Effects of short- and long-acting calcium channel blockers on the relationship between blood pressure and physical activity.

Calcium channel blockers are widely used as antihypertensive drugs. However, there is some controversy as to how they should be used. Our first aim was to clarify how the dihydropyridine calcium channel blocker, benidipine, affects the quantitative relationship between blood pressure (BP) and physical activity. The second aim was to determine whether there is a relationship between systolic blood pressure (SBP) and physical activity in patients with hypertension when treating with a short-acting (nifedipine) or long-acting (benidipine) calcium channel blocker. In Study 1, ambulatory BP and physical activity were measured simultaneously in 27 patients with hypertension before and after 6 months with benidipine. In Study 2, ambulatory BP and physical activity were measured simultaneously in 16 patients with hypertension before (placebo) and after 6 weeks of crossover treatment with nifedipine and benidipine. In Study 1, there was no difference in the SBP change caused by physical activity between the pre- and posttreatment periods. In Study 2, SBP was significantly related to physical activity in the placebo (16/16) and benidipine (16/16) groups but not in the nifedipine (12/16) group. The lowest BP during day-time and nighttime in the nifedipine group were significantly lower than those in the benidipine group. Plasma renin activity (ng/mL/h) was significantly higher in the nifedipine group (1.20+/-1.05) than in the placebo (0.57+/-0.59) and benidipine (0.75+/-0.78) groups. These findings indicate that nifedipine might interfere with the adaptation mechanism of BP changed by physical activity and that the activated renin-angiotensin system might cause cardiac events.

Two novel mutations in the adrenoleukodystrophy gene in two unrelated Japanese families and the long-term effect of bone marrow transplantation.

We identified two novel missense mutations in exon 1 of adrenoleukodystrophy (ALD) gene in two unrelated Japanese families. The first, G(874)C transition results in Arg(163)Pro substitution in the cytoplasmic domain of the ALD protein in adrenomyeloneuropathy family. The second, C(679)G results in Ser(98)Trp substitution in the first transmembrane loop in childhood onset cerebral ALD family. Both mutations cause the substitution of polar amino acid (arginine and serine) with non-polar amino acid (proline and tryptophan). Bone marrow transplantation (BMT) from his non-affected his younger sister was performed on a boy with childhood onset cerebral ALD who showed neurological deficit and brain MRI abnormalities. We evaluated the effect of BMT over a 6-year period in terms of neurological deficit, the level of very-long-chain fatty acids (VLCFA) in plasma and fibroblasts, and brain MRI. After BMT, patient's peripheral white blood cells were replaced by donor's XX ones carrying a normal ALD gene confirmed by in situ hybridization using satellite DNA of the centromere of X and Y chromosomes as probes and the level of VLCFA in lymphocytes was within normal limit. However, his neurological state progressively deteriorated. BMT was not beneficial to him.

CALNUC (nucleobindin) is localized in the Golgi apparatus in insect cells.

A mouse monoclonal antibody 12B1 was raised against Golgi fractions from Sf21 insect cells and selected as Golgi-specific by immunostaining of the cells. The antigen was purified from the cells by immunoaffinity chromatography with the monoclonal antibody, and its N-terminal and internal amino acid sequences were determined. Based on the partial amino acid sequences, cDNA encoding the antigen protein was cloned and sequenced. The amino acid sequence deduced from the cDNA nucleotide sequence showed a homology to those of CALNUC family proteins, CALNUC (or nucleobindin, a calcium-binding Golgi protein with DNA-binding activity) and protein NEFA (a cell surface protein with DNA-binding, EF-hand, and acidic domains). The insect protein had two EF-hand loops at the same sites as the mammalian CALNUC family proteins, but had no leucine zipper which the mammalian homologues commonly have. An electron microscopic immunoperoxidase study demonstrated that the insect protein was localized in the cis-Golgi cisternae and cis-Golgi networks. Since this localization is identical to that of mammalian CALNUC, the insect protein was considered to be a homologue of CALNUC rather than that of NEFA. Assays involving proteinase K digestion, sodium carbonate extraction and Triton X-114 extraction revealed that the insect CALNUC-like protein was a soluble protein tightly associated with the luminal surface of Golgi membranes as reported for mammalian CALNUC. The insect protein was also shown to have calcium-binding activity as does mammalian CALNUC. These data verify that the insect protein is CALNUC. The existence of CALNUC in insect cells suggests that CALNUC is an essential calcium-binding Golgi protein in a wide range of the animal kingdom. A phylogenetic tree analysis, however, suggested that NEFA was derived from CALNUC long after the segregation of a mammalian ancestor from an insect ancestor.

Combination therapy using prednisolone and cyclophosphamide slows the progression of moderately advanced IgA nephropathy.

AIM: We retrospectively examined the effect of combination therapy using prednisolone (PSL) and cyclophosphamide (CPA) on the progression of IgA nephropathy (IgAN) in 45 patients with moderate to severe histological changes. PATIENTS AND METHODS: Patients were recruited from 129 consecutive patients with IgAN seen over 10 years based on semiquantitative histological grading. They were divided into two groups: PSL+CPA group (n = 26, male/female = 11/15, age 40+/-3 years (SEM)) or control group undergone conventional therapy with or without antiplatelet agents (n = 19, male/female = 10/9, age 41+/-3). In PSL+CPA group, PSL and CPA treatment commenced using a dose of 30 and 50 mg/day, respectively. PSL was reduced by 5 mg every month. RESULTS: The clinical parameters at the start of treatment such as age, gender, histological score, blood pressure, urinary protein excretion and serum creatinine concentration (SCr) were not different between the groups. The mean observation period in PSL+CPA group (3.3+/-0.3 years) was not different from the control group (4.0+/-0.7 years). In PSL+CPA group, urinary protein excretion, defined as the ratio of urinary protein to creatinine concentration (UP/UCr), significantly decreased from 3.9+/-0.4 to 1.3 +/-0.2 (p<0.01), whereas it remained high in the control group (3.8+/-0.7 to 2.7+/-0.8). The progression rate (PR), which was determined by the slope of the correlation between time after renal biopsy and reciprocal SCr, was significantly lower in PSL+CPA (0.054+/-0.014) than in the control group (0.172+/-0.032 dl/mg/year, p<0.001). Our results indicated that PSL+CPA combination therapy was effective in slowing the progression of moderately advanced IgAN. CONCLUSION: We suggest that the immunosuppressive treatment with CPA is sometimes necessary to preserve renal function in patients with histologically advanced IgAN.

Sas3 is a histone acetyltransferase and requires a zinc finger motif.

SAS3 was originally isolated as a gene related to SAS2, which encodes a positive regulator of transcriptional silencing in yeast. The Sas3 protein possesses an evolutionally conserved domain that is shared by a group of SAS-like factors. This conserved domain contains an atypical zinc finger motif and a putative acetyl-CoA binding motif. We showed that recombinant Sas3 exhibits histone acetyltransferase (HAT) activity toward acetylate core histones H2A, H3, and H4. This substrate specificity is similar to those of Tip60 and Esa1. Analysis of a series of deletion mutants revealed that the minimum region required for HAT activity is located within amino acid residues 241-577, including the domain conserved in the MYST family proteins. Amino acid substitution mutant analysis showed that both the acetyl-CoA binding motif and the zinc finger motif are required for HAT activity. These results suggest that SAS3 and its family members require the zinc finger motif for their activity.

Nonbacterial prostatitis caused by partial urethral obstruction in the rat.

The pathogenesis of nonbacterial prostatitis (NBP) is not understood mainly due to the lack of appropriate experimental models. We developed a new experimental model of NBP by inducing a partial obstruction of the urethra (PUO) in the rat. Male Wistar rats aged 12 weeks were used. PUO was produced by a nylon ligature on the urethra over a rubber tube. The tube was slipped out after the ligature had been tied. Two rats were examined histologically 6 h, 1 day, 3 days and 7 days after PUO. In another group, two rats were killed at 1, 3 and 7 days after the release of the PUO that had been left in place for 3 days. On day 3, another eight rats with PUO and eight control rats had 2 ml of urine in the bladder replaced by the same volume of lucifer yellow (LY; 10 microg/ml, MW 500), microperoxidase (MP; 20 microg/ml, MW 1900), horseradish peroxidase (HRP; 10 microg/ml, MW 40 000), or saline as control, respectively. Lymphocytic infiltration and interstitial edema were noted in the prostate following PUO, being most prominent on day 3. After the release of the PUO, these inflammatory changes gradually disappeared. Only LY was noted within the prostatic stroma of the rats 2 h after bladder instillation. Intraprostatic urinary reflux may be an etiologic factor in NBP. The present study showed that lower urinary tract obstruction caused NBP in the rat. Penetration of prostatic tissue by low-molecular-weight substances in the urine may trigger NBP.

Neuroprotective effect of mild hypothermia cannot be explained in terms of a reduction of glutamate release during ischemia.

An exogenous glutamate injection into the hypothermic hippocampal CA1 during 5-min ischemia produced the same extent of extracellular glutamate levels as observed in the normothermic CA1 during 5-min ischemia; however, neuronal death was not induced in the hypothermic CA1. Glutamate is released excessively into the extracellular space during ischemia, and is thought to induce brain injury by its neurotoxicity. It has been reported that the massive glutamate release is reduced by mild hypothermia, and it has been proposed that the reduction of ischemia-induced glutamate release exerts the neuroprotective effect on postischemic neuronal death. In the present study, to determine whether the neuroprotective effect of mild hypothermia on postischemic hippocampal CA1 neuronal death is due to the reduction of ischemia-induced glutamate release, gerbils were subjected to 5-min ischemia under hypothermic condition at 31 degrees C and were simultaneously injected exogenously with L-glutamate, so that the hypothermic CA1 around a microdialysis probe was exposed to the same extracellular glutamate levels as seen during normothermic ischemia, and the histological outcome was examined. An injection with 1 mM L-glutamate into the hypothermic CA1 during 5-min ischemia produced a similar extent of increased glutamate (17-fold increase) to that observed in the normothermic CA1 during 5-min ischemia (16-fold increase). However, neuronal death was not induced in the hypothermic CA1. This result indicates that the neuroprotective effect of mild hypothermia cannot be explained in terms of a reduction of glutamate release during ischemia.

Recovery of cardiac norepinephrine concentration and tyrosine hydroxylase activity by the central alpha2-adrenoceptor agonist guanabenz in rats with aortic constriction.

Depletion of cardiac norepinephrine has been reported in cardiac hypertrophy. This depletion causes less support for cardiac output in response to sympathetic nerve activation. The central nervous system is thought to be involved in this abnormality. Correction of this abnormality is expected to restore proper support for the heart. Clipping of the ascending aorta or a sham operation was performed in 10-week-old rats. At 4 weeks after the operation, the left ventricular norepinephrine concentration in clipped rats decreased (p<0.01). The clipped rats and sham-operated rats were treated with either guanabenz (1 mg/kg) or a vehicle for 4 weeks starting from fifth postoperative week. The level of left ventricular norepinephrine increased more in clipped rats treated with guanabenz (469+/-37 ng/g) than in clipped rats treated with a vehicle (325+/-28 ng/g). The norepinephrine concentration in the left ventricle recovered significantly after the treatment with guanabenz (p<0.001). Tyrosine hydroxylase activity in the left ventricle also recovered after treatment with guanabenz (p<0.01). Modulation of sympathetic nerve tone by the alpha2-adrenoceptor agonist restored cardiac norepinephrine concentration and tyrosine hydroxylase activity. This could be a new approach to the treatment of heart failure.

Histological study of urinary bladder transplantation in rats.

Patients who require cystectomy are usually treated with an ileal conduit or intestinal neobladder for urinary control. In some of them, however, the bowel segment cannot be used because of previous abdominal surgery or radiation treatment. Bladder transplantation from cadavers may be beneficial to these patients, if possible. To obtain basic knowledge about bladder transplantation, we developed an animal model of whole bladder transplantation in rats. Male Lewis rats weighing 270-320 g were used as both donors and recipients. Of the 23 recipients, 12 (52.2%) survived 7 days or longer after surgery. At 1 week after transplantation, the bladder showed loss of transitional epithelium and remarkable cellular infiltration. In the bladder at 5 weeks after transplantation, the transitional epithelium regenerated markedly and submucosal cellular infiltration was much improved. Regeneration of some smooth muscle cells was also noted. At 6 months after transplantation, the nerve fibers were recognized in the bladder and the volume of the transplanted bladder was well preserved (1.0-1.3 ml). This article describes an animal model of whole bladder transplantation in the rat which we produced and the results of our study. Because a large number of pure-bred animals can easily be used, we believe our rat model is very useful for basic studies of bladder transplantation.

Organization of the chicken histone genes in a major gene cluster and generation of an almost complete set of the core histone protein sequences.

We present a detailed picture of the disposition of the histone genes in the chicken genome and an almost complete set of the core histone protein sequences. Thirty-nine histone genes, six H1, nine H2A, eight H2B, eight H3 and eight H4, were located within a histone gene cluster of 110 kb, which was covered by five cosmid clones and two lambda clones. Results of our sequence analyses, together with those reported previously, generated a set of the core histone amino acid sequences as follows: three H2A variants, four H2B variants, two H3 variants and an H4 protein.

Molecular structure of the transposable element ninja in Drosophila simulans.

Genetically unstable DNA sequences of 16.1 kb in length were isolated from the white locus of the W(mky) strain of Drosophila simulans. This insertional DNA has some unique characteristics as a transposon. It is found in high numbers in this strain and its revertant strains W(psm1) and W(cho), but not elsewhere, and the sequence is a tandem triplication of a basic repeating unit. In order to determine the structure of the insert as whole and the functional unit as a transposon, we analyzed nine clones isolated from genomic libraries of W(psm1) and W(cho). The repeating unit of the 16.1 kb insertion was the retrotransposon ninja and the DNA sequence of the entire element was determined. The ninja transposon is 6644 bp in length, with a 316 bp long terminal repeat (LTR) on each end. It contains two openreading frames (ORFs), and the pol region is divided between the two ORFs in contrast the organization of other retrotransposons. An alignment analysis of the reverse transcriptase sequences suggested that the ninja element is the first Drosophila retrotransposon belonging to the Pao subgroup.

Initiation of unidirectional ColE2 DNA replication by a unique priming mechanism.

The ColE2 DNA can be replicated in an in vitro system consisting of a crude extract of Escherichia coli cells. DNA synthesis requires a plasmid-coded protein (Rep) and host DNA polymerase I but not host RNA polymerase. Replication starts at a fixed region containing the origin and proceeds unidirectionally. The leading- and lagging-strand DNA fragments synthesized around the origin were identified from early replicative intermediates. The 5' end of the leading-strand DNA fragment was mapped at a unique position in the minimal origin and carried RNA of a few residues. The results suggested that the initiation of the leading-strand DNA synthesis does not require the host DnaG protein. Thus the Rep protein itself seems to be a primase. Synthesis of the primer RNA at a fixed site in the origin region on a double-stranded DNA template is a unique property of the ColE2 Rep protein among other known primases. The 3' end of the lagging-strand DNA fragment was mapped at a unique position just at the end of the minimal origin region. Termination of the lagging-strand DNA fragment at that position seems to be the mechanism of the unidirectional replication of ColE2 plasmid.

[Clean intermittent catheterization in neurogenic bladder patients with vesicoureteral reflux].

BACKGROUND: For neurogenic bladder (NGB) patients with vesicoureteral reflux (VUR), renal deterioration constitutes a primary threat to survival. Although anti-reflux surgery for those patients was reported to be effective, the efficacy of clean intermittent catheterization (CIC) in such patients still remains to be clarified. METHODS: Sixteen neurogenic bladder patients with VUR were treated with CIC. Eight of them had spina bifida and 3 had radical hysterectomy for uterine cancer. Other 5 patients had spinal cord injury, spondyrocace, measles encephalopathy, and 2 unknown diseases, respectively. Hyperactive bladder was noted in 11 patients, whereas hypoactive bladder was noted in 3. Three patients were not evaluated. RESULTS: VUR was improved in only 3 patients (3 ureters), whose VUR grades were not more than III. Nine patients (13 ureters) had to have antireflux surgery. Although 3 of them needed bladder augmentation afterwards, the success rate of the antireflux surgery was 84.6%. CONCLUSION: We concluded that CIC alone was not effective to control VUR in neurogenic bladder patients. Nevertheless, CIC remained a good treatment option when VUR was managed surgically.

Primer RNA synthesis by plasmid-specified Rep protein for initiation of ColE2 DNA replication.

Initiation of in vitro ColE2 DNA replication requires the plasmid-specified Rep protein and DNA polymerase I but not RNA polymerase and DnaG primase. The ColE2 Rep protein binds specifically to the origin where replication initiates. Leading-strand synthesis initiates at a unique site in the origin and lagging-strand DNA synthesis terminates at another unique site in the origin. Here we show that the primer RNA for leading-strand synthesis at the origin has a unique structure of 5'-ppApGpA. We reconstituted the initiation reaction of leading-strand DNA synthesis by using purified proteins, the ColE2 Rep protein, Escherichia coli DNA polymerase I and SSB, and we showed that the ColE2 Rep protein is a priming enzyme, primase, which is specific for the ColE2 origin. The ColE2 Rep protein is unique among other primases in that it recognizes the origin region and synthesizes the primer RNA at a fixed site in the origin region. Specific requirement for ADP as a substrate and its direct incorporation into the 5' end of the primer RNA are also unique properties of the ColE2 Rep protein.

Control of ColE2 plasmid replication: negative regulation of the expression of the plasmid-specified initiator protein, Rep, at a posttranscriptional step.

The incA gene of ColE2 is involved in the copy number control and incompatibility. Two promoters were identified around the incA gene. Transcription of the mRNA for the essential plasmid-coded initiator protein (Rep) mainly starts at a site about 140 bp upstream of the initiation codon of the Rep protein. The second transcript (RNA I) of about 115 nucleotides with two stem-and-loop structures is entirely complementary to the 5' untranslated region of the Rep mRNA. By using translational and transcriptional fusions of the rep gene of ColE2 and the lacZ gene of Escherichia coli, the incA gene product was shown to regulate expression of the rep gene at a posttranscriptional step. The results also suggest that the target of the incA gene product is the 5' untranslated region of the Rep mRNA. Deletion analyses reported here show that a region(s) about 17 to 70 bp upstream of the initiation codon of the Rep protein and another region inside the coding frame are important for efficient production of the Rep protein. This suggests that some additional sequence elements other than the initiation codon and the Shine-Dalgarno region and/or a secondary structure of the Rep mRNA are required for efficient production of the Rep protein. These results show that RNA I is an antisense RNA for the Rep mRNA and imply that it might regulate expression of the rep gene at the initiation step of translation by sequestering such additional sequence elements and/or by disrupting RNA secondary structure. We propose that RNA I represents the incA gene product.

Control of ColE2 plasmid replication: regulation of Rep expression by a plasmid-coded antisense RNA.

We isolated and characterized mutants of ColE2 with increased copy number (cop) and those with reduced sensitivity to the wild-type incA gene (inc). Both types of mutations were single-base substitutions in the incA region and simultaneously increased the plasmid copy number and reduced the inhibitory activity of the incA gene on ColE2 DNA replication. Most of the cop mutations also reduced sensitivity to the wild-type incA gene. These mutations were located in the region specifying the large stem-and-loop structures of RNA I and the 5' portion of the Rep mRNA. All these results indicate that RNA I interacts with the Rep mRNA and thereby inhibits expression of the Rep protein at a post-transcriptional step and that this is probably the only mechanism that controls the ColE2 Rep protein expression. It is suggested that only portions of the nucleotides in the loop region are involved in initial (kissing) interaction of these RNAs. The total level of rep gene expression in the host cells appears to be kept constant (at a level characteristic for each cop allele) irrespective of the actual plasmid copy number above a certain level, when rep gene expression is regulated by the incA gene on the same plasmid. These seem to be the basic mechanisms for the replication control of ColE2.

Exogenous histone H1 injection into mitotic cells disrupts synchronous progression of mitotic events by delaying chromosome decondensation.

At the end of open mitosis, chromosome decondensation, nuclear envelope re-formation and reassembly of interphase microtubules following mitotic spindle dissociation occur coordinately. To determine whether these events progress only synchronously in vivo, we delayed chromosome decondensation by injecting of exogenous proteins into the mitotic rat kangaroo kidney epithelium (PtK2) cells. When histone H1 purified from calf thymus was injected at prometaphase, chromosome condensation was prolonged for several hours, and sister chromatid separation and cytokinesis did not occur. However, interphase microtubules reassembled and lamin B-positive structures re-formed around the condensed chromosomes. Exactly the same results were obtained on injection of bacterially expressed H1. Kinetic experiments showed that there were two types of lamin B-positive structures. One type (type A) was stained uniformly with anti-lamin B antibodies. The other (type B) showed peripheral lamin B staining; that is, the normal interphase staining pattern, and was found to be competent for nuclear protein transport. As the chromosomes decondensed, the amount of type A decreased and that of type B increased. However, even cells containing highly condensed chromosomes had both type A and type B. From these results, we conclude that the re-formation of microtubules and reassembly of a nuclear transport-competent envelope do not depend on chromosome decondensation.

[Effect of enalapril on left ventricular hypertrophy and left ventricular function in patients with hypertension].

The aim of this study was to study the effect of enalapril (E) on left ventricular (LV) mass, LV function and blood renin-angiotensin (RA) in patients with hypertension. Sixteen hypertensives were included in this study (WHO I 8, WHO II 8, 49.5 +/- 10.5 yrs). They were examined for blood pressure and heart rate. Chest X-ray film, echocardiography (echo), X-ray computed tomography (CT) and RA before and after about 6 months of E administration were studied. The LV mass was calculated by CT. The LV function was measured by echo. RAS was unchanged during this study. LV mass was significantly reduced after E (121.4 +/- 25.6 vs 104.6 +/- 13.7 g/cm2). The LV systolic function was unchanged after E, but LV diastolic function improved. It was shown that the long-term administration of E improves LV hypertrophy and LV diastolic function without any change of RAS.

Presence of particular transcription regulatory elements in the 5'-intergenic region shared by the chicken H2A-III and H2B-V pair.

The two chicken histone gene families, H2A and H2B, contain nine and eight members, respectively, within two major histone gene clusters. Six genes each from families H2A and H2B have been found to be closely associated in inverted directions as H2A/H2B gene pairs. Two previously sequenced H2A members (H2A-I and H2A-II) encode the same amino acid (aa) sequence (class I), whereas seven sequenced H2B genes encode three different variants (classes I, II and III). In this study, we first sequenced H2A-III, a member of the H2A family, which is located in inverted orientation and 350 bp upstream from H2B-V, encoding the class-III H2B protein. The protein encoded by H2A-III differs from the class-I H2A protein in a single aa (Ala70-->Pro; class II). As a step toward elucidation of the transcriptional regulation of the H2A and H2B families, we fused this 5'-intergenic region to the cat gene in inverted orientations to generate two chimeric plasmids, pH2A-III-350 and pH2B-V-350. Transient CAT assays using these constructs indicated that the promoter of H2B-V is more active than that of H2A-III. CAT assays with 5'-deletion mutants of H2A-III and H2B-V showed that they each possess particular transcriptional motifs which are located relatively close to, or apart from, their own coding regions. These findings, together with those reported previously on the H2A-V/H2B-II pair, suggest distinct manners of transcription regulation of different members of the chicken histone gene families, H2A and H2B.