Studies of the inheritance of human ribosomal DNA variants detected in two-dimensional separations of genomic restriction fragments.

We have investigated the variation in human ribosomal DNA repeat units as revealed in two-dimensional electrophoretic separates of genomic restriction fragments that were end-labeled at NotI cleavage sites. The transcribed portion of the ribosomal DNA results in approximately 20 labeled fragments visible on each gel as multicopy spots. We have mapped these spots to the sequences responsible for their appearance on the gels, based on their migration positions and direct sequencing of spots, and describe several previously unreported sources of variation. By studying mother/father/child families we gained information on how much of the between-repeats variation is due to differences between and within repeat arrays on homologous chromosomes. Two instances in which a child exhibited more copies of a particular fragment than were present in the parents are described and hypothesized to be due to events such as multiple unequal sister-chromatid exchanges or gene conversions.

Abnormal regulation of transforming growth factor-beta receptors on vascular smooth muscle cells from spontaneously hypertensive rats by angiotensin II.

The effects of angiotensin II (Ang II) on the expression and characteristics of transforming growth factor-beta (TGF-beta) receptors on vascular smooth muscle cells (VSMC) from Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR) were investigated. TGF-beta-induced stimulation of DNA synthesis by VSMC from WKY rats was abolished with Ang II, whereas basal and TGF-beta-stimulated DNA synthesis by VSMC from SHR was increased with Ang II. Ang II stimulated DNA synthesis by VSMC from WKY rats in the presence but not in the absence of neutralizing antibody to TGF-beta1. Antibody to TGF-beta1 enhanced the stimulatory effect of Ang II on DNA synthesis by VSMC from SHR. Ang II increased the specific binding of TGF-beta to VSMC from WKY rats by increasing both the expression of the lower-affinity of TGF-beta receptors as well as the total number of TGF-beta binding sites. In contrast, VSMC from SHR showed a higher affinity and number of TGF-beta receptors in the absence of Ang II than did cells from WKY rats, and these parameters were not affected by Ang II. Ang II increased the expression of TGF-beta type I receptor mRNA in VSMC from WKY rats but had no effect of TGF-beta receptor type I or II mRNA in VSMC from SHR, which predominantly express the type II receptor. These results indicate that an increase in the expression of the TGF-beta type I receptor by Ang II may facilitate the ability of endogenous TGF-beta to counteract the stimulatory effect of Ang II on growth in VSMC from WKY rats, whereas endogenous TGF-beta induced by Ang II cannot counteract the growth-promoting action of Ang II in VSMC from SHR. The abnormal regulation of TGF-beta receptors by Ang II may be associated with the exaggerated growth of VSMC from SHR.

Hammerhead ribozyme targeting human platelet-derived growth factor A-chain mRNA inhibited the proliferation of human vascular smooth muscle cells.

Platelet-derived growth factor (PDGF) A-chain contributes to the pathogenesis of cardiovascular proliferative diseases, such as hypertensive vascular disease, atherosclerosis, and re-stenosis of an artery after angioplasty. To develop a ribozyme against human PDGF A-chain mRNA as a gene therapy for human arterial proliferative diseases, we designed and synthesized a 38-base hammerhead ribozyme to cleave human PDGF A-chain mRNA at the GUC sequence at nucleotide 591. In the presence of MgCl(2), synthetic hammerhead ribozyme to human PDGF A-chain mRNA cleaved the synthetic target RNA to two RNA fragments at a predicted size. Doses of 0.01-1.0 microM hammerhead ribozyme to human PDGF A-chain mRNA significantly inhibited angiotensin II (Ang II) and transforming growth factor (TGF)-beta(1)-induced DNA synthesis in vascular smooth muscle cells (VSMC) from human in a dose-dependent manner. One micromolor of hammerhead ribozyme to human PDGF A-chain mRNA significantly inhibited Ang II-induced PDGF A-chain mRNA and PDGF-AA protein expressions in VSMC from humans. These results indicate that the designed hammerhead ribozyme to human PDGF A-chain mRNA effectively inhibited growth of human VSMC by cleaving the PDGF A-chain mRNA and inhibiting the PDGF-AA protein expression in human VSMC. This suggests that the designed hammerhead ribozyme to PDGF A-chain mRNA is a feasible gene therapy for treating arterial proliferative diseases.

Endogenous angiotensin II suppresses insulin signaling in vascular smooth muscle cells from spontaneously hypertensive rats.

BACKGROUND: Angiotensin II (Ang II) has been reported to inhibit insulin signaling at multiple levels in vascular smooth muscle cells (VSMC) in vitro. We have demonstrated that VSMC from spontaneously hypertensive rats (SHR) produce Ang II in a homogeneous culture. OBJECTIVE: In the current study, we investigated influences of endogenous Ang II on insulin signaling in VSMC from SHR. DESIGN AND METHODS: Phosphatidylinositol 3-kinase (PI3-kinase) activity, insulin receptor substrate-1 (IRS-1) associated tyrosine phosphorylation, and p85 subunit of PI3-kinase were measured in VSMC from SHR and normotensive Wistar-Kyoto (WKY) rats in the absence and presence of Ang II type 1 receptor antagonist RNH6270 and mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK) inhibitor U0126. RESULTS: Insulin treatment increased PI3-kinase activity in VSMC from WKY rats in a dose-dependent manner. In contrast, insulin treatment of VSMC from SHR did not affect PI3-kinase activity. However, co-treatment of VSMC from SHR with RNH6270 and insulin, increased PI3-kinase activity. PI3-kinase activity, IRS-1-associated tyrosine phosphorylation and p85 subunit of PI3-kinase in VSMC from WKY rats decreased in response to treatment with Ang II and returned to control levels upon co-treatment with U0126. Basal levels of PI3-kinase activity, IRS-1-associated tyrosine phosphorylation, and p85 subunit of PI3-kinase were significantly lower in VSMC from SHR than in cells from WKY rats. U0126 treatment of VSMC from SHR significantly increased levels of PI3-kinase activity, IRS-1-associated tyrosine phosphorylation, and p85 subunit of PI3-kinase. CONCLUSION: These results indicate that endogenous Ang II suppresses insulin signaling in VSMC from SHR by activating extracellular signal-regulated kinase. These findings suggest that tissue Ang II may play a role in insulin resistance in hypertension.

Contribution of synthetic phenotype on the enhanced angiotensin II-generating system in vascular smooth muscle cells from spontaneously hypertensive rats.

OBJECTIVE: We have demonstrated that cultured vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats (SHR), but not from normotensive Wistar-Kyoto (WKY) rats, produce angiotensin II (Ang II) in a homogeneous culture with increased levels of angiotensinogen, cathepsin D and angiotensin converting enzyme (ACE) at early passages. In the current study, we investigated how changes in the cell phenotype affect the Ang II-generating system and the growth of VSMC from SHR. DESIGN AND METHODS: We evaluated basal DNA synthesis by [3H]thymidine incorporation, immunofluorescence of alpha-smooth muscle (SM) actin, mRNA expression of phenotype markers such as SM22alpha appeared by contractile phenotype, Ang II-generating system components and growth factors by reverse transcription and polymerase chain reaction analysis, and Ang II levels by radioimmunoassay in quiescent VSMC from WKY/Izumo rats and SHR/Izumo at passages 4, 8 and 12. RESULTS: Basal DNA synthesis in VSMC from WKY rats increased with increasing passage number, whereas in cells from SHR it was markedly higher at early passages and was not affected by the passages. At early passage numbers, immunofluorescence of alpha-SM actin was stronger in VSMC from WKY rats than in cells from SHR, but decreased after several passages. Expression of SM22alpha mRNA was higher in VSMC from WKY rats than in cells from SHR at early passages, and decreased after several passages in cells from both rat strains. Expression of matrix Gla mRNA was higher in VSMC from SHR than in cells from WKY rats at early passage, and increased after several passages in cells from both rat strains. Ang II was not detected at early passages but increased in VSMC from WKY rats with increasing passage, whereas it was detected in VSMC from SHR at early passages and did not change with the passages. Expression of angiotensinogen mRNA was higher in VSMC from SHR than in cells from WKY rats, and was not affected by the passages. Expressions of cathepsin D and ACE mRNA were higher in VSMC from SHR than in cells from WKY rats at early passage, and were increased by the passages in VSMC from WKY rats. Expressions of transforming growth factor-beta1, platelet-derived growth factor A-chain, and basic fibroblast growth factor mRNA were significantly higher in VSMC from SHR than in cells from WKY rats, and were increased by the passages. CONCLUSION: These data indicate that early in culture VSMC from SHR have the synthetic phenotype, whereas VSMC from WKY rats have the contractile phenotype which then changes to the synthetic phenotype after increased passage numbers, with increased expression of cathepsin D and ACE, which produce Ang II, and increased expression of Ang II-related growth factors, which induce the exaggerated growth observed in VSMC from SHR.

Low dose of eicosapentaenoic acid inhibits the exaggerated growth of vascular smooth muscle cells from spontaneously hypertensive rats through suppression of transforming growth factor-beta.

OBJECTIVE: To evaluate effects of eicosapentaenoic acid (EPA), an n-3 polyunsaturated fatty acid, on the exaggerated growth of vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats (SHR). DESIGN: Cultured VSMC were prepared by an explant method from thoracic aortas in 8-week-old male Wistar-Kyoto (WKY)/Izumo rats and SHR/Izumo. Effects of EPA on basal DNA synthesis, expression of growth factors and cyclin-dependent kinase 2 (cdk2) activity were examined in VSMC from WKY rats and SHR. METHODS: The cell cycles were synchronized with serum deprivation, then DNA synthesis in VSMC was measured by [3H]-thymidine incorporation. Fatty acid composition of the phospholipid fraction in VSMC was measured by gas chromatography. Expression of platelet-derived growth factor (PDGF) A-chain, transforming growth factor (TGF)-beta1 and basic fibroblast growth factor (bFGF) mRNAs was evaluated by reverse-transcription and polymerase chain reaction analysis. Cdk2 activity was determined by autoradiography after polyacrylamide gel electrophoresis of VSMC extracts that had been immunoprecipitated with anti-cdk2 antibody and protein A sepharose, and then incubated with 32P-ATP and histone H1. RESULTS: High concentrations (40 and 80 micromol/I) of EPA significantly inhibited basal DNA synthesis in VSMC from both rat strains. Low dose (20 micromol/l) of EPA significantly inhibited basal DNA synthesis in VSMC from SHR, whereas the same dose of EPA stimulated DNA synthesis in VSMC from WKY rats. In analysis of fatty acid composition, low dose of EPA was considerably incorporated in VSMC. Low dose of EPA significantly inhibited angiotensin II- and phorbol ester milisterol-stimulated DNA synthesis in VSMC from both rat strains, whereas EPA did not affect PDGF-AA-stimulated DNA synthesis in VSMC from either rat strain. Low dose of other polyunsaturated fatty acids such as docosahexaenoic acid, arachidonic acid and linoleic acid did not significantly affect basal DNA synthesis in VSMC from either strain. Low dose of EPA significantly inhibited expression of TGF-beta1 mRNA in VSMC from SHR, whereas EPA did not affect expression of PDGF A-chain and bFGF mRNAs in VSMC from SHR. Cdk2 activity in VSMC from SHR was higher than that from WKY rats. Low dose of EPA inhibited cdk2 activity in VSMC from SHR, whereas it stimulated the activity in VSMC from WKY rats. CONCLUSION: Low dose of EPA exerted specific inhibition of the exaggerated growth of VSMC from SHR through the suppression of TGF-beta.

Genetic analysis of children of atomic bomb survivors.

Studies are under way for the detection of potential genetic effects of atomic bomb radiation at the DNA level in the children of survivors. In a pilot study, we have examined six minisatellites and five microsatellites in DNA derived from 100 families including 124 children. We detected a total of 28 mutations in three minisatellite loci. The mean mutation rates per locus per gamete in the six minisatellite loci were 1.5% for 65 exposed gametes for which mean parental gonadal dose was 1.9 Sv and 2.0% for 183 unexposed gametes. We detected four mutations in two tetranucleotide repeat sequences but no mutations in three trinucleotide repeat sequences. The mean mutation rate per locus per gamete was o% for the exposed gametes and 0.5% for the unexposed gametes in the five microsatellite loci. No significant differences in the mutation rates between the exposed and the unexposed gametes were detected in these repetitive sequences. Additional loci are being analyzed to increase the power of our study to observe a significant difference in the mutation rates at the 0.05 level of significance.

Angiotensin II regulates the cell cycle of vascular smooth muscle cells from SHR.

We have demonstrated that spontaneously hypertensive rats (SHR)-derived vascular smooth muscle cells (VSMC) show the exaggerated growth and produce angiotensin II (Ang II). In the current study, we investigated the role of endogenous Ang II in the regulation of the cell cycle in VSMC from SHR. Levels of Ang II in conditioned medium from SHR-derived VSMC cultured without serum were significantly higher than levels in conditioned medium from Wistar-Kyoto (WKY) rat-derived VSMC. Basal DNA synthesis was higher in quiescent VSMC from SHR than that in cells from WKY rats. An Ang II type 1 receptor antagonist, CV11974, significantly inhibited the elevation in DNA synthesis in quiescent VSMC from SHR but did not affect it in cells from WKY rats. Cellular DNA content analysis by flow cytometry revealed that the proportion of cells in S phase was higher, whereas the proportion of cells in G1+G0 phase was lower in VSMC from SHR than those in cells from WKY rats. CV11974 significantly decreased the proportion of cells in S phase and correspondingly increased the proportion of cells in G1+G0 phase in VSMC from SHR, but it did not affect the proportion in cells from WKY rats. Cyclin-dependent kinase 2 (CDK2) activity, which is known to induce the progression from G1 to S phase, was higher in VSMC from SHR than in cells from WKY rats. Expression of CDK2 inhibitor p27(kip1) mRNA was markedly higher in VSMC from SHR than in cells from WKY rats. CV11974 decreased expression of p27(kip1) mRNA in VSMC from SHR, whereas CV11974 increased it in cells from WKY rats. These findings indicate that enhanced production of endogenous Ang II regulates the cell cycle especially in the progression from G1 to S phase, and increases CDK2 activity, which is independent of p27(kip1) in VSMC from SHR.

Role of long-form PDGF A-chain in the growth of vascular smooth muscle cells from spontaneously hypertensive rats.

Vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats (SHR) exhibit exaggerated growth relative to cells from normotensive Wistar-Kyoto (WKY) rats. Platelet-derived growth factor (PDGF) A-chain has been implicated in the exaggerated growth of VSMC from SHR. Two isoforms of PDGF A-chain mRNA that either include (long form) or exclude (short form) exon 6 are produced as a result of alternative splicing. The expression of the long-form PDGF A-chain at the mRNA level and its role in the growth of VSMC from SHR have now been investigated with the use of an antisense oligodeoxynucleotide (ODN) complementary to exon 6 of the PDGF A-chain gene. Reverse transcription-polymerase chain reaction (RT-PCR) analysis with primers encompassing exon 6 of PDGF A-chain mRNA revealed bands corresponding to both long- and short-form PDGF A-chain transcripts in quiescent VSMC from both SHR and WKY rats, with the long-form mRNA more abundant in VSMC from SHR than in cells from WKY rats. Expression of the long-form of PDGF A-chain mRNA was enhanced with angiotensin II and transforming growth factor-beta1 in VSMC from SHR, but not in cells from WKY rats. The antisense ODN significantly inhibited DNA synthesis by VSMC from SHR, but not by cells from WKY rats, in the absence or presence of serum. In addition, the antisense ODN significantly inhibited serum induced proliferation of VSMC from SHR, but not those from WKY rats. The antisense ODN abolished expression of the long-form PDGF A-chain mRNA in VSMC, suggesting that its inhibitory effects on the growth of VSMC from SHR are mediated by depletion of the long-form transcripts. These results indicate that the long-form of PDGF A-chain contributes to the exaggerated growth of VSMC from SHR.

Angiotensin II upregulates transforming growth factor-beta type I receptor on rat vascular smooth muscle cells.

Angiotensin II (Ang II) and transforming growth factor-beta (TGF-beta) modulate cell growth and metabolism. Our objective was to evaluate the effect of Ang II on the characteristics and expression of TGF-beta receptors on vascular smooth muscle cells (VSMC) from Wistar-Kyoto rats. The addition of TGF-beta1 elicited a biphasic response on DNA synthesis in cultured VSMC in the absence of Ang II, but TGF-beta1 did not stimulate DNA synthesis in the presence of Ang II. TGF-beta binding data showed that Ang II increased the specific binding of 125I-TGF-beta1 by enhancing the expression of lower affinity receptors and increasing the number of binding sites. Ang II alone did not stimulate DNA synthesis in these cultures. However, Ang II significantly stimulated DNA synthesis after the inhibition of endogenous TGF-beta with a neutralizing antibody. The DNA synthesis stimulated by phorbol ester milisterol (PMA) was not affected by the TGF-beta neutralizing antibody. Affinity labeling data revealed receptor-ligand complexes of 280, 85, and 70 kDa, corresponding to TGF-beta type III, II, and I receptors, respectively. Incubation of VSMC with Ang II but not with PMA markedly increased the expression of the TGF-beta type I receptor. Reverse transcription and polymerase chain reaction data also indicated that Ang II, but not PMA, significantly increased the expression of TGF-beta type I receptor mRNA. Results suggest that Ang II increases the binding of TGF-beta with upregulation of TGF-beta type I receptor via a C-kinase-independent pathway. The enhanced expression of the TGF-beta type I receptor may counteract Ang II-promoted growth of VSMC.

Interleukin-1beta stimulates galectin-9 expression in human astrocytes.

Galectin-9 is an eosinophil chemoattractant produced by activated T lymphocytes. We have addressed expression of galectin-9 in normal human astrocytes in culture. Expression of galectin-9 mRNA and protein were examined by reverse transcription-polymerase chain reaction (RT-PCR), Western blotting, and immunofluorescent staining. Interleukin-1beta (IL-1beta) was found to enhance the galectin-9 expression in time- and concentration-dependent manners. Galectin-9 protein was detected in the membrane fraction, 105 000 x g precipitate, and immunofluorescent staining revealed diffuse cellular and perinuclear distributions. Dexamethasone pretreatment almost completely suppressed the production. We conclude that astrocytes produce galectin-9 in response to the stimulation with IL-1beta, and this may contribute to inflammatory reactions in the CNS.

Urinary immunoreactive brain natriuretic peptide in patients with renal disease.

Urinary immunoreactive brain natriuretic peptide (BNP) was studied by radioimmunoassay in patients with renal disease. Urinary immunoreactive human BNP excretion measured in 11 normal subjects was 3.82 +/- 0.62 pmol/day (mean +/- SEM). Significantly increased 24-h urinary secretion of immunoreactive human BNP was noted in patients with chronic renal failure (11.07 +/- 1.73 pmol/day, n = 9, P < 0.05 to normal subjects). A significant correlation was noted between 24-h urinary excretion of immunoreactive human BNP and creatinine clearance in patients with various renal diseases (r = -0.43, P < 0.01, n = 45). Gel chromatography of the urine extracts obtained from normal subjects and patients with chronic renal failure showed multiple immunoreactive peaks; two eluting earlier, one in the position of human BNP-32 and others eluting later. Reverse-phase high-performance liquid chromatography of the urine extracts showed a peak in the position of human BNP-32 and a peak eluting earlier. These findings indicate that: (1) immunoreactive human BNP is present in human urine; (2) urinary immunoreactive human BNP consists of multiple components, i.e., human BNP-32 itself or a substance very similar to it, smaller molecular forms which are probably metabolic products of human BNP-32, and larger molecular forms; and (3) 24-h urinary excretion of immunoreactive human BNP is increased in patients with renal dysfunction.

Effect of methylene methylimino linkage of antisense oligonucleotide to the platelet-derived growth factor A-chain on growth of vascular smooth muscle cells from spontaneously hypertensive rats.

Spontaneously hypertensive rats (SHR)-derived vascular smooth muscle cells show exaggerated growth and increased expression of platelet-derived growth factor (PDGF) A-chain mRNA. We examined the effect of methylene methylimino linkage of antisense oligodeoxynucleotide, a novel modification of antisense oligodeoxynucleotide designed to increase nuclease resistance, to PDGF A-chain on the exaggerated growth of vascular smooth muscle cells from SHR. Methylene methylimino-linked oligodeoxynucleotide provided complete resistance against S1 nuclease. Methylene methylimino linkage of antisense oligodeoxynucleotide to PDGF A-chain resulted in a rapid inhibition of basal DNA synthesis of vascular smooth muscle cells from SHR. This inhibition was much greater than that produced by phosphorothioate linkage of antisense oligodeoxynucleotide to PDGF A-chain. The methylene methylimino linkage of antisense oligodeoxynucleotide to PDGF A-chain may prove useful in the treatment of arterial proliferative diseases including hypertension.

Occlusal and TMJ loads in subjects with experimentally shortened dental arches.

To determine whether shortened dental arches (SDAs) cause functional overloading of the teeth and the temporomandibular joints, which has been implicated in periodontal diseases and temporomandibular disorders, we investigated the influences of SDA on occlusal and joint loads. Bite force and masticatory muscle electromyograms were recorded in five dentate subjects who clenched maximally on intra-oral appliances, creating symmetrical SDAs experimentally. Muscular forces estimated from the recorded electromyograms were fed into a finite element jaw model for calculating bite forces and joint loads. Comparison between the measured and the calculated bite forces ensured that the joint loads were representative. The bite force on each tooth increased with missing molar occlusions, while joint loads decreased. The bite force per root surface area was always greatest on the most posterior tooth, and these values were most constant. The findings provide no evidence that SDA causes overloading of the joints and the teeth, which suggests that neuromuscular regulatory systems are controlling maximum clenching strength under various occlusal conditions.

Detection of deletions, insertions and single nucleotide substitutions in cloned beta-globin genes and new polymorphic nucleotide substitutions in beta-globin genes in a Japanese population using ribonuclease cleavage at mismatches in RNA:DNA duplexes.

The applicability of ribonuclease cleavage at mismatches in RNA:DNA duplexes (RNase cleavage method) for determining nucleotide variant rates has been examined in a Japanese population. DNA segments of various lengths obtained from 4 different regions of a normal and 3 thalassemic cloned human beta-globin genes were inserted into transcription vectors. Sense and antisense RNA probes uniformly labeled with 32P were prepared. When RNA probes of 771 nucleotides (nt) or less were hybridized with cloned DNAs and the resulting duplexes were treated with a mixture of RNases A and T1, the length of products agreed with theoretical values. Twelve possible mismatches were examined. Since both sense and antisense probes were used, uncleavable mismatches such as G:T and G:G which were made from one combination of RNA and DNA strands could be converted to the cleavable C:A and C:C mismatches, respectively, by using the opposite combination. Deletions and insertions of 1 (G), 4 (TTCT), 5 (ATTTT) and 10 (ATTTTATTTT) nt were easily detected. A polymorphic substitution of T to C at position 666 of the second intervening sequence (IVS2-666) of the beta-globin gene was detected using genomic DNAs from cell lines established from the peripheral B lymphocytes of 59 unrelated Japanese from Hiroshima or those amplified by polymerase chain reaction (PCR). The frequency of the gene with C at the IVS2-666 (allele C) was 0.48 and that of the gene with T (allele T) was 0.52. The associations of the 2 alleles were in agreement with Hardy-Weinberg proportions. No contradiction to Mendelian inheritance was observed in the results obtained from 11 family studies. Two new polymorphic substitutions of C to A and A to T were detected at nucleotide positions 1789 and 1945 from the capping site, respectively, using genomic DNAs amplified by PCR. The feasibility of the RNase cleavage method combined with PCR for large-scale screening of variation in chromosomal DNA is discussed.

An improved method for the detection of genetic variations in DNA with denaturing gradient gel electrophoresis.

We have examined the feasibility of denaturing gradient gel electrophoresis (DGGE) of RNA:DNA duplexes to detect variations in genomic and cloned DNAs. The result has demonstrated that employment of RNA:DNA duplexes makes DGGE much more practical for screening a large number of samples than that of DNA:DNA heteroduplexes originally developed by Lerman et al. (1986), because preparation of RNA probes is easier than that of DNA probes. Three different 32P-labeled RNA probes were produced. Genomic or cloned DNAs were digested with restriction enzymes and hybridized to labeled RNA probes, and resulting RNA:DNA duplexes were examined by DGGE. The presence of mismatch(es) was detected as a difference in mobility of bands on the gel. The experimental conditions were determined using DNA segments from cloned normal and 3 thalassemic human beta-globin genes. The results of the experiments on the cloned DNAs suggest that DGGE of RNA:DNA duplexes will detect nucleotide substitutions and deletions in DNA. In the course of these studies, a polymorphism due to a single-base substitution at position 666 of IVS2 (IVS2-666) of the human beta-globin gene was directly identified using genomic DNA samples. A study of 59 unrelated Japanese from Hiroshima was made in which the frequency of the allele with C at IVS2-666 was 0.48 and that of the allele with T was 0.52. This approach was found to be very effective for the detection of heritable variation and should be a powerful tool for the detection of fresh mutations in DNA, which occur outside the known restriction sites.

A review of forty-five years study of Hiroshima and Nagasaki atomic bomb survivors. Biochemical genetics study.

After 40 years of studies on the children of atomic bomb survivors and a suitable control population in which no statistically significant genetic effects of A-bomb radiation were observed, two new projects have been initiated in order to detect mutations in the DNA and RNA molecules. Permanent cell lines from peripheral B-lymphocytes from approximately 500 families composed of exposed parents and their children and approximately 500 control families are being established by Epstein-Barr virus transformation. Cells will be sources for DNA and RNA samples in the screening for mutations. After comparison of efficiencies of the scanning techniques, we selected the denaturing gradient gel electrophoresis (DGGE) of DNA fragments amplified by polymerase chain reaction (PCR) for our purpose. A small scale pilot study has started to solve problems and obtain a better efficiency in this approach. Current thinking about the most efficient procedures is presented.

Influence of cisapride on the pharmacokinetics and antihypertensive effect of sustained-release nifedipine.

To investigate the clinical significance of interactions between cisapride and sustained-release nifedipine, we compared the plasma nifedipine concentration and blood pressure after administration of nifedipine alone (20 mg) with those obtained after administration of nifedipine cisapride (2.5 mg) in 20 patients with hypertension. The plasma nifedipine level was not altered by cisapride at one hour after administration, but was significantly increased at two (p < 0.01), three (p < 0.01), and four (p < 0.05) hours when compared with the level measured after nifedipine alone. Cisapride significantly decreased the mean blood pressure at three hours (p < 0.05) after administration of nifedipine. The acetaminophen method was used to determine gastric emptying time. The plasma concentration of acetaminophen at 45 minutes after administration was significantly increased by cisapride, suggesting that enhanced gastrointestinal motility might be the basis for the increase in the plasma nifedipine concentration. These results suggest that enhancement of the antihypertensive effect of nifedipine can occur when the drug is prescribed with cisapride, and that caution is needed when using such a combination therapy.