AIM: To study the enterotoxigenic capacity of 4 strains of Campylobacter jejuni subsp. jejuni using the intestinal loop model. MATERIAL AND METHODS: Rat intestinal loops were inoculated with culture filtrates of the four strains. Enterotoxigenicity was assessed by fluid accumulation, the increase in Na+ and Cl- in the loop fluid, and cAMP increase in loop tissues. An enterotoxigenic Escherichia coil strain and sterile Brucella both were used as positive and negative controls, respectively. RESULTS: The filtrates of two strains produced fluid accumulation in the loops, significantly increased Na+ and Cl- secretion to the intestinal lumen and increased tissue cAMP levels. CONCLUSIONS: Some strains of Campylobacter jejuni subsp. jejuni are able to show enterotoxigenicity in vivo, increasing cAMP levels in the intestinal cells and altering electrolyte exchange mechanisms.     

Analysis on morphology and stress concentration in continuous casting bloom to learn the formation and propagation of internal cracks induced by soft reduction technology

ABSTRACT

The crack formation and propagation were analysed according to actual internal crack morphology and the finite element model simulation of stress concentration. The results showed that most cracks were distributed along the transverse and the longitudinal section of the bloom. Distinct differences between these two major types of cracks were found in dimension and inclination, which were owing to different local stress concentrations. In the longitudinal section of the bloom, shear stress was concentrated in the brittle temperature region, which led to the formation of initial cracks and subsequently cracks propagation. Meanwhile, the maximum tensile stress occurred at the edge between the brittle temperature region and surrounding material, which resulted in crack formation and propagation along the transverse section of the bloom. This phenomenon was due to the obvious bulging deformation of the solidified shell induced by soft reduction.     

The effect of oxidative stress on the production of the recombinant protein, interferon gamma, produced by Chinese hamster ovary cells in stirred-batch culture

BACKGROUND: The uptake of thallium-201 (T1-201) in malignant tumors is associated with malignant potential (metastatic potential and proliferative activity). The grade of accumulation of T1-201 in malignant tumors may provide information regarding prognosis. METHODS: A T1-201 single photon emission computed tomography was conducted 120 minutes after intravenous injection of 111 megabecquerel of T1-201 chloride. The authors calculated the uptake ratio to evaluate the degree of T1-201 uptake in the primary tumor. This ratio was compared with survival time and other prognostic factors. RESULTS: The authors studied 152 patients (125 men and 27 women). The group of patients with the low T1-201 uptake ratio survived longer than the group of patients with the high T1-201 uptake ratio (median survival, 58 weeks vs. 33 weeks; P = 0.0138 by the log rank test). The multivariate analysis confirmed that the T1-201 uptake ratio was an independent prognostic factor for survival. CONCLUSIONS: These results suggest that the T1-201 uptake ratio provides independent and objective prognostic information for patients with lung carcinoma.     

Cytopathic effect, plaque formation, and lysis of Ehrlichia chaffeensis grown on continuous cell lines

The present study describes some unexpected receptor mediated effects of N-methylcarbamylcholine on mouse M1 muscarinic receptor gene transfected cell line (M1Y1) that were not evident from biochemical studies with mouse and rat brain tissue where N-methylcarbamylcholine exhibited only nicotinic properties. Although N-methylcarbamycholine was devoid of muscarinic properties in mouse and rat brain preparations, as determined by phosphoinositide turnover and inhibition of [3H]QNB binding, it exhibited significant muscarinic characteristics in the transfected M1Y1 cell line. At a concentration of 10(-6) M or greater, N-methylcarbamycholine caused a transient increase in intracellular Ca2+ of 50 s duration that was reversible by atropine or pirezepine. The Ca(2+)-transient was not elicited by other nicotinic agents such as nicotine and N,N-dimethylcarbamylcholine, a close analogue of N-methylcarbamylcholine, with comparable affinity for nicotinic receptors and devoid of muscarinic activity. N-Methylcarbamylcholine also stimulated phosphoinositide turnover in M1Y1 cells with an estimated EC50 value 10 times greater than that of carbachol, and the effect was blocked by atropine. Both carbachol and N-methylcarbamycholine inhibited [3H]QNB binding in a concentration-dependent manner; however, the IC50 for carbachol was over two orders of magnitude greater than that observed in mouse and rat brain membranes. In considering possible explanations for the differential characteristics of N-methylcarbamylcholine in mouse and rat brain as compared to the transfected M1Y1 cells, it was concluded that the difference may be attributable to differences in the receptor-transduction coupling efficiency and the microenvironment of the muscarinic receptors.     

In vitro studies of Campylobacter jejuni/coli strains from hens and humans regarding adherence, invasiveness, and toxigenicity

Campylobacter jejuni/coli strains from hens and humans were compared for their ability to adhere to and invade HEp-2-cells and for toxigenicity to CHO-cells. In both hen and human strains, invasiveness was higher among non-toxigenic strains than among toxigenic ones. The frequency of adherence, invasiveness, and toxigenicity was the same in hen and human strains.     

Stimulatory effect of dihydroxyphenyl compounds on the aerotolerance of Spirillum volutans and Campylobacter fetus subspecies jejuni

Administration for different doses of cyproterone acetate (CPA) to male hamsters for 5 weeks slows down the growth of the adrenals and the increase in body weight. This effect is still present 4 weeks after cessation of CPA treatment, though some recovery can be seen. Histological and histometrical evaluations confirm an atrophy of the adrenal, particularly in the zona fasciculata and in higher doses also in the zona reticularis. After operative castration it is the zona reticularis which shows the most marked atrophic changes. It is therefore concluded, that CPA possesses besides its anti-androgenic properties an inhibiting corticoid-like effect on the adreno-pituitary feed-back mechanism.     

Optimisation of continuous casting mould parameters using genetic algorithms and other allied techniques

Abstract

A rigorous optimisation has been carried out for the mould region of the continuous caster, using genetic algorithms, differential evolution, simulated annealing, and the traditional steepest descent method. The optimised predictions of some important casting parameters such as negative strip time, flux pool depth, and vitrification ratio are compared with current industrial practice.     

Etiological mechanism and treatment of Guillain-Barre syndrome-- Campylobacter jejuni enteritis and the development of anti-ganglioside antibody preceding the syndrome

An ulnar dislocation of the trapeziometacarpal joint is reported. On the posteroanterior radiograph of the wrist, the boney alignment appeared normal, and the only sign indicating a dislocation was a slight widening of the joint. By contrast, a true posteroanterior view of the trapezium clearly demonstrated the metacarpal ulnarly displaced. A late tendon reconstruction of the damaged palmar and medial ligaments produced a satisfactory result.     

Ceroid/lipofuscin formation in cultured human fibroblasts: the role of oxidative stress and lysosomal proteolysis

The mechanisms involved in the accumulation of ceroid/lipofuscin within non-dividing cells are not totally understood. Oxidative stress, as well as diminished activity of lysosomal proteolytic enzymes, are known to induce ceroid/lipofuscin accumulation in a variety of cell types. In order to clarify the roles of oxidative stress and lysosomal proteolysis in ceroidogenesis/lipofuscinogenesis, and to study the fate of already formed ceroid/lipofuscin, confluent cultures of AG-1518 human fibroblasts were exposed to oxidative stress (40% ambient oxygen) and/or treated with the thiol protease inhibitor leupeptin for 2 weeks. Both oxidative stress and protease inhibition caused accumulation of ceroid/lipofuscin per se (estimated by fluorescent, confocal and electron microscopy). The combined effect of these factors was, however, almost three times as large as the sum of their isolated effects. The pigment accumulated progressively as long as the oxidative stress and/or protease inhibition acted; was not eliminated after re-establishment of normal conditions; and decreased in amount after subsequent passage. The results suggest that (i) ceroid/lipofuscin forms within secondary lysosomes due to peroxidative damage of autophagocytosed material, and (ii) it is not substantially eliminated from non-dividing cells by degradation or exocytosis.     

Cadmium and cardiac muscle cell - biomarkers of oxidative stress - experimental work

OBJECTIVE: To study in several tissues (heart, kidney and liver) of Halobatrachus didactylus the cellular response induced by an acute exposure to a sublethal cadmium concentration. DESIGN: Fifteen species of H. didactylus (marine teleost) were divided in to three groups: CTRL: control group, the fish were injected with a saline solution; 24 H: 1 mg/kg of cadmium chloride was injected and the fish were sacrificed after 24 hours; 7 D: the fish were subjected to the same cadmium concentration and sacrificed 7 days after injection. INTERVENTIONS: Superoxide dismutase--SOD (McCord & Fridovich, 1969) and catalase--CAT (Clairborne, 1985) activities were determined in the cytosolic and mitochondrial fractions of these three tissues. The lipid degradation products were also determined by the tiobarbithuric acid (TBA) test. MEASUREMENTS AND RESULTS: Cadmium induced an increase in SOD activity in both fractions (cytosolic and mitochondrial) of these H. didactylus tissues. The highest levels of activity observed were located at mitochondrial fraction and in the heart. There was a significant increase in CAT activity in both liver and heart tissue fractions after cadmium exposure. The highest values were observed in the liver. The kidney presented a different response: there was a rise in CAT activity only in the mitochondrial fraction after seven days of exposure. There were no significant changes in lipid degradation products in any of these tissues after cadmium exposure. CONCLUSIONS: The two antioxidant enzymes studied in the heart, kidney and liver of H. didactylus demonstrated a high sensitivity to oxidative stress induced by cadmium and presented a high potential as cellular biological makers. The results indicate membrane lesion caused by lipid peroxidation did not occur, which suggests an efficient response of the cellular protection mechanisms against cadmium cytotoxicity.     

Analysis of a form of oxidative DNA damage, 8-hydroxy-2'-deoxyguanosine, as a marker of cellular oxidative stress during carcinogenesis

8-hydroxy-2'-deoxyguanosine (8-OH-dG) was first reported in 1984 as a major form of oxidative DNA damage product by heated sugar, Fenton-type reagents and X-irradiation in vitro. 8-OH-dG has been detected in cellular DNA using an HPLC-ECD method in many laboratories. Analyses of 8-OH-dG in animal organ DNA after the administration of oxygen radical-forming chemicals will be useful for assessments of their carcinogenic risk. Its analysis in human leucocyte DNA and in urine is a new approach to the assessment of an individual's cancer risk due to oxidative stress. The increase of the 8-OH-dG level in the cellular DNA, detected by HPLC-ECD method, was supported by its immunochemical detection and its enhanced repair activity. The validity of the general use of 8-OH-dG as a marker of cellular oxidative stress is discussed.     

Isolation and identification of Burkholderia pseudomallei from soil using selective culture techniques and the polymerase chain reaction

An environmental soil survey to detect Burkholderia pseudomallei was performed during the dry and wet seasons in Darwin, Northern Territory, Australia. Soil was sampled at regular intervals during a 15-month period at different depths from areas which were representative of the local, soil environment. Selective culture techniques using Ashdown's and Galimand and Dodin's methods and the polymerase chain reaction (PCR) using specific 16S rRNA primers were used to detect and identify the organism and determine its distribution within the soil stratum over the change in seasons. Results showed that Ashdown's method gave higher isolation rates in the dry season, and Galimand and Dodin's method gave higher isolation rates during the wet season. PCR of the soil enrichment proved to be a more sensitive method than culture and was also a useful confirmatory test in determining the identification of isolates where biochemical tests gave inconsistent results. The PCR primers were specific and able to detect 10(1) cfu g-1 soil and 10(4) cfu g-1 of soil using Ashdown's enrichment broth and Galimand and Dodin's broth, respectively. Overall the isolation of B. pseudomallei was greatest during the dry season and at the higher and lower soil depths, which is contradictory to epidemiological evidence that melioidosis occurs primarily during the wet season among patients exposed to contaminated surface soil and water.