A 300 kilobase interval genetic map of rice including 883 expressed sequences.

We have constructed a high resolution rice genetic map containing 1,383 DNA markers at an average interval of 300 kilobases (kb). The markers, distributed along 1,575 cM on 12 linkage groups, comprise 883 cDNAs, 265 genomic DNAs, 147 randomly amplified polymorphic DNAs (RAPD) and 88 other DNAs. cDNAs were derived from rice root and callus, analysed by single-run sequencing and searched for similarities with known proteins. Nearly 260 rice genes are newly identified and mapped, and genomic DNA and cloned RAPD fragments were also sequenced to generate STSs. Our map is the first significant gene expression map in plants. It is also the densest genetic map available in plants and the first to be backed up comprehensively by clone sequence data.

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.

Conversion of gamma-glutamylcysteinylethyl ester to glutathione in rat hepatocytes.

The conversion of gamma-glutamylcysteinylethyl ester (gamma-GCE) to glutathione in a reduced form (GSH) was examined using isolated rat hepatocytes pretreated with diethylmaleate, a GSH-depletor. Incubation of hepatocytes with 0.1 and 5.0 mM gamma-GCE (gamma-GCE-hepatocytes) over a 30-min period resulted in time-dependent increases in intracellular GSH and nonprotein-SH (NP-SH) concentrations. Hepatocytes incubated with 5.0 mM but not 0.1 mM GSH over a period of 30 min showed a time-dependent increase in intracellular GSH concentration. In the gamma-GCE-hepatocytes pretreated with bis-(p-nitrophenyl)phosphate (BNPP), a non-specific esterase inhibitor, an enhancement of intracellular GSH concentration was markedly reduced. gamma-GCE concentration in the gamma-GCE-hepatocytes with BNPP pretreatment was significantly higher than that in the cells without BNPP pretreatment, although there was no difference in the total amount of intracellular NP-SH, i.e., gamma-GCE, GSH, gamma-glutamylcysteine, cysteine ethyl ester, and cysteine between both gamma-GCE-hepatocytes. The present results indicate that gamma-GCE is transported into liver cells more easily than GSH itself, resulting in its conversion to GSH via esterase and glutathione synthetase within the cells.

A high-density rice genetic linkage map with 2275 markers using a single F2 population.

A 2275-marker genetic map of rice (Oryza sativa L.) covering 1521.6 cM in the Kosambi function has been constructed using 186 F2 plants from a single cross between the japonica variety Nipponbare and the indica variety Kasalath. The map provides the most detailed and informative genetic map of any plant. Centromere locations on 12 linkage groups were determined by dosage analysis of secondary and telotrisomics using > 130 DNA markers located on respective chromosome arms. A limited influence on meiotic recombination inhibition by the centromere in the genetic map was discussed. The main sources of the markers in this map were expressed sequence tag (EST) clones from Nipponbare callus, root, and shoot libraries. We mapped 1455 loci using ESTs; 615 of these loci showed significant similarities to known genes, including single-copy genes, family genes, and isozyme genes. The high-resolution genetic map permitted us to characterize meiotic recombinations in the whole genome. Positive interference of meiotic recombination was detected both by the distribution of recombination number per each chromosome and by the distribution of double crossover interval lengths.

Cloning and mapping of telomere-associated sequences from rice.

We have isolated three telomere-associated sequences from rice using cassette-ligation-mediated polymerase chain reaction (PCR). Each of the obtained clones hybridized to the terminal of one or several rice chromosome arms. The telomeres recognized by the clones displayed a high level of polymorphism between two rice varieties, Nipponbare (a japonica variety) and Kasalath (an indica variety). Variability in the chromosome termini was also detected among individual F2 progeny plants, which were derived from a cross between the two rice varieties. One clone containing telomere-associated sequences was located to one end of chromosome 5, and another clone to one end of chromosome 11. For another clone, non-allelic segregation of polymorphic hybridization bands was observed between japonica and indica rice; this clone was mapped to one end of chromosome 12 in japonica and to one end of chromosome 11 in indica rice. This indicates an exchange of termini between nonhomologous chromosomes.

cDNA sequences of three kinds of beta-tubulins from rice.

Complete nucleotide sequences of three kinds of rice beta-tubulin cDNA clones (pTUB22, R1623 and R2242) were determined. Southern hybridization indicated that these beta-tubulins consist of one gene family. Using RFLP mapping, these three beta-tubulin cDNAs were mapped to different chromosomes indicating at least three loci for the beta-tubulin gene. The deduced amino acid sequences of these cDNAs showed a high similarity to other plant beta-tubulins. The asparagine residue located at the 100th amino acid from the N-terminus of plant beta-tubulins was also conserved with these three beta-tubulins. This asparagine is thought to be responsible for the sensitivity against rhizoxin, the toxin of the pathogen of rice seedling blight, Rhizopus sp. a soil-borne microorganism. Expression of the three beta-tubulin genes was analyzed by Northern blotting and all three clones were expressed in root, the possible target tissue of rhizoxin. These results suggest that these clones are candidates of beta-tubulins targeted by rhizoxin.

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.

Skin L-tryptophan-2,3-dioxygenase and rat hair growth.

We have identified a new enzyme, skin L-tryptophan-2,3-dioxygenase (skin TDO), that catalyzes the degradation of L-tryptophan into formylkynurenine in rats. The rate of this degradation peaks in all rats at 5 to 6 weeks after birth, and also, among rats depilated at 8 weeks old, at 10 to 11 weeks after birth. We have also observed that the properties of this enzyme are closer to those of hepatic L-tryptophan-2,3-dioxygenase (hepatic TDO) than to indoleamine 2,3-dioxygenase. Although an intraperitoneal injection of L-tryptophan increased the activity of skin TDO to approximately 2.2 times greater than control values, an intraperitoneal injection of hydrocortisone and alpha-methyl-DL-tryptophan, both compounds known to affect hepatic TDO activity, had no effect on skin TDO activity. The molecular weight of skin TDO was estimated to be 16.0 kDa, which is close to the molecular weight of hepatic TDO, yet a much larger molecule than indoleamine-2,3-dioxygenase. Increased hair growth rates paralleled increased levels of skin TDO activity in 5- to 6-week-old rats, and marked increases in the activity of skin TDO occurred 2 or 3 weeks after depilation. Enzyme activity was also greatest 2 days before the time of maximum hair root length. Therefore, skin TDO may play an important role in the initiation or suppression of rat hair growth.

Cholesterol uptake of isolated rat hepatocytes is accelerated by several kinds of phosphatidylcholine.

Uptake of cholesterol by isolated rat hepatocytes in a serum-free medium was remarkably enhanced by dispersion with several kinds of phosphatidylcholine. Of the various phosphatidylcholines tested, dilinoylphosphatidylcholine had the strongest accelerating effect, while dipalmitoylphosphatidylcholine was the weakest. The abilities to accelerate cholesterol uptake were in proportion to the content of unsaturated fatty acid in the phosphatidylcholine used. It was confirmed by electron microscopy that there is no relation between the size of the cholesterol-phosphatidylcholine complex and uptake. These data suggest that recognition of unsaturated fatty acids in phosphatidylcholine by isolated cells enhances uptake of cholesterol.

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.

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.

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.

Assessment of chemoembolization therapy for primary liver cancer using a stabilized adriamycin-lipiodol suspension.

We formulated a new lipiodol-Adriamycin suspension (ADM/lipiodol, 50 mg/10 ml) that remained stable for 48 h (half-life, 25 +/- 3 days). In five cases of hepatocellular carcinoma (HCC) resected after intra-arterial infusion of this agent, the ADM concentration in the tumor was quite high and the tumor necrosis rate was more than 80% on histological examination. Over a 5-year period, 180 patients with unresectable HCC underwent transcatheter arterial embolization therapy (TAE) in the presence or absence of this agent. The regimens consisted of suspension injection alone (A, n = 54), suspension injection + TAE using gelatin sponge (B, n = 29), TAE followed by suspension injection (C, n = 34), and TAE alone (D, n = 63). The estimated 1-year survival values determined for patients treated with these regimens were 70%, 73%, 43%, and 39% respectively, and the corresponding 3-year survival values were 27%, 31%, 15%, and 10%. The survival achieved using suspension injection was thus superior to that obtained using conventional TAE, and combined therapy with suspension injection followed by TAE seemed to enhance survival, although there were some biases in tumor size and in the stage of tumor progression. For patients with tumors measuring 5 cm or more in diameter, the survival obtained using regimen A was lower than that achieved using regimen D, but the combination of TAE and suspension injection improved the 1-year survival value obtained using regimen D from 34% to 52%. For patients with tumors measuring less than 5 cm in diameter, the survival achieved using regimen A was markedly better than that obtained using regimen D, although no difference was found between the survival value achieved using regimen A and that obtained using regimens B and C. On the basis of these results, our newly formulated ADM-lipiodol suspension was surmised to be effective by itself against relatively small HCC tumors, whereas it enhanced the efficacy of conventional TAE in large lesions.

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.

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.

Early detection of successful coronary reperfusion based on serum concentration of human heart-type cytoplasmic fatty acid-binding protein.

Both human heart-type cytoplasmic fatty acid-binding protein (H-FABPc) and myoglobin are low molecular weight proteins that are abundant in the cytoplasm of myocardial cells. Unlike myoglobin, H-FABPc content in the skeletal muscle is less than in cardiac muscle. To investigate the usefulness of the serum concentration of H-FABPc in the early detection of successful coronary reperfusion, we measured serum concentrations of H-FABPc and myoglobin in 45 patients with acute myocardial infarction treated with intracoronary thrombolysis or direct percutaneous transluminal coronary angioplasty. Coronary angiography was performed every 5 min for reperfusion therapy to identify the onset of reperfusion. Reperfusion, defined as a TIMI grade 2 or 3, was achieved within 60 min of the initiation of reperfusion therapy in 30 patients (the reperfused group), but was not achieved in 15 patients (the non-reperfused group). Blood samples were obtained before initiation of treatment and 15, 30 and 60 min after initiation of treatment in the non-reperfused group. In the reperfused group, samples were obtained before reperfusion and 15, 30 and 60 min after reperfusion. The H-FABPc ratio (the ratio of value after to value before the initiation of treatment or reperfusion) increased sharply after the onset of reperfusion, peaking at 41 +/- 18 min, and decreased rapidly thereafter. The predictive accuracy of an H-FABPc ratio of > 1.8 for the detection of reperfusion within 60 min of the initiation of treatment was 93% at 15 min after reperfusion, 98% at 30 min, and 100% at 60 min. Similar rates of predictive accuracy were observed for a myoglobin ratio > 2.4. The H-FABPc ratio detected successful reperfusion as early as 15 min after the onset of reperfusion and was highly accurate in detecting reperfusion within 60 min of the onset of reperfusion. The predictive accuracy of the H-FABPc ratio was similar to that of the myoglobin ratio for the early detection of successful coronary reperfusion.

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 the L-tryptophan transport system in the liver of growing rats.

In order to characterize the system of L-tryptophan (TRP) transport into liver during the growing period of 10 to 42 days, the changes of tryptophan 2,3-dioxygenase (TDO) activity, levels of serum, liver, brain, and muscle TRP, and the rate and mode of TRP uptake into isolated hepatocytes were examined in male Wistar rats. Liver TDO activity increased rapidly at 16 days of age. A marked and rapid decrease in free serum TRP level occurred before weanling, while a small decrease in total serum TRP level was found after weanling. The change of liver TRP level was similar to that of free serum TRP level and correlated well. There was a significant inverse correlation between liver TDO activity and either free serum TRP level or liver TRP level. A rapid change in TRP level did not occur in brain and muscle during the growing period. The concentrations of brain and muscle TRP correlated better with those of total serum TRP than with those of free serum TRP. The rate of TRP uptake into hepatocytes isolated from rats aged 10 days was lower than that from rats aged 21 and 42 days. The former hepatocytes were lacking in a high-affinity saturable transport component for TRP uptake which was present in the latter ones. The present results indicate that a great change in the system of TRP transport into liver occurs in growing rats, and that in suckling rats a high level of free serum TRP contributes to the efficient transport of the amino acid into the 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.

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.