Changes in gene expression in a porcine preadipocyte cell line during differentiation.

Adipocyte differentiation plays an important role in the formation of fat tissues in pigs and affects meat quality and productivity. Clarification of the nature of the pig genes that participate in adipocyte differentiation will provide a clue to the regulation of fat content and thickness in pig carcases by dietary control; it will also help to find target genes for exploring potentially useful polymorphisms for molecular breeding aimed at fat traits. We constructed a DNA oligomer microarray based on pig transcripts, and we used the array to investigate time-dependent changes in gene expression in the PSPA porcine preadipocyte cell line during differentiation into adipocytes. We selected genes with markedly altered expression (at least fivefold difference in comparison with expression in undifferentiated cells) and classified them into five groups according to gene expression pattern. In the early stage after stimulation of adipocyte differentiation, we observed up-regulation of many genes encoding proteins involved in regulating cell proliferation and transcription. Among the probes corresponding to transcripts that showed marked changes in expression, 27 were located within previously reported QTL regions for traits related to adipose tissues. These results will be valuable resources for finding the genes responsible for fat-related traits that have been identified in previous studies using various pig resource families.

Risk stratification using serum concentrations of cardiac troponin T in patients with end-stage renal disease on chronic maintenance dialysis.

BACKGROUND: It has been recently suggested that cardiac troponin T (cTnT) may be more sensitive than troponin I (cTnI) for subclinical myocardial cell injury in patients on chronic dialysis. METHODS: We prospectively compared the predictive value of cTnT with cTnI, atrial (ANP) and brain natriuretic peptide (BNP) in 100 consecutive outpatients on chronic dialysis without acute coronary syndromes over a period of 3 months, and assessed whether the combination of cTnT with clinical information including age, duration of dialysis, and medical histories was useful for risk stratification of these patients. During the 2-year follow-up period, 19 patients died, mostly due to cardiac causes (53%). RESULTS: The area under the receiver operator characteristic (ROC) curve for the cTnT as predictor of both overall and cardiac death was significantly greater than the area under the cTnI curve (p < 0.0001 and p = 0.01), the BNP curve (p < 0.001 and p < 0.01) or the ANP curve (p < 0.0001 and p < 0.005). In a stepwise multivariate Cox regression analysis, only cTnT (p < 0.05 and p < 0.01) and a history of heart failure requiring hospitalization (p < 0.05 and p < 0.005) were independent predictors of both all cause and cardiac mortality. Using parameters of cTnT > or =0.1 microg/l and/or history of heart failure, the overall and cardiac mortality rate for the low risk group (n=66) were 4.5% and 1.5%, respectively, 40% and 16% for the intermediate risk group (n=25), and 67% and 56% for the high risk group (n=9). CONCLUSION: cTnT concentrations offer a higher prognostic accuracy than cTnI, ANP and BNP in patients on chronic dialysis. The combination of elevated cTnT and a history of heart failure may be a highly effective means of risk stratification of these patients.

Characterization of whole blood aggregation with a new type of aggregometer by a screen filtration pressure method.

A new type of platelet aggregometer of whole blood, based on the screen filtration pressure method, has been developed and characterized. It measures resistance of flow of whole blood samples through a screen of microsieve with 30-microm(2) openings and provides pressure rate as an index of platelet aggregation. On optical microscopic observation, platelet aggregates, but not fibrin fibers, were found to be trapped on microsieves, and the pressure rate and protein amounts on microsieves are correlated. The aggregometer showed good reproducibility for investigations performed on different days. The time course of pressure rates indicated a bell curve change, where the pressure rate was very low immediately after blood collection and gradually increased up to 60 min thereafter. Use of the aggregometer was able to confirm that orally administered aspirin inhibits ADP- and collagen-induced whole blood aggregation as well as platelet aggregation. The results suggest that this platelet aggregometer might be useful in research and clinical diagnosis of thrombotic diseases.

An informative linkage map of soybean reveals QTLs for flowering time, leaflet morphology and regions of segregation distortion.

A genetic linkage map covering a large region of the genome with informative markers is essential for plant genome analysis, including identification of quantitative trait loci (QTLs), map-based cloning, and construction of a physical map. We constructed a soybean genetic linkage map using 190 F2 plants derived from a single cross between the soybean varieties Misuzudaizu and Moshidou Gong 503, based on restriction-fragment-length polymorphisms (RFLPs) and simple-sequence-repeat polymorphisms (SSRPs). This linkage map has 503 markers, including 189 RFLP markers derived from expressed sequence tag (EST) clones, and consists of 20 major linkage groups that may correspond to the 20 pairs of soybean chromosomes, covering 2908.7 cM of the soybean genome in the Kosambi function. Using this linkage map, we identified 4 QTLs--FT1, FT2, FT3, and FT4--for flowering time, the QTLs for the 5 largest principal components determining leaflet shape, 6 QTLs for single leaflet area, and 18 regions of segregation distortion. All 503 analyzed markers identified were located on the map, and almost all phenotypic variations in flowering time were explained by the detected QTLs. These results indicate that this map covers a large region of the soybean genome.

Sperm motion analysis in rats treated with adriamycin and its applicability to male reproductive toxicity studies.

Adriamycin (ADR), an anticancer drug which induces testicular toxicity, was administrated to Slc:SD male rats at doses of 0.5, 1.0, and 2.0 mg/kg intravenously once a week for 4 weeks. The males treated with ADR were mated with untreated females, and sperm analyses (motion, count, and morphology) were performed. Sperm motion was analyzed by Hamilton-Thorne Sperm analyzer (HTM-IVOS) to investigate the useful parameters. Copulated females were necropsied at Day 13 of gestation, and reproductive status was evaluated. In ADR-treated groups, the testicular weight was dose-dependently decreased. Associated with this decrease was a depletion of the number of spermatogonia noted histopathologically at all dosage levels. Sperm morphological abnormalities, which were classified as tailless sperm and/or no-hook head sperm, were increased in both the 1.0 and 2.0 mg/kg groups. The males treated with ADR at 1.0 and 2.0 mg/kg had a decreased number of sperms per cauda epididymis. In sperm motion analysis, decreases in the percentage of motile sperm, percentage of progressive sperm, and sperm velocity (straight line velocity and curvilinear velocity) were noted at 2.0 mg/kg. Impaired fertility was noted at 2.0 mg/kg in the form of decreased numbers of implantations and live embryos, and an increased number of pre-implantation losses. In conclusion, ADR induced deterioration of sperm motion and sperm content, which were responsible for the adverse effect on male fertility. The most sensitive indicators to detect male reproductive toxicity induced by ADR were testicular weight and histopathological findings in the testis. Among the parameters generated by HTM-IVOS, the percentage of motile sperm, the percentage of progressive sperm, and sperm velocity are useful for assessing male fertility.

Development and assessment of enzyme immunoassay for platelet-derived microparticles.

Platelet-derived microparticles (PMPs) are released from platelets through the platelet activation by high shear stress, collagen, or calcium ionophore (A23187). PMPs are observed in patients with acute myocardial infarction, thrombotic thrombocytopenic purpura, hemolytic uremic syndrome, heparin-induced thrombocytopenia and other thrombotic disorders, but the importance of circulating PMPs in the pathogenesis of these diseases is still debated. Numbers of PMPs are usually determined by flowcytometry (FCM), but easier and reproducible PMP assay systems are needed. To develop a better ELISA for PMPs, we used antibodies against the platelet antigens anti-GPIb (NNKY5-5), anti-GPIIb/IIIa (NNKY2-11, anti-CD41), anti-GPIX (KMP-9), and anti-CD9 (NNKY1-19). PMPs were detected with all combinations of these antibodies, but the ELISA having the highest and most specific absorbance was obtained with a combination of KMP-9 (capture antibody) and NNKY5-5 (detecting antibody). PMPs in blood samples were measured by ELISA and FCM. ELISA correlated with PMPs quantitated by FCM. By shaking ELISA plates during incubation, nonspecific binding of platelets was eliminated. The level of PMPs was not increased in diabetes mellitus, thrombotic thrombocytopenic purpura, antiphospholipid syndrome, or sepsis. The concentration of PMP was elevated in hemolytic uremic syndrome. Activated PMPs were absorbed to 0.8 microm filter, but circulating PMPs were not absorbed. These results suggest that activated PMPs are likely to adhere to leukocytes or endothelial cells at the activation site and that the circulating form of PMPs are likely to be a residue of activated PMPs. To detect only the activated form of PMPs, a new ELISA needs to be developed, and it will likely use a combination of antibodies that detect platelet activation markers such as P-selectin (CD62P) or activated GPIIb/IIIa.

Plasma concentration of brain natriuretic peptide as a biochemical marker for the evaluation of right ventricular overload and mortality in chronic respiratory disease.

The purpose of this study was to evaluate whether the plasma brain natriuretic peptide (BNP) concentration is a useful marker of right ventricular (RV) overload and whether it has prognostic value as a predictor of death in patients with chronic respiratory disease (CRD). We measured the plasma BNP and atrial natriuretic peptide (ANP) concentrations in 31 consecutive patients with CRD who underwent right-heart catheterization to evaluate pulmonary hypertension. All patients were followed for >12 months. The plasma BNP concentration closely correlated with the mean pulmonary artery pressure and pulmonary vascular resistance (r=0.62, P<0.0005 and r=0. 85, P<0.0001), and showed a weak linear correlation with cardiac output (r=-0.36, P<0.05). During the follow-up period, 5 (16%) end-stage CRD deaths (4 RV heart failure and 1 respiratory infection) and 2 non-end-stage CRD deaths occurred. In a stepwise multivariate Cox proportional-hazards regression analysis including age, sex, BNP, ANP, hemodynamic variables and the ratio of PaO(2) to fraction of inspired oxygen, only BNP (P<0.05) was an independent predictor of end-stage CRD death. The upward and leftward shift in the receiver operating characteristic curve between patients with end-stage CRD death and those without was greater for BNP than for ANP. Our findings suggest that the plasma BNP concentration may be an inexpensive, simple and useful marker of RV overload and end-stage CRD death in CRD patients. These preliminary results need to be confirmed in a large series of CRD patients.

Spirostanols obtained by cyclization of pseudosaponin derivatives and comparison of anti-platelet agglutination activities of spirostanol glycosides.

Naturally occurring saponins 3 and 4 have a normal type F ring and alpha-arranged CH(3)-21 group. Treatments of pseudosaponin peracetates 18 and 19 derived from 3 and 4, respectively, with alcoholic KOH, followed by acidification with acetic acid, gave spirostanols 20 and 22 having iso type F rings as major products. Structural analyses of sapogenins and saponins derived from pseudo derivatives 11, 12, 18 and 19 were performed by comparisons of their 1H-NMR spectral data and the X-ray analytical data of 3-O-p-bromobenzoyl sarsasapogenin 7, 3-O-acetyl diosgenin 13 and saponin 20. The mechanisms of ring-closure reaction of the side chain at C-22 of pseudosapogenins and pseudosaponins were deduced using stereomodels of the spirostanols derived from 11 under various reaction conditions. Inhibitory activities of saponin diglycosides 3, 4, 20, 21 and 25 on human platelet agglutinations induced by ADP and ristocetin were compared.

Diverse expression profiles of 21 rice peroxidase genes.

Secretory class III plant peroxidases (POXs) catalyze the oxidation of various reductants, and are encoded by a large multigene family. In rice, 42 independent expressed sequence tags for POXs have been identified. By RNA gel blot analysis using specific probes, we show here that 21 rice POX genes are unique in their developmental, organ specific and external stimuli-responsive expression. This would suggest that encoded POX isoenzymes are involved in a broad range of physiological processes in rice plants, individually.

Hd1, a major photoperiod sensitivity quantitative trait locus in rice, is closely related to the Arabidopsis flowering time gene CONSTANS.

A major quantitative trait locus (QTL) controlling response to photoperiod, Hd1, was identified by means of a map-based cloning strategy. High-resolution mapping using 1505 segregants enabled us to define a genomic region of approximately 12 kb as a candidate for Hd1. Further analysis revealed that the Hd1 QTL corresponds to a gene that is a homolog of CONSTANS in Arabidopsis. Sequencing analysis revealed a 43-bp deletion in the first exon of the photoperiod sensitivity 1 (se1) mutant HS66 and a 433-bp insertion in the intron in mutant HS110. Se1 is allelic to the Hd1 QTL, as determined by analysis of two se1 mutants, HS66 and HS110. Genetic complementation analysis proved the function of the candidate gene. The amount of Hd1 mRNA was not greatly affected by a change in length of the photoperiod. We suggest that Hd1 functions in the promotion of heading under short-day conditions and in inhibition under long-day conditions.

An immunohistochemical and ultrastructural study of a follicle-stimulating hormone-secreting gonadotroph adenoma occurring in a 10-year-old girl.

Female gonadotroph adenomas with endocrinological symptoms are uncommon. Six cases of such adenomas have been reported in the literature: two were girls who presented with precocious puberty and four were premenopausal women with accompanying multiple ovarian cysts. We describe here a 10-year-old Japanese girl with a gonadotroph macroadenoma and present detailed morphological findings of the tumor. The patient's chief complaints were nausea, abdominal distention, and abdominal pain. Abdominopelvic ultrasonography and magnetic resonance imaging (MRI) revealed bilateral multiple ovarian cysts. Endocrinological assays showed elevated serum follicle-stimulating hormone (FSH) (33.7 mIU/ml) and estradiol (3840 pg/ml). MRI of the head showed a large pituitary tumor. Two transsphenoidal operations and subsequent radiation therapy were performed. Immunohistochemically, more than half the tumor cells were positive for anti-FSH-beta monoclonal antibody. Ultrastructurally, the tumor cells exhibited a fairly uniform picture of rounded cells. Their nuclei were slightly irregular and contained heterochromatin, and their cytoplasm contained many round, dense core granules, measuring 140-260 nm in diameter, together with well-developed organelles. An in vitro study showed that the tumor cells in primary culture produced FSH (1089.0 mIU/ml). To our knowledge, this is the first immunohistochemical and ultrastructural study of an FSH-secreting gonadotroph adenoma occurring in childhood.

An anti-platelet agent, OPC-29030, inhibits translocation of 12-lipoxygenase and 12-hydroxyeicosatetraenoic acid production in human platelets.

1. In human platelets, arachidonic acid is mainly metabolized by the two enzyme systems; cyclo-oxygenase and 12-lipoxygenase. Cyclo-oxygenase produces prostaglandin H(2) which is further converted to thromboxane B(2). 12-Lipoxygenase synthesizes 12(S)-hydroperoxyeicosatetraenoic acid which is reduced to 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE). 2. An anti-platelet compound, OPC-29030, dose-dependently inhibited 12(S)-HETE production with an IC(50) of 0.06+/-0.01 microM, but not synthesis of thromboxane B(2) in human platelets. Although the compound suppressed 12(S)-HETE production in human platelets, cytosolic 12-lipoxygenase activity was not inhibited up to 10 microM. Essentially identical data were obtained with a 12-lipoxygenase of human erythroleukaemia cells which had megakaryocyte/platelet-like properties. 3. OPC-29030 also suppressed production of 5(S)-HETE, a 5-lipoxygenase product, in rat basophilic leukaemia cells without inhibiting enzyme activity. It has been shown that 5-lipoxygenase binds to membrane 5-lipoxygenase-activating protein (FLAP) to produce 5(S)-HETE, and thus FLAP inhibitor suppresses cellular 5(S)-HETE production. 4. A FLAP inhibitor, L-655,238, suppressed platelet 12(S)-HETE production, but had no effect on the 12-lipoxygenase activity. 5. Western blot analysis showed that platelet 12-lipoxygenase translocated from cytosol to membranes upon thrombin stimulation, and OPC-29030 suppressed this process in a dose-dependent manner. 6. These results suggest that the 12-lipoxygenase of human platelets binds to FLAP or a similar protein, and OPC-29030 suppresses 12(S)-HETE production by inhibiting a certain step of the 12-lipoxygenase translocation.

Arabidopsis-rice: will colinearity allow gene prediction across the eudicot-monocot divide?

With the genomic sequencing of Arabidopsis nearing completion and rice sequencing very much in its infancy, a key question is whether we can exploit the Arabidopsis sequence to identify candidate genes for traits in cereal crops using a map-based approach. This requires the existence of colinearity between the Arabidopsis and cereal genomes, represented by rice, which is readily detectable using currently available resources, that is, Arabidopsis genomic sequence, rice ESTs, and genetic and physical maps. A detailed study of the colinearity remaining between two small regions of Arabidopsis chromosome 1 and rice suggests that at least in these regions of the Arabidopsis genome, conservation of gene orders with rice has been eroded to the point that it is no longer identifiable using comparative mapping. Although our analysis does not preclude that tracts of colinear gene orders may be identified using sequence comparisons or may exist in other regions of the rice and Arabidopsis genomes, it is unlikely that the extent of colinearity will be sufficient to allow map-based cross-species gene prediction and isolation. Our research also highlights the difficulties encountered in identifying orthologs using BLAST searches in incomplete sequence databases. This complicates the interpretation of comparative data among highly divergent species and limits the exploitation of Arabidopsis sequence in monocot studies.

Ommochrome deficiency in an albino strain of a terrestrial isopod, Armadillidium vulgare.

In order to clarify the cause of ommochrome deficiency in an albino strain of the terrestrial isopod, Armadillidium vulgare, levels of xanthommatin, 3-hydroxykynurenine, 3-hydroxyanthranilic acid and tryptophan in whole body extracts of the albino and the wild type individuals were determined together with enzyme activities of kynurenine-3-hydroxylase, kynureninase and tryptophan-2,3-dioxygenase. Xanthommatin could not be detected in the albinos. The levels of 3-hydroxykynurenine and 3-hydroxyanthranilic acid were determined by high-performance liquid chromatography (HPLC) with electrochemical detection and were markedly low in the albinos compared with the wild type individuals. In contrast to those, the tryptophan levels determined by HPLC with fluorescence detection did not differ significantly between the two phenotypes. In the albino A. vulgare, kynurenine-3-hydroxylase activity was lower and kynureninase activity was higher than in the wild type, although the differences were not statistically significant. Tryptophan-2,3-dioxygenase activity in the albinos was less than 10% that in the wild type. Thus, ommochrome deficiency in the albino A. vulgare is considered to be caused by the extremely low activity of tryptophan-2,3-dioxygenase.

Cinnabarinic acid was formed in damaged mitochondria and its effect on mitochondrial respiration.

Tryptophan administration aggravates experimental mouse liver injury caused by carbon tetrachloride when 3-hydroxyanthranilic acid concentration elevates in serum. Tryptophan metabolism is changed by macrophages in injured liver. 3-Hydroxyanthranilic acid may be oxidized to cinnabarinic acid by injured mitochondria in the liver aggravating the state of injured liver. Mitochondria prepared from the liver 24 hr after CCl4 treatment have lost their ability of respiratory control. In consequence, 3-hydroxyanthranilic acid is oxidized to cinnabarinic acid by incubation with these mitochondria, whereas 3-hydroxykynurenine is not. Thus, formed cinnabarinic acid is able to inhibit completely the mitochondrial respiratory control at concentration of 10 microM.

Ommochrome genesis in an albino strain of a terrestrial isopod.

The contents of tryptophan (Trp) metabolites and the activities of the enzymes involved in ommochrome biosynthesis were measured in an albino strain of a terrestrial isopod Armadillidium vulgare. There was little difference between the Trp content in the albino mutant and that in the wild type, although the contents of 3-hydroxykynurenine (3-OH-Kyn), 3-hydroxyanthranilic acid (3-OH-AA) and xanthommatin in the albino were significantly lower than those in the wild type. Tryptophan 2,3-dioxygenase (TDO) activity in the albino was extremely low, while the activities of Kyn-3-hydroxylase and kynureninase did not differ significantly between the two phenotypes. The extremely low activity of TDO is probably one of main reasons why almost no ommochrome pigment is produced in the albino mutant.

Involvement of tryptophan metabolism in the body color of crustacea.

The terrestrial isopod, Armadillidium vulgare is usually grey or black in color, however, red ones are occasionally found in the field. This is caused by the mutation of the ommochrome genesis in the integument. We focused our experiments on the mechanism of pigment genesis in which tryptophan metabolism had been expected to be different from the grey or black wild types. We obtained the result that 3-hydroxyanthranilic acid content was significantly higher in the red phenotype than in the wild type, and kynureninase activity was also higher in the red phenotype.

Kynurenine concentration of serum was increased by exercise.

Kynurenine pathway of tryptophan makes a lot of physiological active substances, such as quinolinate, NAD and so on, suggesting that kynurenine itself may play a very important role physiologically. Therefore, we examined the influence of exercise on serum kynurenine concentration. At first, we assayed kynurenine concentration of students (n = 13) who took part in a rugby camp for three days. The mean value of kynurenine concentration of before and after training were 1.362 +/- 0.306 microM and 1.725 +/- 0.511 microM respectively. These data means that severe exercise rise the serum kynurenine concentration. Then we tried to examine the relationship between the level of exercise and serum kynurenine concentration. Serum kynurenine concentration had significantly increased immediately after the exercise from 1.869 +/- 0.285 microM to 2.138 +/- 0.248 microM of 24 hours later by loading of 65% heart rate max exercise for each subject. These results suggested that at least the severe exercise affect on the tryptophan metabolism. We will discuss the change of serum kynurenine concentration by another sports such as soccer game and 20 km run.

The recovering effect of betaine on carbon tetrachloride-induced liver injury.

The recovering effect of betaine (derivative of choline) on carbon tetrachloride (CCl4)-injured liver was investigated by oral and intraperitoneal administration of betaine at 24 h after acute CCl4 intoxication. The effect of betaine was estimated by the activity of alanine aminotransferase (ALT) released into the serum, histological score, and the incorporation of S-phase indicator (5-bromo-2-deoxyuridine/5-fluoro-2'-deoxyuridine; BrdU/FdU). A significant decrease in the activity of serum ALT was observed by the intraperitoneal (3 mg/kg body) or oral (15 mg/kg body) administration of betaine. Furthermore, an increase in the uptake of BrdU into the hepatocyte nuclear DNA and a reduction in liver necrosis after the oral treatment of betaine (15 mg/kg body) was observed. From these results, the administration of betaine showed a significant effect in recovering CCl4-injured liver.

Ethanolamine stimulates repair processes in acute CCl4 damage of mouse liver.

Ethanolamine, a positively charged hydrophilic component of the phosphatidylethanolamine (PE) which is also a precursor of phosphatidylcholine (PC), enhances the repair processes 24 h after carbon tetrachloride (CCl4) intoxication of mouse liver. Ethanolamine quickly reduced aminotransferase activity released in the serum, increased the hepatocellular nuclear BrdU uptake, a marker of S phase, and the epidermal growth factor (EGF) receptor content in the liver injured with CCl4. Thus, exogenous administration of ethanolamine may function as a liver proliferating factor in CCl4 intoxicated mouse liver.