Impact of frequent Bcl-2 expression on better prognosis in renal cell carcinoma patients.

Previously, we reported that Bcl-2 was frequently expressed in renal cell carcinoma (RCC) specimens, but p53 mutation was a rare event. However, it was unclear whether Bcl-2 positivity was associated with the clinicopathological characteristics and prognosis in RCC. Therefore, we investigated the expression of Bcl-2 protein and its roles in 101 RCC specimens. In addition, the proliferation index (PI), apoptotic index (AI), caspase-3 and p53 expression were examined. The immunohistochemical method was applied for Bcl-2, caspase-3 and p53 protein expression. To investigate the proliferation activity and apoptosis of tumour cells, PI and AI were calculated based on Ki-67 and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labelling (TUNEL)-positive cells, respectively. Bcl-2 expression was detected in 72 out of 101 (71.3%) specimens. Bcl-2 positivity was inversely correlated with PI (P<0.0001) and AI (P=0.0074). Furthermore, Bcl-2 positivity was significantly correlated with better survival (P=0.0014), and was associated with lower stage (P=0.0301) and grade (P=0.0020). In RCC, frequent Bcl-2 expression was correlated with favourable character without higher PI and AI. Thus, Bcl-2 expression might be applied as a novel predictor of better prognosis in RCC patients.

Dietary protein peptic hydrolysates stimulate cholecystokinin release via direct sensing by rat intestinal mucosal cells.

We previously demonstrated that a peptic hydrolysate of guanidinated casein strongly stimulates exocrine pancreatic secretion in chronic bile-pancreatic juice-diverted rats and cholecystokinin (CCK) release from dispersed rat intestinal mucosal cells. These results reveal that the chemically modified protein hydrolysate stimulates CCK secretion and increases pancreatic secretion by a luminal trypsin-independent direct action on the small intestine. In the present study, we examined the direct effect of peptic hydrolysates of naturally occurring dietary proteins, casein, soybean protein isolate (SPI), egg white, and wheat gluten on CCK release under in vitro trypsin-independent conditions. All protein hydrolysates significantly stimulated CCK release from dispersed rat intestinal mucosal cells. Among the hydrolysates treated, SPI hydrolysate was the most effective in stimulating CCK release. The potential of SPI hydrolysate to stimulate CCK release was increased by long-term peptic digestion. However, an SPI-like amino acid mixture did not effect CCK release. In conclusion, peptic hydrolysates of commonly ingested dietary proteins stimulate CCK release via trypsin-independent direct sensing by intestinal mucosal cells.

Various indigestible saccharides enhance net calcium transport from the epithelium of the small and large intestine of rats in vitro.

An Ussing chamber technique was used to determine the effects of six indigestible saccharides on net Ca absorption from the luminal side to the basolateral side of isolated preparations of rat jejunal, ileal, cecal and colonic epithelium in vitro. The concentrations of Ca in the Tris buffer solution on the serosal side and on the mucosal side were 1.25 and 10 mmol/L, respectively. After a 30-min incubation, the Ca concentration in the serosal medium was determined and the net transepithelial Ca transport was calculated. The addition of 0.1-200 mmol/L maltitol, difructose anhydride (DFA)III, DFAIV, raffinose, fructooligosaccharide (FOS) or polydextrose (PD) to the mucosal medium increased the net Ca absorption dose-dependently in the jejunum, ileum, cecum and colon preparations. The threshold concentration required to enhance Ca transport and the extent of enhancement of Ca transport differed among the saccharides tested and among the portions of the intestine examined. Among the saccharides tested, DFA IV had the strongest effect on Ca absorption in the jejunum and cecum. We conclude that indigestible carbohydrates directly affect the epithelial tissue and promote Ca absorption in both the small and large intestine in vitro.

Short-chain fatty acids enhance diffusional ca transport in the epithelium of the rat cecum and colon.

We examined the effect of short-chain fatty acids (SCFAs) on Ca absorption from the large intestine in rats in vitro. An Ussing-type chamber technique was used to determine the net transport of Ca from the luminal side to the basolateral side of isolated epithelium in cecum and colon preparations. The concentration of Ca in the serosal and mucosal Tris buffer solution was 1.25 mM and 10 mM, respectively. Both solutions were warmed at 37 degrees C and bubbled with 95% O2 and 5% CO2. During and after the incubation period (30 min or 60 min), the Ca concentration in the serosal medium was determined and the net transepithelial Ca transport was evaluated. The addition of 80 mM acetic acid, 40 mM propionic acid and 10 mM butyric acid to the mucosal medium increased net Ca absorption (about 300%) in the cecum and colon. An individual application of acetic, propionic or butyric acid (0.01 to 100 mM) to the mucosal medium also increased net Ca absorption at doses of 10 mM and /or 100 mM in the cecum and colon. An increase in solute concentration in the mucosal medium by addition of glycerol or PGE400, or a decrease in pH (7.0-3.0) by addition of HCl did not affect transepithelial Ca transport. We concluded that SCFAs affect the epithelial tissue and promote Ca absorption from the large intestine in vitro. The enhancement of Ca transport induced by SCFAs might be involved in the paracellular transport mechanism.

Establishment of a new cross of the rice blast fungus derived from Japanese differential strain Ina168 and hermaphroditic rice pathogen Guy11.

Mating experiments between Magnaporthe grisea Japanese rice pathogens and Guy11, a hermaphroditic fertile rice pathogen, were done aimed at identification of avirulence genes. A cross named cross 2107 with thirty-six random progenies was obtained. Segregation analyses of genetic markers found that the cross was less suitable for genetic analysis. Backcrosses with cross 2107 progenies and Guy11 were done and another cross named cross 5307 with sixty-five progenies was obtained. A locus controlling kasugamycin resistance named Ksg1R was identified and used for a model case of genetic mapping. Bulked segregant analysis was done to find adjacent RAPD markers for mapping of the gene. Three adjacent markers to Ksg1R were obtained and a genetic map around the Ksg1R was made, but these markers were not located on a single chromosome. These results suggest that genetic analysis to identify a gene locus is available in cross 5307. Infection assay of parental strains of cross 5307 to Japanese differential rice cultivars suggested the possibility of genetic analysis of cultivar specificity toward four rice cultivars: Aichi-asahi, Kusabue, Tsuyuake, and K59.

Characterization of binding between the rat small intestinal brush-border membrane and dietary proteins in the sensory mechanism of luminal dietary proteins.

Dietary proteins are recognized by the gastrointestinal tract to display physiological functions, however, the sensory mechanism of the intestinal mucosa is not known. We examined binding properties between the rat small intestinal brush-border membrane (BBM) and proteins by using a surface plasmon resonance biosensor. BBM and solubilized BBM prepared from the rat jejunum bound to casein immobilized on the sensor surface, but not to bovine serum albumin. The ileal BBM showed less binding to casein than the jejunal BBM. Solubilized BBM binding to immobilized alpha-casein was slightly inhibited by aminopeptidase inhibitors, but still more inhibited by addition of casein with the inhibitors. Guanidinated casein inhibited the solubilized BBM binding to alpha-casein more strongly than casein (casein sodium and alpha-casein) inhibited. Trypsinization of solubilized BBM abolished its binding activity to alpha-casein. These results indicate that some membrane protein, but not aminopeptidases, contained in BBM interacts with dietary proteins, and that guanidinated casein has a higher affinity for BBM than intact casein. These binding intensities for proteins were closely correlated to physiological responsiveness, and are possibly involved in a sensory system for dietary protein in the intestine.

Comparisons of intraosseous graft healing between the doubled flexor tendon graft and the bone-patellar tendon-bone graft in anterior cruciate ligament reconstruction.

PURPOSE: The purpose of this study was to compare intraosseous graft healing between the doubled flexor tendon (FT) graft and the bone-patellar tendon-bone (BPTB) graft in anterior cruciate ligament (ACL) reconstruction. TYPE OF STUDY: Randomized trial. METHODS: A biomechanical and histologic study was conducted with 24 adult beagle dogs. Bilateral ACL reconstructions were performed in each animal. Autogenous doubled FT and BPTB grafts were used for the left and right knees, respectively. Each end of the 2 grafts was tethered with a polyester suture to a screw post with a washer. The animals were then allowed unrestricted activities in their cages. Eight animals were killed at 3, 6, and 12 weeks, respectively. RESULTS: Histologically, the FT graft was anchored to the tunnel wall with newly formed collagen fibers resembling Sharpey's fibers by 12 weeks. These fibers were more abundant in the anterior (ventral) gap than in the posterior (dorsal) gap. In the BPTB graft, the bone plug was anchored with newly formed bone at 3 weeks, although osteocytes in the plug trabeculae were necrotic for 12 weeks. Degeneration of the tendon-bone junction in the plug progressed at 6 weeks. Tensile testing showed that the weakest site was different not only between the 2 grafts but also between the observation periods. In the FT graft, the weakest site was the graft-wall interface at 3 weeks and the intraosseously grafted tendon at 6 weeks. In the BPTB graft, the weakest site was the graft-wall interface at 3 weeks and the proximal site in the bone plug at 6 weeks. The ultimate failure load of the FT graft was significantly inferior (45.8%) to that of the BPTB graft at 3 weeks (P =.021). At 6 weeks, the load of the FT graft was 85% that of the BPTB graft without a significant difference (P =.395). CONCLUSIONS: As to the clinical relevance, the fixation device chosen for soft-tissue fixation appears to be more important than comparing it to the BPTB graft, although this has yet to be conclusively proven.

Induction and catabolite repression mechanisms of cellulase in fungi.

Cellulases are induced in most of fungi only when cellulose or an inducer exists. In Hypocrea jecorina and Penicillium purpurogenum, the respective inducers are sophorose and gentiobiose, which do not have beta-1,4 linkages though cellobiose, which has this linkage, is an inducer in other fungi. beta-Glucosidase, which catalyzes transglucosylation, is the key enzyme in converting cello-oligosaccharides to the inducers for cellulase induction in H. jecorina and P. purpurogenum. There are three states in the regulation of cellulase at the transcriptional level in fungi: expression at a basal level, mass secretion of cellulases induced by inducers, and glucose or catabolite repression. Expression at a basal level allows a small amount of cellulase to hydrolyze cellulose to soluble oligosaccharides or to an inducer if cellulose exists near the mycelia. Once the inducer enters the cell, it triggers full-scale transcription of the cellulase gene mediated by activator proteins and activating elements. After cellulose is degraded a large amount of glucose is liberated, which causes catabolite repression.

H+-ATPase defect in Corynebacterium glutamicum abolishes glutamic acid production with enhancement of glucose consumption rate.

A mutant of Corynebacterim glutamicum ('Brevibacterium flayum') ATCC14067 with a reduced H+-ATPase activity, F172-8, was obtained as a spontaneous neomycin-resistant mutant. The ATPase activity of strain F172-8 was reduced to about 25% of that of the parental strain. Strain F172-8 was cultured in a glutamic-acid fermentation medium containing 100 g/l of glucose using ajar fermentor. It was found that glucose consumption per cell during the exponential phase was higher by 70% in the mutant than in the parent. The respiration rate per cell of the mutant also increased to twice as much as that of the parent. However, the growth rate of the mutant was lower than that of the parent. Under those conditions, the parent produced more than 40 g/l glutamic acid, while the mutant hardly produced any glutamic acid. Instead the mutant produced 24.6 g/l lactic acid as the main metabolite of glucose. Remarkably, the accumulation of pyruvate and pyruvate-family amino acids, i.e., alanine and valine, was detected in the mutant. On the other hand, the parent accumulated alpha-ketoglutaric acid and a glutamate-family amino acid, proline, as major by-products. It was concluded that the decrease in the H+-ATPase activity caused the above-mentioned metabolic changes in strain F172-8, because a revertant of strain F172-8, R2-1, with a H+-ATPase activity of 70% of that of strain ATCC14067, showed a fermentation profile similar to that of the parent. Sequence analyses of the atp operon genes of these strains identified one point mutation in the gamma subunit in strain F172-8.

Cholic acid is accumulated spontaneously, driven by membrane deltapH, in many lactobacilli.

Many lactobacilli from various origins were found to apparently lack cholic acid extrusion activity. Cholic acid was accumulated spontaneously, driven by the transmembrane proton gradient. Accumulation is a newly identified kind of interaction between intestinal microbes and unconjugated bile acids and is different from extrusion and modification, which have been described previously.

Molecular structure of rDNA repeat unit in Magnaporthe grisea.

The 8-kb repeat unit of M. grisea rRNA-encoding DNA (rDNA) was cloned as three subclones, pM50, pM21, and pM86. Nucleotide sequencing of these subclones uncovered the structure of an rDNA repeat unit similar to those of other ascomycetes. The intergenic spacer (IGS) of the rDNA cistron contained a repetitive (R) region, which was rich in two kinds of short tandemly repeated elements.

Prevalence and clinical characteristics of patients with atrial fibrillation: analysis of 20,000 cases in Japan.

In Japan, data on the epidemiological and clinical features of atrial fibrillation (AF) are rather sparse; even less data are available on the risk of thromboembolism in nonvalvular AF. The present study enrolled 19,825 patients who visited the cardiovascular clinics of the 13 hospitals in Hokkaido, Japan, between March and July 1995. The prevalence of AF, the clinical characteristics of AF patients, and the occurrence of ischemic events were examined during the 2 year follow-up period. The prevalence of AF increased with age, and the overall prevalence was 14%. Antithrombotic therapy was used in 57% of AF patients and the incidence of ischemic events during the follow-up period was 4.6% in all AF patients. Warfarin reduced the risk of ischemic events in both the valvular and nonvalvular AF groups. A history of cerebrovascular accidents, advanced age, and the presence of underlying heart disease were each associated with a significantly increased risk of ischemic events in the nonvalvular AF group. These results show a lower incidence of ischemic events and more frequent use of antiplatelet drugs in the nonvalvular AF group. Further prospective studies are needed to determine the best preventive methods for thromboembolic complications in Japanese patients with nonvalvular AF.

Difructose anhydrides: their mass-production and physiological functions.

Difructose anhydrides (DFAs) are the smallest cyclic disaccharides consisting of two fructose residues, and are expected to have novel physiological functions from their unique structures and properties. For mass-production of alpha-D-fructofuranose-beta-D-fructofuranose-2',1:2,3'-dianhydride (DFA III) and beta-D-fructofuranose-beta-D-fructofuranose-2',6:2,6'-dianhydride (DFA IV), Arthrobacter sp. H65-7 and A. nicotinovorans GS-9 were selected as the best producers of inulase II, which produced DFA III from inulin and LFTase, which produced DFA IV from levan. The enzymes were purified and their genes were subsequently cloned and expressed in E. coli at higher levels than in the original bacteria. Thus, it became possible to provide a large amount of DFA III and DFA IV for evaluating their physiological properties. DFA III and DFA IV have half the sweetness of sucrose, but cannot be digested by the digestive system of rats. Their use by the intestinal microorganisms was observed in vivo even though their assimilation could not be detected in vitro. This implied that they were degraded by an unknown system in the intestine. It was also found that they affected calcium absorption mainly in the small intestine through mechanisms different from the known stimulants such as fructooligosaccharides and raffinose.

[Antithrombotic therapy for stroke prevention in patients with atrial fibrillation].

Atrial fibrillation(AF) is a common arrhythmia that is an important independent risk factor for stroke. The overall risk of stroke in AF patients averages about 5%/y, but with wide variation depending on the presence of coexistent thromboembolic risk factors, which include increasing age, history of hypertension, previous stroke or transient ischemic attack(TIA), and diabetes. AF patients with prior stroke or TIA are at highest risk(about 12%/y). Adjusted-dose warfarin(target INR 2.0-3.0) is highly efficacious for preventing stroke in AF patients, and is safe for selected patients. Aspirin has a modest effect on reducing stroke. Warfarin is recommended for high-risk AF patients who can safely receive it. Aspirin may be indicated for those with a low stroke risk and for those who cannot receive warfarin.

Agonist-dependent difference in the mechanisms involved in Ca2+ sensitization of smooth muscle of porcine coronary artery.

This study was undertaken to explore possible signal-transduction mechanisms involved in the Ca2+-sensitizing effects of carbachol and endothelin-1 (ET-1) by using beta-escin-skinned smooth muscle of porcine coronary artery. Pretreatment with C3 exoenzyme of Clostridium botulinum, which selectively inactivates rho p21 by adenosine diphosphate (ADP) ribosylation, resulted in a significant inhibition of ET-1-induced Ca2+ sensitization, but had no effect on carbachol-induced Ca2+ sensitization. Whereas the protein kinase C (PKC) inhibitors calphostin C and staurosporine did not affect the Ca2+-sensitizing effect of carbachol, the tyrosine kinase inhibitors genistein and tyrphostin 25 greatly but incompletely suppressed it. In contrast, the Ca2+-sensitizing effect of ET-1 was significantly inhibited by either calphostin C or genistein. Although the inhibitory effect of calphostin C on ET-1-induced Ca2+ sensitization was less than that of genistein, the effects of calphostin C and genistein were additive. The genistein-sensitive component of ET-1-induced Ca2+ sensitization appeared to include the C3-sensitive one. However, a substantial enhancement by ET-1 of the Ca2+-induced contraction was observed even in the presence of the two inhibitors. In beta-escin-skinned smooth muscle of rabbit mesenteric artery, ET-1-induced Ca2+ sensitization was marginally affected by C3 pretreatment, calphostin C, and genistein. We conclude that, although PKC activation and rho p21 protein-dependent and -independent tyrosine phosphorylation each plays an important role in an increase in myofilament Ca2+ sensitivity, the contributions of these signaling pathways to Ca2+ sensitization are different depending on receptor agonists and tissues used. Furthermore, these data suggest the existence of an as yet undefined signal-transduction mechanism involved in Ca2+ sensitization caused by receptor agonists.

Physical and personality traits of preschool children in Fuzhou, China: only child vs sibling.

Concern about the healthy growth and development of an only child has been voiced since the 1970s, when the Chinese government launched the only child policy. In this study, the physical and personality traits of rural Chinese preschool only-children (onlies) whose age ranged from 3 to 6 years old were evaluated. The sample included 197 onlies and 367 children with siblings who came from seven kindergartens in rural areas in Fuzhou, Fujian province. The results showed no statistically significant differences in height, mass or BMI between the onlies and siblings. Regarding the personality traits, the significant difference was that the onlies exhibited more somatic complaints, however, the data didn't indicate any other undesirable personality traits for the onlies. These results suggest that Chinese preschool children grow normally with or without siblings.

Improvement in fatty acid utilization in relation to a change in left ventricular hypertrophy in spontaneously hypertensive rats.

Although fatty acid metabolism is reportedly impaired in myocardial hypertrophy, it is unclear whether the antihypertensive drugs are associated with improved fatty acid metabolism. In order to evaluate the effects of antihypertensive drugs on fatty acid metabolism and myocardial perfusion, the simultaneous uptake of iodine-125(125I)-15-(p-iodophenyl)-3-(R,S)-methylpentadecanoic acid (BMIPP) and thallium-201 (Tl) were measured in 3 groups of rats: (1) spontaneously hypertensive rats (SHR) without treatment (SHR-N), (2) SHR chronically treated with captopril (SHR-C), and (3) SHR chronically treated with hydralazine (SHR-H). Captopril and hydralazine were administered to their respective groups for 3 weeks from 12 weeks of age. The hearts were removed 10 min after simultaneous intravenous injections of BMIPP and Tl and the 125I and 201Tl counts were measured to calculate the uptake ratio. The systolic blood pressure (SBP) in SHR-N was 222+/-10 mm Hg, whereas the SHR-C and SHR-H groups showed significant SBP reduction (156+/-11, and 158+/-10 mm Hg, respectively) (p<0.01 each). The heart/bodyweight ratio was significantly lower in SHR-C (2.48+/-0.09) than in SHR-N (2.74+/-0.11) (p<0.05). However, there was no significant difference in the heart/bodyweight ratio between SHR-N and SHR-H (2.65+/-0.09). The ratio of BMIPP uptake to Tl uptake (BMIPP/Tl) was significantly higher in SHR-C (0.71+/-0.13) than in SHR-N (0.50+/-0.09) (p<0.05). However, BMIPP/Tl in SHR-H (0.53+/-0.09) was similar to that in SHR-N. These results suggest that captopril improves fatty acid metabolism in the hypertrophied ventricle in SHR. The metabolic alterations may improve with left ventricular hypertrophy regression but are not effected by the reduction of blood pressure only.

Purification and characterization of 2,6-beta-D-fructan 6-levanbiohydrolase from Streptomyces exfoliatus F3-2.

Streptomyces exfoliatus F3-2 produced an extracellular enzyme that converted levan, a beta-2,6-linked fructan, into levanbiose. The enzyme was purified 50-fold from culture supernatant to give a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weights of this enzyme were 54,000 by SDS-PAGE and 60,000 by gel filtration, suggesting the monomeric structure of the enzyme. The isoelectric point of the enzyme was determined to be 4.7. The optimal pH and temperature of the enzyme for levan degradation were pH 5.5 and 60 degrees C, respectively. The enzyme was stable in the pH range 3.5 to 8.0 and also up to 50 degrees C. The enzyme gave levanbiose as a major degradation product from levan in an exo-acting manner. It was also found that this enzyme catalyzed hydrolysis of such fructooligosaccharides as 1-kestose, nystose, and 1-fructosylnystose by liberating fructose. Thus, this enzyme appeared to hydrolyze not only beta-2,6-linkage of levan, but also beta-2,1-linkage of fructooligosaccharides. From these data, the enzyme from S. exfoliatus F3-2 was identified as a novel 2,6-beta-D-fructan 6-levanbiohydrolase (EC 3.2.1.64).

Molecular cloning and high-level expression in Escherichia coli of fungal alpha-galactosidase from Absidia corymbifera IFO 8084.

A cDNA encoding the alpha-galactosidase of Absidia corymbifera IFO 8084 was cloned and sequenced. The cloned DNA has a single open-reading frame consisting of 2190 base pairs, and the deduced amino acid sequence revealed that the mature enzyme consisted of 730 amino acid residues with a molecular mass of 82,712 Da. The native structure of the alpha-galactosidase of A. corymbifera IFO 8084 was determined to be a tetramer. Comparison with amino acid sequences of other alpha-galactosidase showed high homology with sequences of members of family 36. An expression vector, pET32Trx/galalpha, was constructed by introducing the cDNA coding region into a thioredoxin fusion system, pET32-Ek/LIC. The resulting transformant, pET32Trx/galalpha, overproduced the active enzyme as a thioredoxin fused form in the host Escherichia coli. By using His-binding metal affinity chromatography, recombinant alpha-galactosidase was purified to homogeneity in a single step. The purified recombinant fusion alpha-galactosidase showed properties very similar to the native alpha-galactosidase from A. corymbifera IFO 8084.

Microbial demetallization of crude oil: nickel protoporphyrin disodium as a model organo-metallic substrate.

A soil isolate designated as YA-1 strain was selected for its ability to degrade nickel protoporphyrin disodium (NiPPDS). The strain was capable of utilizing NiPPDS as the sole source of carbon. This strain, a gram-negative aerobic rod, was identified as Pseudomonas azelaica YA-1 based on the result of its 16S rRNA analysis. Product analyses by HPLC showed that this strain can decompose the porphyrin ring to which a metal ion is bound. However, the use of whole bacterial cells cannot result in extensive NiPPDS degradation; therefore, the YA-1 enzyme was extracted and purified. This NiPPDS-degrading enzyme named as protoporphyrinase was purified from P. azelaica YA-1 by ammonium sulfate fractionation and sequential chromatographies using DEAE Toyopearl 650 M, CM Toyopearl 650 M and Biogel P-60 columns, with a yield of 11.3% based on the enzyme activity and an overall purification of 498-fold. The molecular weight of this enzyme is estimated to be 39,000 Da by SDS-PAGE and 34,000 Da by gel filtration. The optimum pH and temperature for the enzyme were 7.0 and 30 degrees C, respectively. The activity was stable at pH 2.0-11.0 and at temperatures below 50 degrees C. The enzyme activity was inactivated by ferric chloride, potassium ferricyanide, ZnCl2 and CdCl2.