Targeted mutation in the Fas gene causes hyperplasia in peripheral lymphoid organs and liver.

Fas, a type I membrane protein that transduces an apoptotic signal, is expressed in lymphocytes as well as in various tissues such as the liver, lung and heart. The mouse lymphoproliferation (lpr) mutation is a leaky mutation in Fas. By means of gene targeting, we generated a mouse strain which is completely deficient in Fas. In addition to the massive production of lymphocytes, the Fas-null mice showed substantial liver hyperplasia, which was accompanied by the enlargement of nuclei in hepatocytes. The Fas system seems to play a role in the apoptotic process to maintain homeostasis of the liver as well as the peripheral lymphoid organs.

Effect of exercise training on the density of endothelial cells in the white adipose tissue of rats.

We examined the effects of a 9-week exercise training (TR) in Wistar male rats, beginning at 4 weeks of age, on the density of endothelial cells (ECs) in epididymal white adipose tissue (WAT) and the mRNA expression of angiogenic factors in adipose tissue stromal vascular fraction (SVF) cells. The number of ECs and mRNA expressions were assessed by lectin staining and real-time reverse transcriptase-polymerase chain reaction, respectively. Compared with control (CR) rats, TR rats gained weight more slowly and had significantly lower final weight of WAT due to the reduction in the size and the number of adipocytes. TR significantly increased the number of ECs per square millimeter and per adipocyte (1.37- and 1.23-fold, respectively) in WAT. This is probably because the number of adipocytes is fewer while the number of ECs is constant in the WAT of TR rats, because the regression line of TR rats for adipocyte number-dependent EC number was shifted toward the left without significant differences in the slopes between groups. TR also induced the upregulation of mRNA expression of vascular endothelial growth factor (Vegf)-A and Vegf-receptor-2 in SVF cells, thereby retaining a constant number of ECs in the WAT.

Selective apoptosis of CD4+CD8+ thymocytes by the anti-Fas antibody.

Fas is a cell surface protein that mediates apoptosis. A mouse mutant, lpr (lymphoproliferation), has a mutation in the Fas gene. In this report, we studied the expression and function of Fas in various subpopulations of mouse thymocytes. Abundant expression of Fas was detected on CD4+CD8+ double positive as well as CD4+ or CD8+ single positive thymocytes in wild-type mice. Little or low levels of Fas were expressed in CD4-CD8- double negative thymocytes except for the CD4-CD8-CD3+ phenotype, which expresses Fas as abundantly as double positive or single positive subsets. On the other hand, no Fas expression was detected in any population of thymocytes from lpr mice. When the wild-type thymocytes were treated with the agonistic anti-Fas antibody, double positive cells from the wild-type mice were selectively killed by apoptosis, whereas, the single positive cells were resistant to its cytolytic activity despite their abundant expression of Fas. Unlike the apoptosis of thymocytes induced by glucocorticoid or T cell activator, the Fas-induced apoptosis of thymocytes was enhanced by metabolic inhibitors such as cycloheximide. Furthermore, intraperitoneal administration of the anti-Fas antibody into mice caused rapid apoptosis of thymocytes in vivo.

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.

Solubilization and characterization of the acceptor for Clostridium botulinum type B neurotoxin from rat brain synaptic membranes.

The acceptor for Clostridium botulinum type B neurotoxin was solubilized from rat brain synaptic membrane with nonionic detergent, nonanoyl-N-methylglucamide (MEGA-9). The solubilized acceptor was assayed for the binding activity by precipitating the acceptor with acetone in the presence of phosphatidylcholine. 125Ilabeled neurotoxin specifically bound to the lipid vesicles having incorporated the acceptor together with gangliosides. The lipid vesicles having incorporated either the acceptor or gangliosides alone showed extremely low binding activity. The treatment of the solubilized acceptor with lysyl endopeptidase and glycopeptidase F but not with sialidase resulted in decreased toxin binding, indicating that the putative acceptor is a glycoprotein accompanying an N-linked carbohydrate moiety. The observations suggest also that a protein acceptor/ganglioside complex may be required to form the functional toxin receptor.

Properties of a protease-sensitive acceptor component in mouse brain synaptosomes for Clostridium botulinum type B neurotoxin.

To characterize an acceptor for Clostridium botulinum type B neurotoxin, its binding kinetics were examined with mouse brain synaptosomes treated with various enzymes. The amount of 125I-labelled neurotoxin bound to synaptosomes decreased upon treatment with lysyl endopeptidase, neuraminidase, or phospholipase C. The binding of the neurotoxin was partially recovered by incubation of neuraminidase-treated synaptosomes with ganglioside GT1b or GD1a. Gangliosides incorporated into untreated, lysyl endopeptidase-treated, and phospholipase C-treated synaptosomes had no effect on the binding of the neurotoxin. These results may suggest that type B neurotoxin binds to gangliosides in cooperation with a certain protease-sensitive substance on the neural membranes.

Heat and acid sensitivity of motile Aeromonas: a comparison with other food-poisoning bacteria.

The present study was undertaken to compare the heat and acid sensitivity of aeromonads with those of other food-poisoning bacteria. It became obvious that aeromonads were more sensitive to heat than Escherichia coli O157:H7, Staphylococcus aureus, and Salmonella typhimurium. Aeromonads were killed in peptone water within 2 min at 55 degrees C, while the other bacteria survived heating at 55 degrees C for more than 15 min. Aeromonas cells were also less resistant to heat in hamburger steaks. These findings suggest that Aeromonas infection can easily be prevented by heat treatment, although correct handling of food is required to avoid recontamination since aeromonads are very common in various kinds of food. E. coli, S. aureus and S. typhimurium cells survived in buffer at pH 3.2 and in foods seasoned with vinegar. By contrast, Aeromonas cells were found to be highly sensitive to acid. However, the resistance of Aeromonas to acid may be sufficient to allow it to infect the gastrointestinal tract since Vibrio parahaemolyticus, which causes numerous outbreaks of food-poisoning every year in Japan, was susceptible to acid to the same extent as Aeromonas.

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.

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.

Hypertrophic cardiomyopathy in two kittens.

Two 2-month-old kittens presented with a loud cardiac murmur. One cat showed severe signs of heart failure such as respiratory effort and exercise intolerance. Echocardiography revealed left ventricular concentric hypertrophy and severe left ventricular outflow obstruction. They died at 5 and 12 months of age, respectively. Necropsy and histopathology confirmed hypertrophic cardiomyopathy.

[Anti-GD1b IgG antibody-related Guillain-Barré syndrome initially mimicking brainstem infarction].

We described a 58-year-old woman with Guillain-Barré syndrome, who initially showed rapid progression of brainstem infarction-like signs. She developed superficial sensory disturbance on the left side, dysarthria, and left-predominant limb weakness within a few hours. She showed bilateral extensor plantar responses and head CT scan detected no abnormality. It was difficult to be distinguished from brainstem infarction until symmetrical limb weakness and generalized areflexia appeared. Serum anti-GD1b IgG antibody with cross-reactivity with GM1b was detected. Cerebrospinal fluid examination revealed albuminocytologic dissociation on day 5. After 5 sessions of immunoadsorption therapy, her symptoms gradually lessened. Anti-GD1b antibody has been detected in patients with sensory ataxic neuropathy. Our patient, however, was characterized with early involvement of brainstem with ataxia of cerebellar type. Our case suggests that anti-GD1b antibody-associated neuropathy has a broad spectrum of clinical features, which are related to cross-reactivity of this antibody.

[A case presenting with Raeder's syndrome-like symptoms due to vertebral artery aneurysm].

A 50-year-old man complained of headache around his left orbit, left frontal pain and paresthesia associated with left incomplete Horner syndrome. MRI demonstrated a mass at the level of medulla oblongata. Left vertebral angiogram revealed an aneurysm of left vertebral artery. Following the removal of the aneurysm, these Raeder's syndrome-like symptoms improved. Therefore, they were probably caused by a compression of the spinal tract of the trigeminal nerve and the central sympathetic tract by the aneurysm. This is the first report of Reader's syndrome-like symptoms caused by vertebral artery aneurysm, thus indicating that MRI and cerebral angiogram are necessary for differential diagnosis of this syndrome.

Effects of exercise training on adipogenesis of stromal-vascular fraction cells in rat epididymal white adipose tissue.

AIM: Previous studies have shown that exercise training reduced white adipose tissue (WAT) mass compared to that in sedentary controls, and that the smaller mass contained fewer adipocytes. However, the effect of exercise training on adipogenesis is not completely clear. Therefore, we re-examined the effect of exercise training on adipocyte numbers in WAT and, if such an effect was found tested the adipogenic responses of stromal-vascular fraction (SVF) cells containing adipose tissue-derived stem cells (ADSC) in epididymal WAT from exercise-trained (TR) rats. METHODS: Wistar male rats were divided into two groups: control (C) and TR. The TR rats were subjected to exercise on a treadmill for 9 weeks. SVF cells containing ADSC were separated from epididymal WAT by centrifugation. Expression of adipocyte differentiation-related genes and adipogenesis of SVF cells were examined. RESULTS: In SVF cells of TR rats, the expression of peroxisome proliferator-activated receptor γ (PPARγ) and that of PPARγ target lipogenic genes was dramatically downregulated, whereas that of preadipocyte factor-1 gene was significantly upregulated. Lipid accumulation in SVF cells of TR rats after the induction of adipocyte differentiation was significantly suppressed in comparison with that of C rats. Moreover, increased expression of hypoxia-inducible factor-1α (HIF-1α) protein was observed in SVF cells of TR rats. Pre-treatment of YC-1, a potent HIF-1α inhibitor, in SVF cells of TR rats restored adipogenesis. CONCLUSION: These results suggest that exercise training suppresses the ability of SVF cells to differentiate into adipocytes, and that underlying mechanisms involve the upregulation of HIF-1α expression.

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.

Antigenic structure of Clostridium botulinum type B neurotoxin and its interaction with gangliosides, cerebroside, and free fatty acids.

A fragment distinct from the heavy and light chains was obtained by treatment of Clostridium botulinum type B neurotoxin with chymotrypsin. Enzyme-linked immunosorbent assay and immunoblotting analysis with monoclonal antibodies showed that the fragment consisted of the light chain and part of the heavy chain (H-1 fragment) linked together by a disulfide bond. Monoclonal antibodies reacting to the heavy chain but not to the fragment were thought to recognize the epitopes on the remaining portion (H-2 fragment) of the heavy chain, being easily digested by chymotrypsin. Thus, the antigenic structure of type B neurotoxin resembles those of type A and E neurotoxins. The chymotrypsin-induced fragment bound to cerebroside and free fatty acids but not to gangliosides. The manner of binding of type B neurotoxin to gangliosides and free fatty acids was different from those of type A and E neurotoxins. Such differences in the reactivities to lipids may be related to the finding that each neurotoxin binds to a type-specific site on the neural membrane.

[Assessment of dentofacial growth changes using quadrilateral analysis].

This study was investigated to demonstrate the normal standards of the quadrilateral analysis and thereafter to clarify the mode of growth change by assessment of skeletal, dental and soft tissue proportion by the means of quadrilateral analysis. The materials used in this study consisted of cephalograms of 30 girls with normal occlusion. Cephalograms were taken longitudinally from first year grade of elementary school to third year grade of middle high school. For the group of each year grade, mean facial diagram, mean values of conventional cephalometric analysis and those of quadrilateral analysis were made, and compared and discussed for each other. The results were as follows: 1) The mandible grows forward with the decrease of gonial angle and forward rotation of mandibular plane. 2) In quadrilateral analysis, both jaws grow proportionally to the 6 year grade of elementary school, and after then mandible grows more forward than maxilla. 3) Though occlusal plane tended to rotate counterclockwisely to the palatal plane, it fell at right angle with A'B'. 4) Upper centrals tended to incline labially to the cranial base from first year grade to 6 year grade of elementary school and after that stage it remained stable. Lower centrals varied to third year grade of elementary school, but after that stage it remained stable. 5) Distances of upper and lower incisors to A'B' showed comparatively constant increase after the full eruption of permanent incisors. 6) Distance of nose tip to A'B' increased the most in the soft tissue landmarks. Subnasale soft pogonion line was considered to be effective base line of soft tissue profile because the angle with A'B' was stable along the growth and the distances of that to upper and lower lips were stable.