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Biomedical subjects

K Harada

Publications and source records attributed to K Harada.

At least 73 records · Page 4Linked to original sources
PubMed2001

Abnormal elution behavior of ornitine derivatized with 1-fluoro-2,4-dinitrophenyl-5-leucinamide in advanced Marfey's method.

During the course of establishing the advanced Marfey's method that has been developed to non-empirically determine the absolute configuration of constituent amino acids in a peptide using LC-MS, we encountered the "ornitine mystery" in the di-DLA (2,4-dinitrophenyl-5-leucinamide) derivative such that the elution order of ornitine (Orn) was opposite (D-->L) in spite of their relatively long retention time. In order to resolve this problem, the elution behavior of several mixed DLA and DPEA (2,4-dinitrophenyl-5-phenylethylamine) derivatives with different absolute configurations was carefully observed during HPLC. The length of the methylene chain in basic amino acids was obviously critical for this behavior, because Dab (2,4-diamino-n-butyric acid) and lysine (Lys) did not exhibit this abnormality. The presence of the carboxyamide moiety at the omega position was also essential for this phenomenon, because it was never observed in the DPEA derivatives at the omega position. Furthermore, it was found that the following combination of absolute configurations of Orn and DLA at the omega position only induced this abnormality: D-Orn and L-DLA, and L-Orn and D-DLA. This suggested that the structural interaction such as hydrogen bonding between the carboxyamide of DLA at the omega position and carboxylic acid at the alpha position in these derivatives reduced their retention power on the reversed-phase column.

Amino Acids↗
PubMed2001

In vitro effects of E3040, a dual inhibitor of 5-lipoxygenase and thromboxane A(2) synthetase, on eicosanoid production.

In vitro pharmacological profiles of E3040, 6-hydroxy-5, 7-dimethyl-2-(methylamino)-4-(3-pyridylmethyl) benzothiazole were investigated. Against the 5-lipoxygenase activity of rat basophilic leukemia cells, E3040 and zileuton (a 5-lipoxygenase inhibitor) had an IC(50) of 0.23 and 0.93 microM, respectively. Against the thromboxane A(2) synthetase activity of human platelets, E3040 had an IC(50) of 0.01 microM, which was comparable to that of OKY-1581 (sodium (E)-3-[4-(3-pyridylmethyl) phenyl]-2-methylacrylate, a thromboxane A(2) synthetase inhibitor). Against cyclooxygenase activity of sheep seminal vesicles, E3040 showed no inhibition (IC(50), >300 microM). Sulfasalazine and 5-aminosalicylic acid, therapeutic drugs for inflammatory bowel disease, inhibited 5-lipoxygenase activity with an IC(50) of 293 and 970 microM, respectively. Sulfasalazine inhibited thromboxane A(2) synthetase activity with an IC(50) of 20 microM. In rat peritoneal leukocytes, E3040 inhibited leukotriene B(4) and thromboxane B(2) production with an IC(50) of 0.17 and 0.24 microM, respectively. E3040 inhibited leukotriene B(4) production in human neutrophils and thromboxane B(2) production in human platelets (IC(50) of 0.21 and 0.09 microM, respectively). These results indicated that E3040 potently inhibited 5-lipoxygenase and thromboxane A(2) synthetase and blocked leukotriene B(4) and thromboxane B(2) production in rat peritoneal and human blood cells.

Animals↗
PubMed2001

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.

Chromosome Mapping↗
PubMed2001

Cloning of hypoxia-inducible factor 1alpha cDNA from chick embryonic ventricular myocytes.

Hypoxia-inducible factor 1 (HIF-1) is composed of HIF-1alpha and arylhydrocarbon nuclear receptor translocator (ARNT), which belong to the basic-helix-loop-helix-Per/ARNT/Sim (bHLH-PAS) family of transcription factors. HIF plays key roles in oxygen homeostasis and embryonic cardiovascular development. In this study, we have cloned cDNAs encoding the chick HIF-1alpha from cultured embryonic ventricular myocytes (CEVM) and then examined its expression in various embryonic tissues. The deduced amino acid sequence of the chick HIF-1alpha cDNA showed 79% identity with that of the human HIF-1alpha cDNA. In contrast, sequence homology between the chick HIF-1alpha and endothelial PAS protein 1 (EPAS1), another member of the bHLH-PAS proteins, was only low (49%). HIF-1alpha mRNA was expressed abundantly in CEVM, but scarcely in the liver, which was quite different from expression pattern of EPAS1 mRNA. These data suggest that HIF-1alpha may be involved in embryonic cardiovascular development. HIF-1alpha and EPAS1 may play distinct roles during developmental stages.

Amino Acid Sequence↗
PubMed2001

Interaction of p130 with, and consequent inhibition of, the catalytic subunit of protein phosphatase 1alpha.

The protein p130 was originally isolated from rat brain as an inositol 1,4,5-trisphosphate-binding protein with a domain organization similar to that of phospholipase C-delta1 but which lacks phospholipase C activity. Yeast two-hybrid screening of a human brain cDNA library for clones that encode proteins that interact with p130 has now led to the identification of the catalytic subunit of protein phosphatase 1alpha (PP1calpha) as a p130-binding protein. The association between p130 and PP1calpha was also confirmed in vitro by an overlay assay, a "pull-down" assay, and surface plasmon resonance analysis. The interaction of p130 with PP1calpha resulted in inhibition of the catalytic activity of the latter in a p130 concentration-dependent manner. Immunoprecipitation and immunoblot analysis of COS-1 cells that stably express p130 and of mouse brain extract with antibodies to p130 and to PP1calpha also detected the presence of a complex of p130 and PP1calpha. The activity of glycogen phosphorylase, which is negatively regulated by dephosphorylation by PP1calpha, was higher in COS-1 cells that stably express p130 than in control COS-1 cells. These results suggest that, in addition to its role in inositol 1,4,5-trisphosphate and Ca(2+) signaling, p130 might also contribute to regulation of protein dephosphorylation through its interaction with PP1calpha.

Catalysis↗
PubMed2001

Apoptotic and necrotic brain lesions in a fatal case of carbon monoxide poisoning.

A 41-year-old man was accidentally exposed to carbon monoxide (CO) gas and found in a state of cardiopulmonary arrest while he took bath. After admission, he was resuscitated and underwent artificial ventilation in a comatose state and died about 19h later. Computed tomography (CT) examination disclosed bilateral low density area in the basal ganglia and the thalamus, a well-known finding in the CO intoxication. Necropsy, histological examination, DNA ladder assay gave the first line of evidence for the presence of apoptosis as well as necrosis in the human case of CO intoxication. TdT-mediated dUTP-biotin nick-end labeling (TUNEL) positive apoptotic cells were more predominant in the CA2 area than in CA1 area. There is general co-relation between the ratio of TUNEL-positive cells and the DNA laddering on the agarose gel. Basal ganglia and thalamus, which showed bilateral low density area in CT, were revealed to be severe edema. The two types of cell death occurred in the cortex, basal ganglia, hippocampus, thalamus, and cerebellum. Hypoxia caused by CO-hemoglobin formation alone cannot explain the phenomena.

Adult↗
PubMed2001

Asialoglycoprotein receptor deficiency in mice lacking the major receptor subunit. Its obligate requirement for the stable expression of oligomeric receptor.

The asialoglycoprotein receptor is an abundant hetero-oligomeric endocytic receptor that is predominantly expressed on the sinusoidal surface of the hepatocytes. A number of physiological and pathophysiological functions have been ascribed to this hepatic lectin (HL), the removal of desialylated serum glycoproteins and apoptotic cells, clearance of lipoproteins, and the sites of entry for hepatotropic viruses. The assembly of two homologous subunits, HL-1 and HL-2, is required to form functional, high affinity receptors on the cell surface. However, the importance of the individual subunits for receptor transport to the cell surface is controversial. We have previously generated HL-2-deficient mice and showed that the expression of HL-1 was significantly reduced, and the functional activity as the asialoglycoprotein receptor was virtually eliminated. However, we failed to detect phenotypic abnormalities. To explore the significance of the major HL-1 subunit for receptor expression and function in vivo, we have disrupted the HL-1 gene in mice. Homozygous HL-1-deficient animals are superficially normal. HL-2 expression in the liver is virtually abrogated, indicating that HL-1 is strictly required for the stable expression of HL-2. Although these mice are almost unable to clear asialo-orosomucoid, a high affinity ligand for asialoglycoprotein receptor, they do not accumulate desialylated glycoproteins or lipoproteins in the plasma.

Animals↗
PubMed2001

Monocyte chemotactic protein-1, -2, and -3 are distinctively expressed in portal tracts and granulomata in primary biliary cirrhosis: implications for pathogenesis.

The monocyte chemotactic proteins (MCPs) form a distinct structurally related subclass of C-C chemokines. MCPs select specific target cells due to binding to a distinct set of chemokine receptors and because of their effects on monocytes, and may participate in the process of granuloma formation during bacterial and/or mycobacterial infections. The aetiology of primary biliary cirrhosis (PBC) is still unclear, although bacterial infection and autoimmune processes have been implicated. In this study, the expression of three of the most potent monocyte chemoattractants, MCP-1, -2, and -3, was examined in patients with PBC and the data were compared with results for other liver diseases including primary sclerosing cholangitis (PSC), chronic viral hepatitis C, hepatic sarcoidosis, and normal liver. MCP-1 was expressed mainly in biliary epithelial cells of all liver specimens, irrespective of the cause of disease. Some mononuclear leukocytes in the portal tract expressed MCP-1 in all the disease groups examined and there were no significant differences in frequency between these groups. In contrast, more than 80% of PBC livers showed MCP-2- and MCP-3-positive mononuclear leukocyte infiltration in portal tracts, particularly around the bile ducts, whereas such cells were far less frequent in the other disease groups or in normal livers. Epithelioid granulomata of PBC patients contained MCP-2- and MCP-3-positive cells at their edge. In double staining experiments, more than 60% of the MCP-positive mononuclear cells co-expressed CD68, suggesting that a proportion of MCP-2- and MCP-3-positive cells are derived from monocytes. These monocytes expressing MCP-2 and MCP-3 may be responsible for the chemotactic activity of more monocytes. Such an expression pattern of MCP-1, -2 and -3 in portal tracts seems to be distinctive for PBC. This pattern underlines the importance of MCP-1, -2, and -3 in the recruitment of monocytes and possibly T lymphocytes into portal tracts, around the injured bile ducts, and into epithelioid granulomata in PBC. The data further implicate bacterial materials derived from bile in the overall pathogenesis of PBC.

Adult↗
PubMed2001

Frequent molecular identification of Campylobacter but not Helicobacter genus in bile and biliary epithelium in hepatolithiasis.

Bacterial infection of the biliary tree and bile stasis may be causally related to hepatolithiasis, but which bacterial species are involved and their roles in the pathogenesis of hepatolithiasis have not been ascertained. Recently, the Helicobacter genus was detected in human bile and biliary mucosal samples by molecular techniques, and its association with several biliary diseases has been suggested. The Campylobacter genus, which is closely related to the Helicobacter genus, has also recently been identified as causative of human gastrointestinal diseases. This study attempted to elucidate whether Helicobacter and/or Campylobacter bacteria are present in bile samples and biliary mucosal specimens from hepatolithiasis patients and whether they are involved in the pathogenesis of hepatolithiasis. The 16S rRNA gene of the Helicobacter and of the Campylobacter genus was examined by polymerase chain reaction in DNA samples extracted from bile and/or microdissected biliary epithelium from 69 patients with hepatolithiasis and control patients with choledocholithiasis, cholecystolithiasis, and normal gall bladders. The Helicobacter genus was detected in 1 of 8 (13%) biliary epithelial samples in hepatolithiasis and 1 of 10 (10%) bile samples in choledocholithiasis. The Campylobacter genus was detected in 3 of 14 (21%) bile samples and 5 of 8 (63%) epithelial samples in hepatolithiasis, and in 2 of 15 (13%) bile samples and 1 of 8 (13%) epithelial samples in cholecystolithiasis. The detection rate for Campylobacter in biliary epithelium of hepatolithiasis was significantly higher than in the bile or biliary epithelium of control groups (p<0.05). By a phylogenetic analysis based on nucleotide sequences, the Campylobacter genuses detected in hepatolithiasis were clustered with C. rectus or C. showae. The frequent detection of the Campylobacter 16S rRNA gene in bile, and especially in biliary epithelium of hepatolithiasis, suggests a pathogenetic relationship with Campylobacter infection.

Aged↗
PubMed2001

PCR and in situ hybridization studies of telomerase subunits in human non-neoplastic livers.

Telomerase, a ribonucleoprotein enzyme associated with cellular immortality, consists of human telomerase RNA component (hTERC), human telomerase protein 1 (hTEP1), and human telomerase reverse transcriptase (hTERT). In this study, the expression of these subunits was examined in non-neoplastic livers [13 cases of chronic viral hepatitis (CVH), 16 of primary biliary cirrhosis (PBC), two of primary sclerosing cholangitis, and six normal livers], using the reverse transcription-polymerase chain reaction (RT-PCR), nested PCR, and in situ hybridization (ISH). Six hepatocellular carcinoma (HCC) cases and one colonic cancer were used as positive controls. Telomeric repeat amplification protocol (TRAP) assay disclosed distinct telomerase activity in all positive controls and weak telomerase activity in non-neoplastic livers in 4 of 13 CVH cases and 5 of 16 PBC cases. By RT- and nested PCR, both hTERC and hTEP1 mRNA were detectable in all non-neoplastic liver tissues; ISH revealed hTERC and hTEP1 mRNA in the periportal and periseptal hepatocytes and inflammatory mononuclear cells in those cases examined. ISH revealed hTERT mRNA only in a few infiltrating mononuclear cells in 3 of 13 CVH and 2 of 16 PBC livers and these five cases were also positive by TRAP assay. In four of these five cases, hTERT mRNA was also detectable by nested PCR, suggesting that hTERT mRNA in the non-neoplastic liver is expressed by infiltrating mononuclear cells. Biliary epithelial cells were totally negative for these human telomerase subunits. Three subunits were constantly detected in all positive controls by ISH as well as by RT- and nested PCR. The finding that hTERC and hTEP1 mRNA, but not hTERT mRNA, were detectable in the non-neoplastic hepatocytes suggests that telomerase is present but not activated and that additional factor(s) are necessary for the expression of hTERT mRNA in the hepatocytes, along with immortalization and neoplastic transformation.

Adult↗
PubMed2001

Miniaturized pyrosequencer for DNA analysis with capillaries to deliver deoxynucleotides.

As the human genome project proceeds, various types of DNA analysis tools are required for life sciences and medical sciences including DNA diagnostics. For example, a small DNA sequencer for sequencing a short DNA is required for bed-side DNA testing as well as DNA analysis in a small laboratory. Here, a new handy DNA sequencing system (pyrosequencer) based on the detection of inorganic pyrophosphate (PPi) released by polymerase incorporation is demonstrated. The system uses the bioluminescence detection system. The key point for the miniaturized DNA sequencer is to make a deoxynucleotide triphosphate (dNTP) delivery system small and inexpensive. It has been realized by using narrow capillaries to connect a reaction chamber and four dNTP reservoirs. Each dNTP is introduced into the reaction chamber by applying a pressure to the reservoir. Compared with other microdispensers, it is much cheaper and easier. By optimizing the conditions, an excellent sequencing ability is achieved while it is a simple and inexpensive system. In most cases, more than 40 bases can be successfully sequenced. A homopolymeric region, which can not be easily sequenced by a conventional gel-based DNA sequencer, is readily sequenced with this system. The new system is successfully applied to sequence a GC rich region or a region close to a priming region where misreading frequently occurs. A rapid analysis for a short DNA was easily achieved with this small instrument.

Base Sequence↗
PubMed2001

Identification of recurrent chromosomal rearrangements and the unique relationship between low-level amplification and translocation in glioblastoma.

To elucidate the structural abnormalities and the relationship between chromosome structural disorders and DNA copy number aberrations in tumor cells, we applied the techniques of spectral karyotyping (SKY), comparative genomic hybridization (CGH), and fluorescence in situ hybridization (FISH), using yeast artificial chromosome (YAC) probes for nine human glioblastoma cell lines. One striking finding was that independently derived cell lines had the same recurrent marker chromosomes. Seven recurrent chromosomes were detected by these cytogenetic methods. In particular, cell lines U251, SNB-19, and U373-MG showed very similar karyotypes. It is also interesting that regions of DNA amplification were found translocated and/or inserted at a high rate (91.7%). In all, there were 12 amplified loci in five of the nine cell lines. These amplified chromosomal bands were scattered on the chromosomes, including the normal chromosome, with one exception (7q32-qter in U373-MG). FISH with YAC clones mapping to these chromosomal regions as DNA probes often showed DNA probe signals not only at original chromosomal sites but also in translocated or inserted segments. This form of DNA amplification was characterized by low-level increases (four- to 10-fold) and by translocation or insertion of the relevant chromosomal locus. These studies shed light on typical derivative chromosomes and the relationship between DNA amplification and chromosomal translocation in glioblastoma.

Chromosome Aberrations↗
PubMed2001

The toxicities of a polyunsaturated fatty acid and a microcystin to Daphnia magna.

Polyunsaturated fatty acids (PUFAs) are essential components of zooplankton diets. However, studies with PUFAs from cyanobacteria indicate toxic properties. We investigated the toxicity of the PUFA gamma-linolenic acid and the cyanobacterial peptide toxin microcystin-LR to Daphnia. The PUFA was acutely toxic at a concentration of 9 micrograms ml-1. The effect of microcystin-LR was not statistically significant at the concentration used (3 micrograms ml-1), but an additive effect with the PUFA was observed. Relative to LC50-values of well-known pollutants, the PUFA was intermediately toxic. The activity equaled that of microcystin-LR, which is commonly treated as one of the most potent cyanobacterial toxins. Our results suggest that the toxic properties of PUFAs deserve more attention.

Animals↗
PubMed2001

Co-production of microcystins and aeruginopeptins by natural cyanobacterial bloom.

The relationship between Microcystis composition and the production of microcystins and nontoxic peptides in bloom cells, which was regularly collected in Lake Suwa, Japan, in the summer season from 1991 to 1994, was investigated. In order to determine the structures of the nontoxic peptides, we collected large amounts of bloom materials from the same lake on July 23, 1991, and isolated three nontoxic peptides. They were named as aeruginopeptins 917S-A, -B, and -C, and their structures were mainly determined by a mass spectrometry/mass spectrometry (MS/MS) technique as 19-membered cyclic depsipeptides possessing the Ahp (3-amino-6-hydroxy-2-piperidone) moiety. An analysis of the microcystins and aeruginopeptins in the collected blood cells and their Microcystis composition suggested that the M. aeruginosa large cell size produces both microcystins and aeruginopeptins, and the production of both compounds is genetically closely related.

Cell Size↗
PubMed2001

Functional consequences of the mutations in human cardiac troponin I gene found in familial hypertrophic cardiomyopathy.

Functional consequences of the six mutations (R145G, R145Q, R162W, DeltaK183, G203S, K206Q) in cardiac troponin I (cTnI) that cause familial hypertrophic cardiomyopathy (HCM) were studied using purified recombinant human cTnI. The missense mutations R145G and R145Q in the inhibitory region of cTnI reduced the intrinsic inhibitory activity of cTnI without changing the apparent affinity for actin. On the other hand, the missense mutation R162W in the second troponin C binding region and the deletion mutation DeltaK183 near the second actin-tropomyosin region reduced the apparent affinity of cTnI for actin without changing the intrinsic inhibitory activity. Ca(2+) titration of a fluorescent probe-labeled human cardiac troponin C (cTnC) showed that only R162W mutation impaired the cTnC-cTnI interaction determining the Ca(2+) affinity of the N-terminal regulatory domain of cTnC. Exchanging the human cardiac troponin into isolated cardiac myofibrils or skinned cardiac muscle fibers showed that the mutations R145G, R145Q, R162W, DeltaK183 and K206Q induced a definite increase in the Ca(2+)-sensitivity of myofibrillar ATPase activity and force generation in skinned muscle fibers. Although the mutation G203S also showed a tendency to increase the Ca(2+) sensitivity in both myofibrils and skinned muscle fibers, no statistically significant difference compared with wild-type cTnI could be detected. These results demonstrated that most of the HCM-linked cTnI mutations did affect the regulatory processes involving the cTnI molecule, and that at least five mutations (R145G, R145Q, R162W, DeltaK183, K206Q) increased the Ca(2+) sensitivity of cardiac muscle contraction.

Actins↗
PubMed2001

Endogenous nitric oxide and endothelin-1 in persistent pulmonary hypertension of the newborn.

UNLABELLED: We studied changes in endogenous nitric oxide (NO) synthesis and endothelin-1 (ET-1) production in infants with persistent pulmonary hypertension of the newborn (PPHN). We determined concentrations of serum NO metabolites, i.e., nitrites and nitrates (NOx), and of plasma ET-1 in five infants with PPHN (PPHN group) and in 25 healthy full-term neonates (control group). In both groups, serum NOx concentrations increased over time and plasma ET-1 concentrations decreased with age. The differences in serum NOx concentrations between groups were not significant at < 12 h and 24 h of age; however, they were significantly higher in the PPHN group than in the control group at 5 days of age. The differences in plasma ET-1 concentrations between groups were not significant at 5 days of age, but were significantly higher in the PPHN group than in the control group at < 12 h and 24 h of age. CONCLUSION: Limited endogenous nitric oxide synthesis and elevated endogenous endothelin-1 production during the first few days of life may contribute to pulmonary hypertension in infants with persistent pulmonary hypertension of the newborn.

Age Factors↗