Multiplex real-time PCR for exhaustive detection of diarrhoeagenic Escherichia coli.

AIMS: The source and routes of diarrhoeagenic Escherichia coli (DEC) have not been clarified because it is difficult to detect these organisms in samples with numerous coliform bacteria. We have developed multiplex real-time PCR assays for exhaustive detection of DEC. METHODS AND RESULTS: Primers and TaqMan probes were designed to amplify and quantify one gene (eae, stx1, stx2, elt, est, virB, aggR, astA, and afaB) from each of seven pathotypes of DEC, in duplex or triplex reactions under the same PCR cycling conditions. Specificity was confirmed using 860 strains including 88 DEC strains. The fluorescence threshold cycle and DNA concentrations correlated with decision coefficients of more than 0.99. Subsequently, meat samples and enrichment broths were spiked with DEC and the assays used to detect the genes. The detection limits varied from 7.1 x 10(2) to 1.1 x 10(4) CFU ml(-1), depending on the target genes. All meat samples spiked with a variety of DEC (more than 10 CFU 10 g(-1)) were found to be positive by the method. CONCLUSIONS: The present system allows for the efficient and simultaneous determination of various DEC pathotypes. SIGNIFICANCE AND IMPACT OF THE STUDY: This system makes epidemiological investigations for DEC sensitive and quick, and is a useful tool to clarify the source and routes of DEC.

Prominent expression of glial cell line-derived neurotrophic factor in human skeletal muscle.

Glial cell line-derived neurotrophic factor (GDNF) has been shown to exert neurotrophic effects on motor neurons as well as mesencephalic dopaminergic neurons. Because GDNF promotes survival of motor neurons in vivo and in vitro and rescues motor neurons from naturally occurring cell death, the potential use of GDNF for treatment of motor neuron diseases has been a major focus of recent research. The expression of GDNF in humans, however, has not been fully examined. In the present study, we examined the expression of GDNF in adult human muscle by Northern blot, reverse transcriptase polymerase chain reaction (RT-PCR), and immunohistochemical analyses to address physiological roles of GDNF in humans. Northern blot analysis demonstrated high expression of GDNF mRNA in human skeletal muscle when compared to that of mouse. Intense GDNF immunoreactivity was observed in the vicinity of plasma membranes of skeletal muscle, particularly at neuromuscular junctions. GDNF immunoreactivity was also observed within the axons and surrounding Schwann cells of peripheral nerves. However, RT-PCR detected expression of GDNF mRNA only in skeletal muscle, and not within the anterior horn cells of human spinal cord. These results suggest that GDNF is produced by skeletal muscle and taken up at the nerve terminals for retrograde transport by axons. Thus, GDNF in human skeletal muscle may be involved in promoting motor neuron survival as a target-derived neurotrophic factor.

Adhesion to and invasion of human colon carcinoma Caco-2 cells by Aeromonas strains.

The enteropathogenicity of Aeromonas strains that showed mannose-resistant adhesion to INT407 cells was evaluated by infecting Caco-2 cells and observing them by light and electronmicroscopy. Five of six strains adhered in large numbers to Caco-2 cells in the presence of mannose and caused cytopathic effects. Two strains of Aeromonas spp. seemed to invade Caco-2 cells, as membrane-bound bacteria were seen within the cytoplasm of these cells; however, staining by acridine orange-crystal violet appeared to show intracellular fluorescent bacteria in three strains. Fimbriae did not appear to play an important role in adhesion because fimbrial structures were not seen by transmission electronmicroscopy. Adhesion of four strains was inhibited by the addition of L-fucose. The strains were negative in the fluorescence actin staining test, which in enteropathogenic Escherichia coli strains correlates with the ability to attach and efface intestinal microvilli. The DNA of the Aeromonas strains did not hybridise with the E. coli eae and ipaB probes, associated with attaching and effacing ability and invasion, respectively. These results give support to the enteropathogenicity of adhesive strains of Aeromonas spp., although the mechanisms of adhesion, and possibly invasion, remain to be elucidated.

Inactivation of herpes viruses by high hydrostatic pressure.

The effects of high hydrostatic pressure on herpes simplex virus type 1 (HSV-1) and human cytomegalovirus (HCMV) were examined. Pressure at more than 300 MPa for 10 min at 25 degrees C inactivated these virions and drastically inhibited their infection to cultured cells, and at greater than 400 MPa, reduced infective titers of HSV-1 and HCMV by more than 7 and 4 logs, respectively. Electron microscopic examination illustrated coincidentally that high pressure at 300 MPa damaged the virus envelope and prevented the virus particles from binding to the cells. The findings suggest that treatment at high hydrostatic pressure is promising as a means of inactivating HSV-1, HCMV and other enveloped viruses.

Expression of human GFR alpha-1 (GDNF receptor) at the neuromuscular junction and myelinated nerves.

Motor neurons have been known to require a wide variety of neurotrophic factors for their survival. As one of the target-derived trophic factors, glial cell line-derived neurotrophic factor (GDNF) has been shown to exert its effects on motor neurons via a receptor complex including GDNF receptor alpha 1 (GFR alpha-1). Immunoreactivity of GFR alpha-1 was observed at myelinated peripheral nerves and neuromuscular junction (NMJ) of human skeletal muscles. Reverse transcriptase polymerase chain reaction (RT-PCR) analyses showed that mRNA of GFR alpha-1 existed in the ventral horn of human spinal cord, but not in the skeletal muscles. The results suggested that GFR alpha-1 might play a key role for uptake and internalization of GDNF at the human NMJ.

Up-regulation of glial cell line-derived neurotrophic factor (GDNF) expression in regenerating muscle fibers in neuromuscular diseases.

Glial cell line-derived neurotrophic factor (GDNF) has been shown to exert a target-derived trophic factor for motor neurons. Immunohistochemical analyses revealed that expression of GDNF in regeneration muscle fibers was up-regulated in polymyositis (PM) and Duchenne type muscular dystrophy (DMD). Reverse transcriptase polymerase chain reaction (RT-PCR) analyses showed that the full length GDNF was up-regulated in PM and DMD muscle; normal muscle exhibited mostly truncated GDNF. The results indicate that the GDNF expression is regulated in regeneration of human skeletal muscle.

Influence of blood proteins on biomedical analysis. VIII. Attempts at purification of gliclazide-degrading factor in human serum.

Gliclazide(hypoglycemic drug having sulfonylurea structure)-degrading activity was found in fraction M(macroglobulin, Fr. M) obtained from pooled human serum by gel filtration using a Sephadex G-150 column. The main degrading activity was in the fraction eluted from the Fr. M-subjected DEAE-cellulose column with 0.4 M phosphate buffer (pH 5.2), and the gliclazide-degrading protein localized around alpha 2 to beta-globulin on an electrophoretic pattern using a cellulose acetate membrane. The degrading activity was enhanced about two-fold by lyophilizing Fr. M solution containing a higher sodium phosphate (Na2HPO4-NaH2PO4), over 0.27 M. This indicates that the appearance and enhancement of the degrading activity required the combination of the lyophilization of the sample solution and a certain initial concentration of sodium phosphate prior to lyophilization.

Listeria monocytogenes isolated from cold-smoked fish products in Osaka City, Japan.

Listeria monocytogenes contamination of ready-to-eat seafood products commercially available in Osaka was examined between 1999 and 2000. L. monocytogenes was isolated from 12 (13%) of the 95 products tested. All positive samples were from cold-smoked fish with 9 being obtained during the summer. Thirteen isolates of L. monocytogenes were typed by pulsed-field gel electrophoresis (PFGE) and polymerase chain reaction (PCR)-based typing methods. Isolates of the same serotype originating from the same manufacturer gave similar DNA profiles, irrespective of the type of sample or date of isolation. The finding suggest that persistent strains in each manufacturing facility proliferate during the summer and contaminate products during manufacturing processes.

Pharmacokinetics of gliclazide in healthy and diabetic subjects.

The pharmacokinetics of total and free gliclazide, 1-(3-azabicyclo[3,3,0]oct-3-yl)-3-(p-tolylsulfonyl)urea, a potential hypoglycemic drug, was studied in healthy (n = 12) and diabetic (n = 12) subjects. The serum level of gliclazide was determined by a high-performance liquid chromatographic method (HPLC). The free fraction of gliclazide was obtained from serum by an ultrafiltration technique using a collodion membrane. The mean adsorption of gliclazide to the membrane was approximately 50% when the membrane was used more than twice. Therefore, the gliclazide level in the filtrate was corrected by doubling the apparent value. The ratio of gliclazide-protein binding remained constant at approximately 92% in serum after administration to healthy and diabetic subjects. The mean pharmacokinetic parameters of elimination rate (ke), time to reach the peak level (tmax), elimination half-life (t 1/2), and volume of distribution (Vd) were 0.07 h-1, 2.8 h, 12.3 h, and 17.4 L, respectively. The parameters did not differ significantly between healthy and diabetic subjects or between single and successive administrations; moreover, they did not differ between the free and total drug level. Although there were intersubject variations, the therapeutic effects of oral administration of gliclazide on serum glucose and insulin levels were found in four diabetic patients. The results of this study show that the pharmacokinetics of the total gliclazide level reflect those of the free gliclazide in serum.

Phage typing and DNA-based comparison of strains of enterohemorrhagic Escherichia coli O157 from apparently sporadic infections in Osaka City, Japan, 1996.

A marked increase in sporadic cases of enteritis due to enterohemorrhagic Escherichia coli serogroup O157 occurred in Osaka City, Japan, during 1996. To elucidate why the number of cases had increased, the isolates were classified using phage typing, random amplified polymorphic DNA analysis, and pulsed-field gel electrophoresis (PFGE). Fifty-seven percent of the isolates (105/184) belonged to the same phage type (PT-32) and gave the same PFGE pattern; the clone had been isolated during a 3-week period, with a peak on July 15. It was concluded that the majority of the cases identified in July 1996 formed an outbreak, although epidemiological links to a possible common source were not established. The possibility that this outbreak was part of a huge regional outbreak including children at primary schools in Sakai City was discussed.

An electromyographic analysis of the hip abductors during load carriage: implications for hip joint protection.

Decreasing the relative force demands on the hip abductor muscles may reduce hip joint forces. The purpose of this study was to use surface electromyography (EMG) to determine the relative demand on the hip abductor muscles as subjects walked and carried single hand-held loads of multiple weights. Thirty healthy, college-aged subjects carried single hand-held loads by their side. The loads weighed between 3 and 30% of body weight. Loads were carried in a position either ipsilateral or contralateral to a given hip side. Normalized EMG (%EMG) was collected during the middle stance phase of walking. The amount of %EMG remained statistically equal to or less than the no-load EMG baseline for all ipsilateral-held loads and greater than the no-load EMG for all contralateral-held loads above 3% body weight. Load positions and weights that generated %EMG levels less than or equal to the no-load baseline most likely offer a degree of hip joint protection for persons with hip disability.

Prevalence of enteric bacteria that inhibit growth of enterohaemorrhagic Escherichia coli O157 in humans.

Enterohaemorrhagic Escherichia coli O157 (O157) is infectious to humans, particularly children, at very low doses and causes not only haemorrhagic colitis but also other serious symptoms. To investigate an association between intestinal bacterial flora and resistance to such infections, we screened faecal samples for the presence of enteric bacteria that are able to suppress the growth of O157. Samples from 303 individuals, 35 children (aged < or =6 years) and 268 adults (aged 20-59 years), were examined. Colonies with different appearances on sorbitol MacConkey agar medium were screened for the production of bacteriocins inhibitory for O157 in an overlay agar plate assay. O157-inhibiting strains were isolated from 52 individuals. The prevalence of these bacteria tended to rise with age, and was significantly higher among 40- to 59-year-old adults (23/101, 22.8%) than among children (3/35, 8.6%; P<0.05). To test the hypothesis that these bacteriocin-producing strains contribute to resistance against O157 in human adults, we examined faecal samples of 25 healthy O157 carriers. Inhibitory bacteria were more prevalent among the latter (9/25, 36.0%) than among age-matched subjects who did not carry O157 (49/268, 18.3%). It appears, therefore, that inhibitory bacteria in the human gut may play a role in inhibiting propagation of O157 and/or suppressing expression of virulence factors by this pathogen.

Cellular differentiation in the process of generation of the eukaryotic cell.

Primitive atmosphere of the earth did not contain oxygen gas (O2) when the proto-cells were generated successfully as the result of chemical evolution and then evolved. Therefore, they first had acquired anaerobic energy metabolism, fermentation. The cellular metabolisms have often been formed by reorganizing to combine or recombinate between pre-existing metabolisms and newly born bioreactions. Photosynthetic metabolism in eukaryotic chloroplast consists of an electron-transfer photosystem and a fermentative reductive pentose phosphate cycle. On the other hand, O2-respiration of eukaryotic mitochondrion is made of Embden-Meyerhof (EM) pathway and tricarboxylic acid cycle, which originate from a connection of fermentative metabolisms, and an electron-transfer respiratory chain, which has been derived from the photosystem. These metabolisms already are completed in some evolved prokaryotes, for example the cyanobacterium Chlorogloea fritschii and aerobic photosynthetic bacteria Rhodospirillum rubrum and Erythrobacter sp. Therefore, it can be reasonably presumed that the eukaryotic chloroplast and mitochondrion have once been formed as the result of metabolic (and genetic) differentiations in most evolved cyanobacterium. Symbiotic theory has explained the origin of eukaryotic cell as that in which the mitochondrion and chloroplast have been derived from endosymbionts of aerobic bacterium and cyanobacterium, respectively, and has mentioned as one of the most potent supportive evidences that amino acid sequences of the photosynthetic and O2 -respiratory enzymes show similarities to corresponding prokaryotic enzymes. However, as will be shown in this discussion, many examples have shown currently that prokaryotic sequences of informative molecules are conserved well not only in those of the mitochondrial and chloroplast molecules but also in the nuclear molecules. In fact, the similarities in sequence of informative molecules are preserved well among the organisms not only in phylogenetically close relationships but also under highly selective pressure, that is under a physiological constraint for the species in their habitats. Therefore, the similarities in amino acid sequences of proteins between the prokaryotes and the organelles are not necessarily direct evidence for their phylogenetical closeness: it gives still less evidence for a symbiotic relationship between the prokaryotes and the organelles. The metabolic compartmentalization of the membranes is an important tendency in cellular evolution to guarantee high specificity and rate of the metabolisms. It is suggested from the data that the intracellular membranes are not static but undergo dynamic turnover. Furthermore, these facts strongly support the Membrane Evolution Theory which was proposed by one of the authors in 1975.

Three different forms of tubular structures associated with the replication of bovine rotavirus in a tissue culture system. Brief report.

We demonstrated by electron microscopy that three different forms of tubular structures are present both within infected cells and in cultured fluid of bovine rotavirus in a tissue culture system.