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Biomedical subjects

K Harada

Publications and source records attributed to K Harada.

At least 631 records · Page 35Linked to original sources
PubMed1994

Characterization of insoluble macromolecular Sn(II) complex and its application to the 99mTc labeling of human serum albumin-bearing mercapto groups.

A chelating ion exchange resin containing aminophosphonic acid groups was used as a polymer matrix for the preparation of an insoluble macromolecular Sn(II)(R-Sn) complex. Sn(II), which strongly bound to the surface of the polymer matrix by chelation, retained the ability to reduce 99mTc in the R-Sn complex. Human serum albumin-bearing-mercapto groups (HMA) was labeled with 99mTc at pH 2-3 using the R-Sn complex or SnCl2 as reducing agent. The 99mTc-HMA labeled with the R-Sn complex resulted in a higher sustained level of radioactivity in the blood of mice than the 99mTc-HMA labeled with SnCl2. These results suggest that the use of the R-Sn complex minimized Sn(II) contamination of the 99mTc labeling solution and can be used effectively as a reducing agent for 99mTc labeling of proteins containing sulfhydryl groups.

Animals↗
PubMed1994

Molecular analysis of Gpdh null mutations that arose in mutation accumulation experiments in Drosophila melanogaster.

In order to clarify the cause of null mutations in enzyme loci, the molecular structure of six null mutations in the Gpdh locus (encoding alpha GPDH: alpha glycerol-3-phosphate dehydrogenase (NAD+), E.C. 1.1.1.8; map position at 2-17.8) that arose in mutation accumulation experiments was examined. A restriction map analysis showed that five of the mutations are insertional mutations whereas the sixth is a deletion. The Gpdh regions of these null mutations were then cloned and sequenced. The inserted DNA fragments are all internally deleted P elements measuring 1.1 kb in length. Two are a KP element and two others are a HP element. All the insertions occur in the region near the initiation signal of transcription. The deletion encompasses the seventh and eighth exons over a length of 1.1 kb. These results therefore indicate that the null mutation rate at the Gpdh locus is largely influenced by P elements.

Animals↗
PubMed1994

Simultaneous stereoinversion and isomerization at specific aspartic acid residues in alpha A-crystallin from human lens.

We characterized the primary structure of alpha A-crystallin from the lens of the human eye by detailed analyses of the amino acid sequence, mass, and stereoisomers, and found the biologically uncommon D- and beta-aspartic acid (Asp) residues in the protein. The stereoconfiguration of the Asp151 and Asp58 residues in alpha A-crystallin from old subjects (mean age: 80 years) was inverted to the D-isomer, and the residues were simultaneously isomerized to beta-aspartyl residues, which may occur via a succinimide intermediate. This is thought to be the first observation of stereoinversion of amino acids in protein in vivo. It is noteworthy that similar stereoinversion was observed in the same residues of alpha A-crystallin from young subjects (age: 11 months), with simultaneous isomerization, although the extent of isomerization was low compared with that in the aged. The conversion may take place in the early life of the lens and the resulting isomers may accumulate with aging. We also found that terminal serine173 was cleaved in aged alpha A-crystallin. Since the cleavage was not observed in young alpha A-crystallin, it may result from the in vivo aging process. The present findings also make it necessary to revise the amino acid sequence of human alpha A-crystallin presented previously: human alpha A-crystallin is composed of 173 (formerly reported as 172) amino acid residues, and the sequence from residues 153 to 155 is THA (formerly reported as -HT).

Aging↗
PubMed1994

Mouse 230-kDa bullous pemphigoid antigen gene: structural and functional characterization of the 5'-flanking region and interspecies conservation of the deduced amino-terminal peptide sequence of the protein.

The 230-kDa bullous pemphigoid antigen is a hemidesmosomal protein of the cutaneous basement membrane zone. The primary sequences deduced from full-length human cDNAs predict that this molecule consists of a central rod region and flanking globular domains. To get insight into regulation of the 230-kDa bullous pemphigoid antigen gene (BPAG1), and to evaluate evolutionary conservation of the amino-terminus of the protein, we screened a mouse genomic DNA library with a 0.3-kb cDNA corresponding to the 5' end of the human 230-kDa bullous pemphigoid antigen cDNA. A positive clone was isolated, and Southern analysis of the clone with the 0.3-kb cDNA allowed isolation of a 3.0-kb Hind III fragment containing the 5' end of the coding sequence. Alignment of the sequences of this subclone and human BPAG1 sequences revealed that this fragment contained 2466 bp of 5'-flanking DNA, upstream from the ATG translation initiation site, and 258 bp of translatable sequences that encode a putative polypeptide of 86 amino acids at the amino-terminus of the protein. This deduced polypeptide showed 91% homology with the corresponding human sequence. The TATAAA and CCAAT consensus sequences, as well as several putative cis-regulatory elements, were identified in the 5'-flanking region of the mouse DNA. To test the functional promoter activity of the 5'-flanking DNA, three mouse BPAG1 promoter/CAT reporter gene constructs, with the promoter segments spanning from -1133, -525, and -213 to -1, were developed. Transient transfections of mouse transformed keratinocytes (Pam 212 cells) with these constructs revealed clearly detectable CAT activities, indicating that the 5'-flanking region contains a functional promoter. Furthermore, these experiments suggested that the upstream sequences contain upregulatory elements, as well as elements that confer, at least in part, tissue specificity to the expression of the mouse 230-kDa BPA gene.

Amino Acid Sequence↗
PubMed1994

Cross-reactivity and neutralizing ability of monoclonal antibodies against microcystins.

Monoclonal antibodies (MAbs) against the microcystin-leucine-arginine variant (MCYST-LR), a cyclic peptide toxin of the freshwater cyanobacterium Microcystis aeruginosa, were prepared from cloned hybridoma cell lines. The specificity of the MAbs and their ability to neutralize the toxin were investigated by an indirect enzyme-linked immunosorbent assay (ELISA) and by a neutralizing test in mice, respectively. All MAbs reacted with MCYST-LR and also with the microcystin-arginine-arginine variant (MCYST-RR), 3,7-didesmethylmicrocystin (MCYST-3, 7-dDMLR) and 7-desmethylmicrocystin (MCYST-7-DMLR). Furthermore, the antibodies reacted with cell-extracts of toxic and non-toxic M. aeruginosa strains. The MAbs can apparently recognize the common configuration, but not the variant-specific structure, in the microcystin molecules. The non-toxic strains apparently contain some substance(s) related antigenically to microcystin. The in vivo toxin-neutralizing ability of MAbs was minimal.

Animals↗
PubMed1994

High detection rate of breast cancer by mass screening using mammography in Japan.

A trial of mass screening for breast cancer using both mammography and physical examination (independently) was carried out in women over 50 years of age in Tokushima Prefecture. Breast cancer was detected in eight of a total of 950 examinees by mammography alone, and no cases of breast cancer were detected by physical examination. The detection rate of breast cancer was 0.84%, which is very much higher than that obtained by conventional mass screening using physical examination alone. The detection rate increased especially in the sixth and seventh decades of life. All eight detected breast cancers were in the early stage. Based on these results, it is recommended to employ mammography in breast cancer screening for asymptomatic women aged over 50 years.

Adult↗
PubMed1994

Central nervous system abnormalities in chromosome deletion at 11q23.

Two Japanese pediatric patients with terminal deletion of the long arm of chromosome 11 are described. Both had the morphological abnormalities of the 11q deletion syndrome, such as prominent epicanthal folds, broad flat nasal bridge with short, upturned nose, short philtrum with carp-shaped mouth, cardiac anomalies and nonprogressive moderate psychomotor developmental delay. Patient 1 is the first case to be reported with 11q deletion with serial magnetic resonance (MR) examinations of cerebral white matter. The initial MR imaging studies demonstrated multiple areas of T1 and T2 prolongation in the cerebral white matter in both patients at the ages of 2 5/12 and 2 1/12 years, respectively. A second MR imaging, performed 1 year after the first in Patient 1, demonstrated slight improvement of the lesions. Neither patient showed clinical deterioration. These results suggest that the lesions were caused by delayed myelination, rather than by demyelination. It is suggested that an unknown factor which is important for myelination is located on the long arm of chromosome 11: perhaps the neural cell adhesion molecule (NCAM).

Abnormalities, Multiple↗
PubMed1994

A multicenter, randomized, controlled trial of intravenous gamma globulin therapy in children with acute Kawasaki disease.

We studied the effect of intravenous, polyethyleneglycol-treated, human immunoglobulin, administered at 200 mg/kg per day (group A: n = 147; male 86, female 61; age < 1 year, 50) or 400 mg/kg per day (group B: n = 152; male 87, female 65; age < 1 year, 52) for five consecutive days and compared it with freeze-dried, sulfonated human immunoglobulin [group C: n = 152; male 87, female 65; age < 1 year, 51), administered at 200 mg/kg per day for five consecutive days, on the prevention of coronary artery abnormalities in Kawasaki disease. Echocardiograms were interpreted blindly and independently. Proportions of 87.1%, 95.4%, and 82.3% in groups A, B, and C, respectively, had no coronary artery abnormalities. The confidence limits of difference between the proportions of groups A and C, groups B and C, and groups B and A were -4.4% and 10.4%, 7.8% and 15.9%, and 4.0% and 10.8%, respectively. Duration of fever and serum immunoglobulin G (IgG) levels were correlated with the prevalence of coronary artery abnormalities. We concluded that intravenous, polyethyleneglycol-treated, human immunoglobulin and freeze-dried, sulfonated human immunoglobulin had clinically equivalent effects on coronary artery abnormalities, and that five daily doses of 400 mg/kg of intravenous, polyethyleneglycol-treated, human immunoglobulin is more effective than that of 200 mg/kg gamma globulin.

Acute Disease↗
PubMed1994

[M-typing of Streptococcus pyogenes by latex agglutination using monoclonal antibody. Streptococcal Diseases Study Group].

The Lancefield's serotyping system of Streptococcus pyogenes is based on the M protein. The typing sera used for this system are prepared in rabbits immunized with whole organisms of specific serotypes. To remove cross reactive antibodies, the sera should be extensively absorbed with organisms of selected heterologous serotypes. In this study, the possibility of using monoclonal antibody (MAb) as a type specific reagent was discussed. MAb which specifically reacted to M type 12 protein of S. pyogenes were produced by cell fusion. This specific MAb reacted to hot acid extracted M type 12 protein in ELISA but didn't react in agglutination and precipitation. Latex beads were sensitized with MAb and examined by the coagglutination method. The latex reagent could detect a very small amount of M type 12 protein, so it could be used for M typing of S. pyogenes which produced a small amount of M 12 protein.

Animals↗
PubMed1994

Expression of the Bacillus subtilis spoIVCA gene, which encodes a site-specific recombinase, depends on the spoIIGB product.

The Bacillus subtilis spoIVCA gene encodes a site-specific recombinase which creates a sigK gene by DNA rearrangement. We have determined the transcription initiation point of the spoIVCA gene and found that (i) the spoIVCA promoter contains sequences which are similar to -10 and -35 regions of promoters recognized by sigma E and (ii) mutation of spoIIGB, which encodes pro-sigma E, blocked the expression of spoIVCA.

Bacillus subtilis↗
PubMed1994

Structure and regulation of the human interferon regulatory factor 1 (IRF-1) and IRF-2 genes: implications for a gene network in the interferon system.

Interferon regulatory factor 1 (IRF-1) and IRF-2 are structurally similar DNA-binding factors which were originally identified as regulators of the type I interferon (IFN) system; the former functions as a transcriptional activator, and the latter represses IRF-1 function by competing for the same cis elements. More recent studies have revealed new roles of the two factors in the regulation of cell growth; IRF-1 and IRF-2 manifest antioncogenic and oncogenic activities, respectively. In this study, we determined the structures and chromosomal locations of the human IRF-1 and IRF-2 genes and further characterized the promoters of the respective genes. Comparison of exon-intron organization of the two genes revealed a common evolutionary structure, notably within the exons encoding the N-terminal portions of the two factors. We confirmed the chromosomal mapping of the human IRF-1 gene to 5q31.1 and newly assigned the IRF-2 gene to 4q35.1, using fluorescence in situ hybridization. The 5' regulatory regions of both genes contain highly GC-rich sequences and consensus binding sequences for several known transcription factors, including NF-kappa B. Interestingly, one IRF binding site was found within the IRF-2 promoter, and expression of the IRF-2 gene was affected by both transient and stable IRF-1 expression. In addition, one potential IFN-gamma-activated sequence was found within the IRF-1 promoter. Thus, these results may shed light on the complex gene network involved in regulation of the IFN system.

Animals↗
PubMed1994

Left ventricular preload reserve in preterm infants with patent ductus arteriosus.

The left ventricular Frank-Starling response was studied in 15 preterm infants, less than 1500 g birth weight, and in 16 fullterm infants with patent ductus arteriosus. Left ventricular end diastolic volume (LVEDV), stroke volume, and cardiac output were calculated from biplane echocardiographic images with a modified Simpson's rule, and the left ventricular function curve was obtained by standardising with birth weight and body length. In the relationship between LVEDV and stroke volume, the slope of the regression line was significantly milder in preterm than in fullterm infants; however, there was no significant difference in the relationship between LVEDV and cardiac output. The heart rate was significantly higher in preterm than in fullterm infants. Our data indicated that the premature infants had less left ventricular reserve capacity to respond to the increased preload through the left-to-right ductal shunting than the mature ones, and that the high pulse rate made it possible to generate adequate cardiac output in premature infants.

Cardiac Output↗
PubMed1994

Differential modulation by adenylate cyclase of Ca2+ and delayed K+ current in ventricular myocytes.

This study was designed to investigate the differential modulation of the L-type Ca2+ (ICa) and the delayed rectifier K+ (IK) currents by direct activation of adenylate cyclase in guinea pig ventricular preparations. Action potentials were measured with conventional microelectrodes in excised papillary muscles. Isoproterenol significantly shortened the action potential duration at 90% repolarization (APD90) at 0.1 nM but significantly prolonged it at a higher concentration (10 nM). A water-soluble forskolin derivative, 6-(3-dimethylaminopropionyl) forskolin (NKH-477), slightly but significantly shortened APD at 12 nM but not at a higher concentration (120 nM). Effects of isoproterenol and NKH-477 on ICa and IK were also investigated by use of the whole cell voltage-clamp technique in single ventricular cells. Isoproterenol increased not only IK but also ICa at the same threshold concentration (0.3 nM). In contrast, the threshold concentration of NKH-477 for increasing IK (approximately 1 nM) was clearly lower than that for increasing ICa (10 nM). These results indicate that ICa and IK channels could be differentially regulated during beta-adrenoceptor stimulation.

Action Potentials↗
PubMed1994

Basic FGF enhances endothelium-dependent relaxation of the collateral-perfused coronary microcirculation.

The effect of chronic, periadventitial administration of basic (b) fibroblast growth factor (FGF) on endothelial dysfunction in the collateral-dependent and normally perfused coronary microcirculation was examined. Ameroid constrictors were placed on the proximal left circumflex coronary artery (LCX) in 23 pigs. In 11 pigs, bFGF was released from calcium alginate microcapsules into the perivascular space of the proximal left anterior descending coronary artery (LAD) and LCX. After 5-8 wk, coronary arterial microvessels (80-170 microns) were studied in a pressurized (40 mmHg) no-flow state with video microscopy. Receptor-mediated endothelium-dependent relaxations to ADP and serotonin were reduced while contraction to acetylcholine was enhanced in the collateral-dependent LCX microvessels of non-bFGF-treated control hearts. Relaxation of vessels to the non-receptor-mediated, endothelium-dependent agent A-23187; endothelium-independent relaxation to nitroprusside; and contraction to KCl were similar in all groups. Chronic treatment with bFGF normalized responses to ADP, serotonin, and acetylcholine in the collateral-dependent LCX region but had no effect on the responses of vessels in the normally perfused LAD region. Arteriolar density in the collateral-perfused LCX region of bFGF-treated hearts was markedly increased (4-fold compared with that in untreated hearts, suggesting a link between the angiogenic effect of bFGF and its action on endothelial preservation. Thus the periadventitial, sustained delivery of bFGF preserves receptor-mediated, endothelium-dependent responses in the collateral-dependent LCX region but has no effect on responses of microvessels in the normally perfused LAD region or on non-receptor-mediated endothelium-dependent relaxation.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed1994

Role of cytokine-induced neutrophil chemoattractant, a member of the interleukin-8 family, in rat experimental colitis.

A study was made of the role of cytokine-induced neutrophil chemoattractant (CINC), regarded as a member of the interleukin-8 family, in rat experimental colitis induced by trinitrobenzene sulfonic acid and ethanol. Colonic myeloperoxidase (MPO) activity, a marker of tissue neutrophil infiltration, was observed to reach a plateau from 24 h to 1 week following the induction of colitis; tissue CINC levels, as measured by enzyme-linked immunosorbent assay, rose rapidly, peaking at 12 h before the rise in myeloperoxidase activity. The time-course of tissue leukotriene B4, another chemoattractant, was followed by that of MPO activity. Neutrophil accumulation into tissue in this model would thus appear to be under the control of CINC. Anti-CINC was also noted to inhibit 32.9 to 58.1% of chemotactic activity determined by bioassay during the same period, this being further evidence that CINC is a major chemotactic agent in this model. The present results indicate that CINC may have a crucial role in initiating neutrophil infiltration in experimental colitis.

Animals↗
PubMed1994

Overexpression of human lipoprotein lipase enhances uptake of lipoproteins containing apolipoprotein B-100 in transfected cells.

To investigate the role in lipoprotein metabolism of lipoprotein lipase (LPL) secreted by tissues, we established two cell lines. Fusion plasmids containing either human LPL cDNA or antisense LPL cDNA under control of the cytomegalovirus promoter were transfected into Chinese hamster ovary (CHO) cells, designated as CHO-LPL and CHO-anti-LPL, respectively. CHO-LPL constitutively produced a high level of LPL, whereas CHO-anti-LPL produced a minimal level. When very-low-density lipoprotein (VLDL) was incubated with CHO-LPL, VLDL triglycerides were hydrolyzed, intermediate-density lipoprotein (IDL) was produced, and apolipoprotein E contents increased. CHO-LPL took up and degraded 125I-VLDL at 37 degrees C four times more strongly than did CHO-anti-LPL. Whereas the degradation of apolipoprotein E-deficient VLDL was only 12% that of normal VLDL in CHO-LPL, structural changes of the lipoprotein, including apolipoprotein E expression on the lipoprotein surface, may be important for the cellular uptake of VLDL. Furthermore, we found that binding at 4 degrees C of VLDL and LDL to CHO-LPL was greater than to CHO-anti-LPL, and this binding difference was abolished by washing the cells with heparin. This suggests that cell surface LPL plays a role in the binding of lipoproteins to the cells. We conclude that both the composition of VLDL particles and their cellular binding are influenced by LPL secreted by cells, both of which may enhance the cellular uptake of VLDL.

Animals↗