Epicatechin downregulates adipose tissue CCL19 expression and thereby ameliorates diet-induced obesity and insulin resistance.

BACKGROUND AND AIMS: Epicatechin (EC) intake has been suggested to be beneficial for the prevention of cardiovascular disorders, and it is well known that adipose tissue inflammation is one of the major risk factors for coronary heart diseases. The purpose of the present study was to determine the in vitro and in vivo effects of EC on adipose tissue inflammation and obesity. METHODS AND RESULTS: DNA microarray analysis was performed to evaluate the effects of EC on gene expression in adipocytes co-cultured with bacterial endotoxin-stimulated macrophages. To determine the in vivo effects of the catechin, C57BL/6 mice were fed either a high-fat diet (HFD) or HFD combined with EC, and metabolic changes were observed EC suppressed the expression of many inflammatory genes in the adipocytes co-cultured with endotoxin-stimulated macrophages. Specifically, EC markedly suppressed chemokine (CC motif) ligand 19 (CCL19) expression. The target cell of EC appeared to macrophages. The in vivo study indicated that mice fed the EC-supplemented HFD were protected from diet-induced obesity and insulin resistance. Accordingly, the expression levels of genes associated with inflammation in adipose tissue and in the liver were downregulated in this group of mice. CONCLUSIONS: EC exerts beneficial effects for the prevention of adipose tissue inflammation and insulin resistance. Since we previously reported that mice deficient in the CCL19 receptor were protected from diet-induced obesity and insulin resistance, it can be concluded that the beneficial effects of EC could be mediated, at least in part, by marked suppression of CCL19 expression.

Gate-Tunable Spin-Charge Conversion and the Role of Spin-Orbit Interaction in Graphene.

The small spin-orbit interaction of carbon atoms in graphene promises a long spin diffusion length and the potential to create a spin field-effect transistor. However, for this reason, graphene was largely overlooked as a possible spin-charge conversion material. We report electric gate tuning of the spin-charge conversion voltage signal in single-layer graphene. Using spin pumping from an yttrium iron garnet ferrimagnetic insulator and ionic liquid top gate, we determined that the inverse spin Hall effect is the dominant spin-charge conversion mechanism in single-layer graphene. From the gate dependence of the electromotive force we showed the dominance of the intrinsic over Rashba spin-orbit interaction, a long-standing question in graphene research.

Experimental Demonstration of Room-Temperature Spin Transport in n-Type Germanium Epilayers.

We report an experimental demonstration of room-temperature spin transport in n-type Ge epilayers grown on a Si(001) substrate. By utilizing spin pumping under ferromagnetic resonance, which inherently endows a spin battery function for semiconductors connected with a ferromagnet, a pure spin current is generated in the n-Ge at room temperature. The pure spin current is detected by using the inverse spin-Hall effect of either a Pt or Pd electrode on n-Ge. From a theoretical model that includes a geometrical contribution, the spin diffusion length in n-Ge at room temperature is estimated to be 660 nm. Moreover, the spin relaxation time decreases with increasing temperature, in agreement with a recently proposed theory of donor-driven spin relaxation in multivalley semiconductors.

Endothelial Insulin Resistance Exacerbates Experimental Periodontitis.

Epidemiological studies suggest that the severity of periodontitis is higher in people with diabetes than in healthy individuals. Insulin resistance might play a crucial role in the pathogenesis of multiple diabetic complications and is reportedly induced in the gingiva of rodents with type 2 diabetes; however, the molecular mechanisms underlying the pathogenesis of diabetes-related periodontitis remain unclear. Therefore, we aimed to investigate whether endothelial insulin resistance in the gingiva may contribute to the pathogenesis of periodontitis as well as elucidate its underlying molecular mechanisms. We demonstrated that insulin treatment downregulated lipopolysaccharide (LPS)-induced or tumor necrosis factor α (TNFα)-induced VCAM1 expression in endothelial cells (ECs) via the PI3K/Akt activating pathway, resulting in reduced cellular adhesion between ECs and leukocytes. Hyperglycemia-induced selective insulin resistance in ECs diminished the effect of insulin on LPS- or TNFα-stimulated VCAM1 expression. Vascular endothelial cell-specific insulin receptor knockout (VEIRKO) mice exhibited selective inhibition of the PI3K/Akt pathway in the gingiva and advanced experimental periodontitis-induced alveolar bone loss via upregulation of Vcam1, Tnfα, Mcp-1, Rankl, and neutrophil migration into the gingiva compared with that in the wild-type (WT) mice despite being free from diabetes. We also observed that insulin-mediated activation of FoxO1, a downstream target of Akt, was suppressed in the gingiva of VEIRKO and high-fat diet (HFD)-fed mice, hyperglycemia-treated ECs, and primary ECs from VEIRKO. Further analysis using ECs transfected with intact and mutated FoxO1, with mutations at 3 insulin-mediated phosphorylation sites (T24A, S256D, S316A), suggested that insulin-mediated regulation of VCAM1 expression and cellular adhesion of ECs with leukocytes was attenuated by mutated FoxO1 overexpression. These results suggest that insulin resistance in ECs may contribute to the progression of periodontitis via dysregulated VCAM1 expression and cellular adhesion with leukocytes, resulting from reduced activation of the PI3K/Akt/FoxO1 axis.

Comparison of haemolytic activity between Fusobacterium necrophorum subsp. necrophorum and Fusobacterium necrophorum subsp. funduliforme in vitro and in vivo.

Haemolytic activity of two subspecies of Fusobacterium necrophorum was compared in vitro and in vivo. F. necrophorum subsp. necrophorum (Fnn) showed a stronger activity than F. necrophorum subsp. funduliforme (Fnf) in vitro. Haemolytic activity of Fnn and Fnf was 57.97%+/-1.90 and 17.33%+/-1.44, respectively, compared to complete haemolysis by distilled water. In the mice injected with Fnn, haemolysin was detected in the liver at a titre of from 1 : 16 to 1 : 128, and Fnn was recovered from all mice at a viable bacterial count of 10(5) to 10(6) cells per gram liver tissue. In the mice injected with Fnf, haemolysin titre was <1 : 2 to 1 : 32. No liver abscess was formed. The viable count of recovered bacteria was 10(3) to 10(5) cells per gram, except for two mice in which no Fnf was detected. The results suggest that haemolysin might be a virulence factor in this species.

Interactions between Fusobacterium necrophorum hemolysin, erythrocytes and erythrocyte membranes.

The interactions between the hemolysin of Fusobacterium necrophorum subsp. necrophorum, erythrocytes and erythrocyte membranes were studied as an attempt to determine the initial characteristics leading to hemolysis. The spectrum of erythrocyte sensitivity indicated that horse, dog and mouse erythrocytes were highly sensitive whereas those of cattle, sheep, goat and chicken were insensitive to the hemolysin. Binding of hemolysin to horse and dog erythrocytes or their ghosts was more pronounced than to those of cattle and sheep as detected by a decrease of hemolytic activity from hemolysin preparations. The kinetics of hemolysis revealed that lysis is preceded by a prelytic phase characterized by binding of hemolysin to erythrocytes. Treatment of horse erythrocytes with hemolysin at various temperatures prior to incubation at 37 degrees C also revealed that this binding prelytic phase is temperature independent. This was followed by a temperature dependent lytic stage since erythrocytes pretreated with hemolysin and incubated at 4 degrees C showed no hemolysis. An inverse relation was found between erythrocyte concentration and hemolytic activity suggesting a multiple-hit mechanism of hemolysis.

The erythrocyte receptor for Fusobacterium necrophorum hemolysin: phosphatidylcholine as a possible candidate.

An attempt was made to determine the receptor for the hemolysin of Fusobacterium necrophorum using horse erythrocyte or its membranes as target. The spectrum of erythrocyte sensitivity has indicated that horse, dog and mouse erythrocytes are highly sensitive whereas cattle, sheep, goat and chicken red blood cells are insensitive to this hemolysin. A high correlation between sensitivity and phosphatidylcholine content of the erythrocyte membranes was noted. Binding of hemolysin to horse erythrocyte membranes was reduced significantly by prior treatment of membranes with phospholipase A2 but not with phospholipase C. Pretreatment of erythrocyte membranes with pronase, proteinase K, trypsin or neuraminidase did not alter binding of hemolysin to the membranes, suggesting that protein or sialyl residues are not involved as receptors. Gas liquid chromatography analysis showed that the fatty acid profile from hydrolysis of bovine liver phosphatidylcholine by hemolysin and phospholipase A2 were similar. In conclusion, this report presents evidence that phosphatidylcholine may be acting as a possible receptor for the hemolysin of F. necrophorum.

Studies on the factors affecting the hemolytic activity of Fusobacterium necrophorum.

The in vitro activity of the hemolysin of Fusobacterium necrophorum was determined using the hemolysis of horse erythrocytes as an assay. The effects of medium composition and pH on hemolysin production were investigated. Calf serum and casitone stimulated a comparatively higher hemolytic activity in F. necrophorum subsp. necrophorum and F. necrophorum subsp. funduliforme, respectively. However, sugars, such as glucose, galactose and fructose were inhibitors of hemolytic activity. The spectrum of erythrocyte sensitivity to the hemolysin indicated that horse and quail erythrocytes were more sensitive to the hemolysin of both F. necrophorum subsp. necrophorum and subsp. funduliforme, than were cat, dog, rabbit, pigeon and human erythrocytes. Cat erythrocytes were however insensitive to the hemolysin of subsp. funduliforme. Cattle, sheep and chicken erythrocytes were insensitive to the hemolysin of the two subspecies. Medium pH near neutral were more effective in enhancing hemolytic activity, and hemolytic activity was positively correlated with growth. In general, F. necrophorum subsp. necrophorum was more hemolytic than subsp. funduliforme.

Aggregation of bovine platelets by Fusobacterium necrophorum.

Washed cell suspensions of biovar A strains of Fusobacterium necrophorum aggregated cattle platelets, but similar suspensions of biovar B strains did not. Platelets were also aggregated by heat-treated bacterial cells or the lipopolysaccharide of biovar A. No platelet aggregation occurred in the presence of the cell-free culture supernatant of biovar A and of all samples prepared from biovar B. Scanning electron microscopy revealed that aggregated platelets were not damaged. Platelet aggregation was inhibited by EDTA, aspirin and quinacrine, and lag time was retarded by these inhibitors, indicating the reaction was a Ca(2+)-dependent, cyclo-oxygenase sensitive event. Platelet aggregation may be a virulence marker, probably mediated by the lipopolysaccharide of F. necrophorum biovar A strains.

The effects of physical and chemical agents on the secretion and stability of a Fusobacterium necrophorum hemolysin.

The effect of various agents as enhancers or inhibitors of hemolysin secretion by Fusobacterium necrophorum was investigated. The hemolysin secreted in phosphate buffered saline (PBS) alone was inactivated shortly after secretion. Tween-80 or albumin preserved the hemolytic activity in PBS in which cultures of F. necrophorum had been suspended. Hemoglobin was found to enhance hemolysin secretion. However, higher concentrations diminished secretion. Chloramphenicol, an inhibitor of protein synthesis, exhibited no effect on the hemolytic activity. However, the addition of sodium azide, an energy metabolic inhibitor, significantly reduced the hemolytic activity. Lower temperatures and pH above 9 and below 6 all yielded a low hemolytic activity. Cells suspended in Tween-80 prior to sonication yielded a substantial amount of extracellular hemolytic activity with low intracellular activity detected. However, cells suspended in PBS alone yielded a low extracellular activity but rather a high intracellular activity. The same spectrum of red blood cells of different species were found to be sensitive to both the extracellular and intracellular hemolysins.

Establishment of Bcgr congenic mice and their susceptibility/resistance to mycobacterial infection.

Bcg congenic mice were developed by using C57BL/6 and DBA/2 strains of mice as progenitors. They were obtained by introgressively backcrossing the Bcgr marker of DBA/2 onto C57BL/6. After twenty successive backcrossings, the heterozygous resistant mice were mated with each other to obtain homozygous mice as the Bcgr congenic mice. The results of immunogenic and genetic markers coupled with those of an mixed lymphocyte reaction, all confirmed that the newly developed mice were highly congenic. These congenic mice were found to be resistant to in vivo infections by Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium bovis BCG.

Stability and stabilization of Fusobacterium necrophorum hemolysin.

The stability and stabilization of the hemolytic activity of Fusobacterium necrophorum subsp. necrophorum and Fusobacterium necrophorum subsp. funduliforme were monitored over a period of four weeks using culture supernatants. The hemolytic activity was completely lost after one week at room temperature and 37 degrees C. After a two-week storage at 4 degrees C and -80 degrees C only trace activity was detected with -80 degrees C being the better of the two conditions. The addition of cysteine monohydrochloride, bovine serum albumin or Tween 80 as stabilizers, however, led to the detection of a considerable amount of the hemolytic activity in the sample stored at 4 degrees C and - 80 degrees C throughout the period investigated. The hemolytic activity appeared to be more stable in the presence of Tween 80 at -80 degrees C. Cysteine monohydrochloride was found to crystallize at - 80 degrees C and was therefore ineffective as a stabilizer at this temperature. Hemoglobin was also ineffective as a stabilizer.

The effect of Bcg gene on antigen presentation of spleen adherent cells and peritoneal macrophages from Mycobacterium bovis BCG-infected Bcg(s) and Bcg(r) mice.

To determine the role of the Bcg gene in the resistance and susceptibility of BCG-infected C57BL/6 (Bcg(s)) and its Bcg(r) congenic mice, the antigen presenting ability of spleen adherent cells and peritoneal macrophages, delayed type hypersensitivity (DTH) and lymphocyte blastogenic responses were investigated. The results obtained indicate that the DTH and lymphocyte blastogenic responses in Bcg(r) congenic mice were higher than in the Bcg(s) mice. Stimulation of spleen adherent cells with live Mycobacterium bovis BCG or PPD-BCG resulted in a higher antigen presenting ability in Bcg(r) than in Bcg(s) mice. However, comparatively low responses were associated with M. avium stimulation, with those in Bcg(r) being higher than in Bcg(s). I-A expression was also comparatively higher in Bcg(r) than in Bcg(s) mice. This study demonstrates that the Bcg gene seems to exhibit some effect on the antigen presenting ability of macrophages in immune responses of the congenic mice.

Differentiation of Fusobacterium necrophorum subspecies from bovine pathological lesions by RAPD-PCR.

Nineteen strains from bovine abscesses identified as Fusobacterium necrophorum by the VPI method were examined by other methods. The API 20A test kit characterized all 19 strains as F. necrophorum. Seven of the strains had haemagglutinating activity and were classified as F. necrophorum subspecies necrophorum, and the remaining, 12 nonhaemagglutinating strains, were classified as F. necrophorum subspecies funduliforme. We used RAPD-PCR with a 10-mer oligonucleotide primer, W1L-2, to confirm this differentiation of the two subspecies. These results suggest that random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) with a suitable primer can be used as a new tool for the differentiation of F. necrophorum subspecies isolated from bovine pathological lesions.

Lymphoproliferative responses in pigs infected with Mycobacterium avium.

Naturally infected cases of swine mycobacteriosis were divided into two groups, localized infection (LI) and disseminated infection (DI). Lymphoproliferative response (LPR) was then examined to estimate their immunological states. Both control and LI groups showed strong response to Concanavalin A (Con A) and phytohemagglutinin (PHA) in the LPR, and lymphocytes recovered from the LI responded well to purified protein derived from M. avium (PPD). On the other hand, the DI group showed weak response to both Con A and PHA, despite their strong response to PPD stimulation. These data suggest that the low LPR to Con A and PHA observed in the DI groups was probably not due to the general unresponsiveness of T-cells.

Isolation of Pasteurella multocida during an outbreak of infectious septicemia in Japanese quail (Coturnix coturnix japonica).

In May 1994, about fifty Japanese quails out of ninety being bred for experimental purposes at Miyazaki University died of acute septicemia within a few days. At autopsy, there were no gross pathological lesions, however, severe bacteremia was observed in all cases. Bacterial examination revealed the presence of Pasteurella multocida in blood and several organs in pure culture and they were of Carter's capsular type A, Heddleston's type 3-4 and Namioka's type O-8-9. The LD50 of bacteria in quails and mice were 4.3 x 10(4) cfu and 3.9 x 10(2) cfu, respectively. All of the three chickens experimentally infected with 4 x 10(4) of the isolate died within 20 hr after the infection and several bacteria were recovered from their blood and organs. This, to our knowledge, is the first report on an outbreak of fowl cholera in Japanese quails in Japan.

Platelet abnormalities in a dog suffering from gangrenous mastitis by Staphylococcus aureus infection.

Severe gangrenous mastitis due to Staphylococcus aureus infection was diagnosed in a 7 year-old intact female beagle which was presented with swelling of mammary glands after dystocia. Leukocytosis (25,200-48,600/microliters), decreased platelets (107,000-179,000/microliters), and abnormal platelet pattern continued during the critical condition. Consistent with platelet pattern, large platelets were observed in the blood smear. The number of leukocytes and platelets rapidly returned to normal during treatment, and the platelet pattern was also restored. The number and pattern of platelet may provide a clue for the evaluation of the clinical condition and/or severity of the lesions in the dog with mastitis.

[Detection of Emericella nidulans from bedding materials in horse breeding environment and its significance as a causative agent of guttural pouch mycosis in horses].

Sixty-six new and used samples of horse bedding materials: 60 rice straws, 2 wheat straws, 2 timothy hays and 2 wood chips, were collected from horse breeding stables of 33 farms in Japan and examined for the presence of Emericella nidulans (E. nidulans; anam. Aspergillus nidulans). The incidence of E. nidulans in the bedding materials was 75.8% and there was no significant difference in detection of the fungus between the new and used materials (25 out of the 33 samples, respectively). The growth of E. nidulans isolated in sterilized rice straw culture was accelerated by the addition of water up to about Aw 0.94, which as determined to be the most favorable moisture content. The addition of 0.3% urea solution onto the sterilized rice straw culture also appeared to very effectively enhance its conidial and ascocarp formation. A significant influence of urea on conidial and ascocarp formation of E. nidulans isolates was confirmed by their cultures on a synthetic medium which had urea as the sole nitrogen. These results suggest that severe contamination of E. nidulans on new bedding materials can be hazardous and its proliferation can readily occur at the stable due to the enhancing effect of urine. This analysis is meaningful to elucidate a reservoir of E. nidulans as the causative agent of guttural pouch mycosis in horses.