Pharmacogenetic characterization of sulfasalazine disposition based on NAT2 and ABCG2 (BCRP) gene polymorphisms in humans.

The role of breast cancer resistance protein (BCRP), an efflux ABC transporter, in the pharmacokinetics of substrate drugs in humans is unknown. We investigated the impact of genetic polymorphisms of ABCG2 (421C>A) and NAT2 on the pharmacokinetics of sulfasalazine (SASP), a dual substrate, in 37 healthy volunteers, taking 2,000 mg of conventional SASP tablets. In ABCG2, SASP AUC(0-48) of C/C, C/A, and A/A subjects was 171 +/- 85, 330 +/- 194, and 592 +/- 275 microg h/ml, respectively, with significant differences among groups. In contrast, AUC(0-48) of sulfapyridine (SP) tended to be lower in subjects with the ABCG2-A allele as homozygosity. In NAT2, AUC(AcSP)/AUC(SP) was significantly higher in rapid than in intermediate and slow acetylator (SA) genotypes. We successfully described the pharmacokinetics of SASP, SP, and N -acetylsulfapyridine (AcSP) simultaneously by nonlinear mixed-effects modeling (NONMEM) analysis with regard to both gene polymorphisms. The data indicate that SASP is a candidate probe of BCRP, particularly in its role in intestinal absorption.

Ultrasound biomicroscopic study of ciliary body changes in the post-treatment phase of Vogt-Koyanagi-Harada disease.

AIMS: To investigate the usefulness of ultrasound biomicroscopy for evaluating changes in the ciliary body in patients with Vogt-Koyanagi-Harada disease. METHODS: Ultrasound biomicroscopy was used to evaluate 14 eyes of seven patients diagnosed with Vogt-Koyanagi-Harada disease. Cross sectional images of the ciliary body and thickness of the pars plana 3.0 mm posterior to the scleral spur were examined. Predicted thickness of the pars plana was obtained by multiple linear regression analysis of thickness in the acute phase and in the remission phase. RESULTS: In the active phase, the cross sectional images showed a shallow anterior chamber in eight of the 14 eyes, ciliochoroidal detachment in five eyes, and a thickened ciliary body in all 14 eyes. Internal reflectivity of the ciliary stroma was low, with ciliary processes being unclear in 13 eyes. One month after steroid treatment, slit lamp examination findings were normal in 14 eyes. 10 eyes of five patients were examined by ultrasound biomicroscopy at this stage. Ciliochoroidal detachment was no longer seen in any eye. Internal reflection of the ciliary stroma became relatively homogeneous, and the ciliary processes were seen, though not clearly. However, the pars plana remained thickened. The actual thickness was greater at 1 month after steroid treatment than the predicted thickness for the remission phase. In the remission phase, the internal reflection was homogeneous and the ciliary processes were delineated clearly in all 14 eyes. CONCLUSION: Objective, quantitative evaluation of the ciliary body is possible with ultrasound biomicroscopy during the course of Vogt-Koyanagi-Harada disease. Ultrasound biomicroscopy is useful in determining disease activity in the anterior segment and in monitoring the clinical course, and it may improve evaluation of the efficacy of treatment.

Genetically modified Shiga toxin 2e (Stx2e) producing Escherichia coli is a vaccine candidate for porcine edema disease.

Porcine edema disease (ED) is an enterotoxaemia in pigs after weaning, caused by Shiga toxin 2e (Stx2e) producing Escherichia coli. Recently in Japan, outbreaks of ED are re-emerging in pig production. In this study we constructed a mutant that retained immunogenicity but lost Vero cell cytotoxicity, which produced genetically modified toxin (Stx2e*) by replacing glutamate with glutamine at position 167 and arginine with leucine at position 170 of the A subunit. The stx(2e)* gene was replaced with the stx(2e)gene of the wild type virulent strain by homologous recombination. As the parent wild strain was pathogenic to pigs but the mutant was not, the mutant named as YT106 was given to the pigs to examine its protective immunity against ED. All 20 pigs vaccinated with YT106 survived, but only eight of the 20 non-vaccinated pigs survived after the challenge with a wild strain. Also, the eight pigs that survived had decreased rates of gain relative to those of the controls. Blood IgG and intestinal IgA titres increased 3.3 and 1.6 times more than the control, respectively, showing that YT106 might be a good candidate of a live attenuated vaccine strain to protect against ED.

Antibacterial activity of chaff vinegar and its practical application.

Since enterohemorrhagic Escherichia coli O157, Salmonella, etc., sometimes contaminate animal feces and may cause infectious diseases to humans, it is important to remove pathogenic bacteria from domestic animal waste. For the purpose, we examined the antibacterial activity of chaff vinegar. We found that the chaff vinegar inhibited the growth of pathogenic bacteria immediately in vitro but not efficiently spores and lactic acid bacteria. Further, it removes bacteria, especially Enterobacteriaceae, from animal feces and the surface of the concrete-floor in the cattle barn. Chaff vinegar is advertised as a natural chemical substance for a soil conditioner, to promote the composting and to deodorize their smell. Chaff vinegar may be useful for organic agriculture without enteric pathogenic bacteria.

Regulation of insulin secretion by overexpression of Ca2+/calmodulin-dependent protein kinase II in insulinoma MIN6 cells.

Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) may play a key role in Ca2+-induced insulin secretion. We have previously reported that treatment of insulinoma MIN6 cells with secretagogues activated CaM kinase II and increased the phosphorylation of synapsin I, followed by insulin secretion. Here, we identified isoforms of CaM kinase II in MIN6 cells and rat islets. Immunoblot analysis suggested that the major isoforms of CaM kinase II were beta'e and delta2 at the protein level in MIN6 cells. Only the beta'e isoform was detected in rat islets by both RT-PCR and immunoblot analysis. We transiently overexpressed beta'e and delta2 isoforms in MIN6 cells and confirmed that treatment of cells with tolbutamide and glucose activated the isoforms. Immunoblot analysis with an antibody against synapsin I phosphorylated by CaM kinase II demonstrated that treatment with tolbutamide and glucose rapidly increased phosphorylation of synapsin I and that phosphorylation was potentiated by overexpression of the isoforms. The secretagogue-induced insulin secretion was potentiated by overexpression of the isoforms. Our results further support our conclusion that activation of CaM kinase II and the concomitant phosphorylation of synapsin I contribute to insulin secretion from pancreatic beta-cells.

Cloning from insulinoma cells of synapsin I associated with insulin secretory granules.

Synapsin I is a synaptic vesicle-associated protein involved in neurotransmitter release. The functions of this protein are apparently regulated by Ca2+/calmodulin-dependent protein kinase II (CaM kinase II). We reported evidence for CaM kinase II and a synapsin I-like protein present in mouse insulinoma MIN6 cells (Matsumoto, K., Fukunaga, K., Miyazaki, J., Shichiri, M., and Miyamoto, E. (1995) Endocrinology 136, 3784-3793). Phosphorylation of the synapsin I-like protein in these cells correlated with the activation of CaM kinase II and insulin secretion. In the present study, we screened the MIN6 cDNA library with the full-length cDNA probe of rat brain synapsin Ia and obtained seven positive clones; the largest one was then sequenced. The largest open reading frame deduced from the cDNA sequence of 3695 base pairs encoded a polypeptide of 670 amino acids, which exhibited significant sequence similarity to rat synapsin Ib. The cDNA contained the same sequence as the first exon of the mouse synapsin I gene. These results indicate that synapsin Ib is present in MIN6 cells. Synapsin I was expressed in normal rat islets, as determined by reverse transcriptase-polymerase chain reaction analysis. Immunoblot analysis after subcellular fractionation of MIN6 cells demonstrated that synapsin Ib and delta subunit of CaM kinase II co-localized with insulin secretory granules. By analogy concerning regulation of neurotransmitter release, our results suggest that phosphorylation of synapsin I by CaM kinase II may induce the release of insulin from islet cells.

[Mechanism and clinical usefulness of S4-S1 interval in heart failure associated with left ventricular inflow pattern].

The interval between S4 and S1 detected by auscultation or phonocardiography is prolonged by exacerbation and shortened by improvement of heart failure. The timing of S4, S1, and the terminal point of the A wave of transmitral inflow velocities on pulsed Doppler echocardiography (At) was studied to elucidate the mechanism of the prolongation of the S4-S1 interval on exacerbation of heart failure. The study population consisted of 30 patients, nine with old myocardial infarction, six with dilated cardiomyopathy, six with hypertensive heart disease, nine with chronic hemodialysis, and 17 normal subjects. The interval from the peak of the A wave by apexcardiography and At to the onset of main vibration of S1 were measured as the S4-S1 interval and At-S1 interval, respectively. The P-Q interval and Q-S1 interval were also measured. Both intervals were compared during exacerbation and improvement of heart failure. Patients with P-Q prolongation were excluded. The S4-S1 interval was 102 +/- 24 msec during exacerbation of heart failure or before hemodialysis, and shortened to 76 +/- 18 msec after improvement of heart failure or after hemodialysis. The At-S1 interval was concordantly shortened from 59 +/- 31 msec to 30 +/- 23 msec (p < 0.001). However, both the P-Q interval and Q-S1 interval were not significantly changed before and after improvement of heart failure. The timing of S4 becomes parallel to that of At earlier during the exacerbation of heart failure. Thus, S4-S1 interval is a convenient and useful index to investigate patients with heart failure.

[Estimation of left ventricular systolic function based on the electrocardiograms in cases with left bundle branch block].

The electrocardiographic features indicating left ventricular dysfunction were studied in 32 consecutive patients having left bundle branch block including 10 with idiopathic genesis without significant underlying disease, 6 with dilated cardiomyopathy, 8 with old myocardial infarction, and 8 with hypertensive heart disease. The patients were divided into two groups; those with favorable left ventricular systolic function and those with poor left ventricular systolic function evaluated by using non-invasive methods. Electrocardiographic findings were compared between these two groups. Ten patients had favorable and 22 poor left ventricular systolic function. One or more of the following electrocardiographic findings were observed in the poor group, but none in the favorable group: low voltage in the limb leads, prolonged intraventricular conduction (QRS duration wider than 0.17 sec), transitional zone between V5 and V6, depression of the ST-J point by more than 0.2 mV in V6, reverse progression of the R wave in V1-V5, marked left axis deviation (axis beyond: 30 degrees), left atrial overload (positive Morris index), PQ prolongation, and abnormal Q waves in I, aVL, V6. No significant differences in the distribution of these findings was observed in any of the underlying diseases. The clinical background of patients with left bundle branch block who had no significant underlying disease showed favorable left ventricular systolic function except the patients above 80 years of age, who showed poor left ventricular systolic function. In contrast, patients with underlying mild hypertensive heart disease may have a favorable left ventricular systolic function. Thus, left ventricular systolic function in patients with left bundle branch block may be suspected by observing these electrocardiographic findings.

Cloning of the Candida albicans homolog of Saccharomyces cerevisiae GSC1/FKS1 and its involvement in beta-1,3-glucan synthesis.

Saccharomyces cerevisiae GSC1 (also called FKS1) and GSC2 (also called FKS2) have been identified as the genes for putative catalytic subunits of beta-1,3-glucan synthase. We have cloned three Candida albicans genes, GSC1, GSL1, and GSL2, that have significant sequence homologies with S. cerevisiae GSC1/FKS1, GSC2/FKS2, and the recently identified FKSA of Aspergillus nidulans at both nucleotide and amino acid levels. Like S. cerevisiae Gsc/Fks proteins, none of the predicted products of C. albicans GSC1, GSL1, or GSL2 displayed obvious signal sequences at their N-terminal ends, but each product possessed 10 to 16 potential transmembrane helices with a relatively long cytoplasmic domain in the middle of the protein. Northern blotting demonstrated that C. albicans GSC1 and GSL1 but not GSL2 mRNAs were expressed in the growing yeast-phase cells. Three copies of GSC1 were found in the diploid genome of C. albicans CAI4. Although we could not establish the null mutation of C. albicans GSC1, disruption of two of the three GSC1 alleles decreased both GSC1 mRNA and cell wall beta-glucan levels by about 50%. The purified C. albicans beta-1,3-glucan synthase was a 210-kDa protein as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and all sequences determined with peptides obtained by lysyl endopeptidase digestion of the 210-kDa protein were found in the deduced amino acid sequence of C. albicans Gsc1p. Furthermore, the monoclonal antibody raised against the purified beta-1,3-glucan synthase specifically reacted with the 210-kDa protein and could immunoprecipitate beta-1,3-glucan synthase activity. These results demonstrate that C. albicans GSC1 is the gene for a subunit of beta-1,3-glucan synthase.

[Prognostic importance of pseudonormalized left ventricular inflow pattern especially for sudden cardiac death].

The prognostic value of left ventricular inflow velocities by the pulsed Doppler method was studied in 32 patients with congestive heart failure including 18 with old myocardial infarction, 9 with dilated cardiomyopathy, and 5 with hypertensive heart disease, who initially revealed pseudonormalized left ventricular inflow pattern. Pulsed Doppler echocardiography, apexcardiography, and phonocardiography were performed at 3- to 9-month intervals. The prognosis was evaluated for two groups of patients with persistent or transient pseudonormalized inflow patterns. Survival rates at 6 months and 2 years in the total patient population were 78% and 47%, respectively. Twenty-one patients had the pseudonormalized left ventricular inflow pattern (persistent group), while the other 11 patients had a changed pattern (transient group). The survival rate at 2 years was 37% for the persistent group, and 82% for the transient group. Significant differences in patient profiles and the initial data between the two patient groups were the presence of cardiogenic shock (10/21 in persistent group vs 1/11 in transient group; p < 0.001) and the value of left ventricular end-diastolic wall stress (159 +/- 62 g/cm2 in persistent group vs 135 +/- 42 g/cm2 in transient group; p < 0.05). There were no significant differences in NYHA class, the values of left ventricular end-diastolic pressure, left ventricular ejection fraction. A/E ratio, or deceleration half time of left ventricular inflow velocities. Left ventricular end-diastolic wall stress in patients with persistent pseudonormalized left ventricular inflow pattern was significantly increased, and may be related to decreased preload reserve. Atrial fibrillation and atrioventricular dissociation were recorded prior to the development of ventricular fibrillation in two patients with sudden cardiac death. Abrupt loss of atrial contribution as well as ventricular arrhythmias may be a trigger of sudden death. Evaluation and follow-up of the pseudonormalized left ventricular inflow pattern is a sensitive indicator for the management of patients with congestive heart failure.

Differential regulation of c-fos gene expression by two types of human endothelin receptor in Chinese hamster ovary cells.

To investigate the nuclear signalling pathway induced by endothelin (ET) isopeptides, we have established permanent Chinese hamster ovary (CHO) cell lines, CHO-ETA/fos-lacZ and CHO-ETB/fos-lacZ, that produce both a c-fos-beta-galactosidase fusion protein and either the type A or the type B human ET receptor. These cell lines permitted a colorimetric measurement of c-fos expression, which was induced by the signal transduction system with ET receptors and ET isopeptides. We found that the ET-1-dependent c-fos expression was so efficient that it could respond to low concentrations (even a physiological concentration) of ET-1. For example, CHO-ETA/fos-lacZ and CHO-ETB/fos-lacZ responded to ET concentrations of 5 x 10(-9) M and 5 x 10(-13) M respectively. Using this highly sensitive system, the H-7 sensitive protein kinase was found to be involved in signal transduction mediated by ETA, and also partly in the ETB-mediated pathway. These lines of evidence suggest that c-fos expression occurs through at least two different pathways, depending on the concentration of ET in plasma.

Underwinding of DNA on binding of yeast TFIID to the TATA element.

The TATA box-binding factor TFIID is an essential component for the initiation of transcription by eukaryotic RNA polymerase II. We investigated the effect of DNA supercoiling on TFIID: promoter interactions using recombinant yeast (ry) TFIID. DNase I footprinting analysis showed that ryTFIID has a higher affinity for the adenovirus major late promoter in the negatively supercoiled state than that in the relaxed state. On the contrary, its affinity for the Drosophila hsp70 promoter is constant irrespective of DNA topology. Binding of ryTFIID to these promoters induced underwinding of duplex DNA. The functional TATA box and active ryTFIID are essential for the underwinding. The step was facilitated by negative supercoiling of DNA on the adenovirus major late promoter but not on the Drosophila hsp70 promoter.

[Mechanism of discrepancy between mean pulmonary capillary wedge pressure and left ventricular end-diastolic pressure].

It is well known that discrepancies between mean pulmonary capillary wedge pressure (man-PCWP) and left ventricular end-diastolic pressure (LVEDP) exist in the pathological heart with sinus rhythm. We discussed the mechanism of these discrepancies in the aspect of increased LV end-diastolic stiffness. Fifty-two patients observed in this study included 23 with old myocardial infarction (OMI), 4 with hypertrophic cardiomyopathy and 9 with hypertensive heart disease (LVH group), 6 with dilated cardiomyopathy (DCM), and 10 normal subjects (N). All 52 patients had sinus rhythm. Those with significant mitral and aortic regurgitation were excluded. End-diastolic LV stiffness was evaluated by the ratio of increases in LV pressure and volume during atrial systole (delta P/delta V), as proved by cardiac catheterization and cine-angiography. Discrepancies between m-PCWP and LVEDP were 5.9 +/- 4.3 mmHg in OMI group, 4.5 +/- 4.6 mmHg in LVH group, 5.8 +/- 4.5 mmHg in DCM, and 1.6 +/- 1.8 mmHg in N group. These discrepancies correlated well with delta P/delta V (r = 0.74). More significant discrepancies were observed in patients with so-called pseudo-normalized left ventricular inflow velocities proved by pulsed Doppler echocardiography, and in patients with marked concentric LV hypertrophy with increased delta P/delta V. In clinical observation, symptoms of heart failure may be determined by m-PCWP rather than LVEDP. We concluded that discrepancies between m-PCWP and LVEDP were caused by the booster pump function of the left atrium against increased LV end-diastolic stiffness. By the use of apexcardiogram and echocardiogram including the pulsed Doppler method, it was possible to predict these discrepancies non-invasively.

[Preload dependence of atrial contribution: correlation with left ventricular endo-diastolic wall stress].

The correlation between left ventricular end-diastolic wall stress (sigma ed) and atrial contribution was investigated using echocardiography including the pulsed Doppler method and cardiac catheterization with cineangiography in 21 patients with coronary artery disease, 4 with hypertensive heart disease, 3 with hypertrophic cardiomyopathy, 3 with dilated cardiomyopathy, 4 with aortic regurgitation, and 10 normal subjects. The ratio of peak velocities (A/R) in the rapid filling phase (R) and atrial contraction phase (A) was used as the index of atrial contribution. Left ventricular end-diastolic stiffness (delta P/delta V) was calculated by the ratio of increases in left ventricular pressure and volume during the atrial contraction phase. The correlations between A/R and sigma ed, mean pulmonary capillary wedge pressure (m-PCWP), and left ventricular end-diastolic pressure (LVEDP) were analyzed in the 35 patients. Correlations between delta P/delta V and sigma ed and A/R were also analyzed. A/R was inversely correlated with sigma ed (r = -0.75), m-PCWP (r = -0.62) and LVEDP (r = -0.59), suggesting that A/R is influenced significantly by preload. A/R was also inversely correlated with delta P/delta V (r = -0.72). delta P/delta V was significantly correlated with sigma ed (r = 0.92), suggesting that sigma ed is a factor for determining left ventricular end-diastolic stiffness. Left ventricular volume can be increased in patients with low sigma ed by atrial contraction, achieving good atrial contribution. Left ventricular volume will be little increased by atrial contraction in patients with elevated sigma ed despite significant increase of left ventricular end-diastolic pressure, resulting in diminished atrial contribution.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular characterization of the 5'-flanking region of human genomic ETA gene.

A clone containing the promoter, the exon 1 and exon 2 of human genomic ETA gene was isolated and sequenced. The transcription initiation site and TATA box were identified at 528 bp upstream of the initiation codon and 24 bp upstream of the transcription initiation site, respectively. The 534 bp fragment containing 5'-flanking sequence and most of the exon 1 of the human ETA gene showed a promoter activity corresponding to 55% of SV40 early promoter activity when placed upstream of the luciferase gene and transfected into CHO cells. Thus, the TATA box which consists of the TAAAAA sequence is functional for transcription of the human ETA gene. The coding region of the human ETA receptor, therefore, starts from exon 2 which contained the first and the second transmembrane regions. The human ETA gene was demonstrated to be a single pair of copy by Southern blot analysis, and it was localized in chromosome 4 by analysis with a panel of human-rodent somatic cell hybrid lines.

Truncation of N-terminal extracellular or C-terminal intracellular domains of human ETA receptor abrogated the binding activity to ET-1.

We have investigated the function of N-terminal and C-terminal domains of the human ETA receptor by expressing truncated mutants in COS-7 cells. Three kinds of ETA receptors truncated in the N-terminal extracellular or C-terminal intracellular domains were produced. Deletion of the entire extracellular N-terminal or intracellular C-terminal domain completely inactivated the ET-1 binding activity. However, the deletion of one half of the N-terminal extracellular domain of the ETA receptor, missing one of two N-linked glycosylation sites, maintained complete binding activity. Specific monoclonal antibodies detected all the truncated ETA receptors in the cell membrane fraction of transfected COS-7 cells. The size of the ETA receptor was heterogeneous due to differential glycosylation and distributed in 48K, 45K and 42K dalton bands in Western blot analysis. These results demonstrated that a part of the N-terminal domain in close proximity to the first transmembrane region is required for the ligand binding activity of the ETA receptor, and the C-terminal domain is perhaps necessary as an anchor for maintenance of the binding site.

DNA supercoiling facilitates the assembly of transcriptionally active chromatin on the adenovirus major late promoter.

Assembly of nucleosomes on the adenovirus major late promoter blocked initiation of transcription by RNA polymerase II. However, the formation of transcription preinitiation complexes prevented subsequent assembly of promoter sequences into nucleosomes and allowed transcription on the chromatin templates. When the formation of preinitiation complexes was in competition with nucleosome assembly, transcription on linear or relaxed closed circular DNA was inactivated by nucleosome assembly over the promoter region. However, transcription on partially supercoiled DNA (mean superhelical density of -0.036) remained active because the rapid formation of preinitiation complexes prevented subsequent assembly of promoter sequences into nucleosomes.

Robot system for preparing lymphocyte chromosome.

Towards the automatization of the scoring of chromosome aberrations in radiation dosimetry with the emphasis on the improvement of biological preparations, the conventional culture and harvesting method was modified. Based on this modified method, a culture and harvest robotic system (CHROSY) for system (CHROSY) for preparing lymphocyte chromosome was developed. The targeted points of the modification are as in the following. 1. Starting culture with purified lymphocytes in a fixed cell number. 2. Avoiding the loss of cells in changing the liquids following centrifugalization. 3. Keeping the quantity of the liquids to be applied to the treatments of cells fixed. 4. Building a system even a beginner can handle. System features are as follows. 1. Operation system: Handling robot having 5 degrees of freedom; a rotator incubator with an automatic sliding door; units for setting and removing pipette tips; a centrifuge equipped with a position adjuster and an automatic sliding door; two aluminum block baths; two nozzles as pipettes and aspirators connected to air pumps; a capping unit with a nozzle for CO2 gas; a compressor; and an air manipulated syringe. 2. Control system: NEC PC-9801RX21 with CRT; and program written in Basic and Assembly languages on MS-DOS. It took this system 2 hours and 25 minutes to harvest 2 cultures. A fairly good chromosome slide was made from the sample harvested by CHROSY automatically.