Circulating CD34-Positive Cells Are Associated with Handgrip Strength in Japanese Older Men: The Nagasaki Islands Study.

BACKGROUND: Handgrip strength is a simple measurement of overall muscular strength and is used to detect sarcopenia. It also predicts adverse events in later life. Many mechanisms of sarcopenia development have been reported. A hypertensive status impairs endothelial dysfunction, which might deteriorate skeletal muscle if vascular angiogenesis is not maintained. OBJECTIVES: This study investigated muscle strength and circulating CD34-positive cells as a marker of vascular angiogenesis. DESIGN: Cross-sectional study. PARTICIPANTS: 262 male Japanese community dwellers aged 60 to 69 years. MEASUREMENTS: The participants' handgrip strength, medical history, and blood samples were taken. We stratified the participants by hypertensive status to investigate the association between handgrip strength and circulating CD34-positive cells according to hypertensive status. Pearson correlation and linear regression analyses were used. RESULTS: In the Pearson correlation analysis, handgrip strength and the logarithm of circulating CD34-positive cells were significantly associated in hypertensive participants (r=0.22, p=0.021), but not in non-hypertensive participants (r=-0.01, p=0.943). This relationship was only significant in hypertensive participants (ß=1.94, p=0.021) in the simple linear regression analysis, and it remained significant after adjusting for classic cardiovascular risk factors (ß=1.92, p=0.020). The relationship was not significant in non-hypertensive participants (ß=-0.09, p=0.903). CONCLUSIONS: We found a positive association between handgrip strength and circulating CD34-positive cells in hypertensive men. Vascular maintenance attributed by circulating CD34-positive cells is thought to be a background mechanism of this association after hypertension-induced vascular injury in skeletal muscle.

Reduced vascular reserve measured by stressed single photon emission computed tomography carries a high risk for stroke in patients with carotid stenosis.

AIM: A few studies have observed reduced vascular reserve measured by single photon emission computed tomography (SPECT) to be a risk factor for stroke in patients with carotid artery occlusion, but stenosis has been excluded from these former studies. This study has evaluated the prognosis of reduced vascular reserve in patients with stenosis, and the effect of carotid endarterectomy (CEA) on these patients. METHODS: Forty patients diagnosed as having >70% stenosis of the carotid artery at the University of Tokyo Hospital, between 2001 and 2004, underwent acetazolamide-stress SPECT test first. A resting SPECT study was performed on a different day from the stressed SPECT study. The patients were grouped as having reduced vascular reserve or normal vascular reserve from the SPECT results. Analysis of risk factors and the stroke-free curve analysis for reduced vascular reserve was performed. RESULTS: Of the 40 patients, 24 (60%) had reduced vascular reserve and 18 underwent CEA. The mean follow-up period was 21.5+/-15.5 months (mean+/-SD). Four strokes occurred during follow-up: in 1 patient with CEA and 3 without CEA. All stroke patients had reduced vascular reserve. The patients with reduced vascular reserve without any surgery had a significantly lower stroke-free rate compared with those with normal vascular reserve or reduced vascular reserve, but also receiving CEA. CONCLUSIONS: We propose performing SPECT tests in patients with severe carotid stenosis regardless of symptoms, and performing CEA on those with a reduction in vascular reserve.

Carotid endarterectomy may reduce the high stroke rate for patients with the disease of abdominal aorta and peripheral arteries.

AIM: The prevalence of carotid stenosis is reported to be high among patients with arteriosclerosis, but the hazards of carotid stenosis and the benefits of carotid endarterectomy (CEA) on long-term event-free survival are still unknown. The aim of this prospective study was to screen preoperative patients with arterial disease for carotid stenosis, and to determine whether CEA had any effect on stroke during the postoperative follow-up period. METHODS: From 1999 to 2003, 406 consecutive preoperative patients with arterial disease underwent routine carotid duplex scan. Patients with known carotid stenosis and those due to undergo operation in emergency were excluded from the study. CEA was performed before or simultaneously with vascular surgery if necessary. The prevalence and risk factors for carotid stenosis were studied, and the patients were followed up for stroke or death. RESULTS: Among the 406 patients examined, 19.4% had greater than 50% stenosis and 11.3% had greater than 70% stenosis. The risk factors for carotid stenosis were having occlusive arterial disease (P=0.0001), and history of stroke (P=0.0038). Long-term follow-up study revealed that patients with greater than 70% carotid stenosis without CEA had a higher tendency for stroke or death, but the stroke rate in patients with severe stenosis who underwent CEA remained low, as in patients with less than 70% stenosis. CONCLUSIONS: Patients with greater than 70% carotid stenosis, diagnosed before arterial operation who did not undergo CEA, had a higher risk for stroke during the postoperative follow-up period. However, their risk could be reduced by performing CEA before or simultaneously with scheduled vascular surgery.

Microbicidal efficacy of ozonated water against Candida albicans adhering to acrylic denture plates.

BACKGROUND/AIMS: Ozone is known to act as a strong antimicrobial agent against bacteria, fungi, and viruses. We examined the effect of ozonated water on Candida albicans on acrylic denture plate. METHODS: The heat-cured acrylic resins were cultured with C. albicans. After treatment of flowing ozonated water, the number of attached C. albicans was counted. In some experiments, the test samples were treated with ozonated water in combination with ultrasonication. RESULTS: After exposure to flowing ozonated water (2 or 4 mg/l) for 1 min, viable C. albicans cells were nearly nonexistent. The combination of ozonated water and ultrasonication had a strong effect on the viability of C. albicans adhering to the acrylic resin plates. There were no significant differences in antimicrobial activity against C. albicans between plates immersed in ozonated water with ultrasonication and those treated with commercially available denture cleaners. In addition, electron microscopic analysis revealed that small amounts of C. albicans remained on the plate after exposure to flowing ozonated water or immersion in ozonated water with ultrasonication. CONCLUSION: Our results suggest that application of ozonated water may be useful in reducing the number of C. albicans on denture plates.

Efficacy of ozone on survival and permeability of oral microorganisms.

In the present study, we examined the effect of ozonated water on oral microorganisms and dental plaque. Almost no microorganisms were detected after being treated with ozonated water (4 mg/l) for 10 s. To estimate the ozonated water-treated Streptococcus mutans, bacterial cells were stained with LIVE/DEAD BacLight Bacterial Viability Kit. Fluorescence microscopic analysis revealed that S. mutans cells were killed instantaneously in ozonated water. Some breakage of ozonated water-treated S. mutans was found by electron microscopy. When the experimental dental plaque was exposed to ozonated water, the number of viable S. mutans remarkably decreased. Ozonated water strongly inhibited the accumulation of experimental dental plaque in vitro. After the dental plaque samples from human subjects were exposed to ozonated water in vitro, almost no viable bacterial cells were detected. These results suggest that ozonated water should be useful in reducing the infections caused by oral microorganisms in dental plaque.

Enantioselective transesterification using immobilized Aspergillus oryzae overexpressing lipase.

In the present study, we used gene manipulation to construct a recombinant Aspergillus oryzae strain overexpressing lipase and investigated its application to the optical resolution of chiral compounds. A. oryzae niaD300, which was derived from the wild-type strain RIB40, was used as the host strain. The tglA gene, which encodes a triacylglycerol lipase, was cloned from the A. oryzae niaD300 chromosomal genome, then reintroduced, with and without a secretion-signal sequence, into the genome and expressed under the control of the improved glaA promoter of plasmid pNGA142. The resulting recombinant strain overexpressing A. oryzae lipase was immobilized within biomass-support particles and used as a whole-cell biocatalyst. The immobilized lipase-overexpressing strain with secretion-signal sequence showed high activity and was used to selectively synthesize (R)-1-phenylethyl acetate from (RS)-1-phenylethanol and vinyl acetate. After 48 h reaction at 30 degrees C with molecular sieve 4A, the yield and enantiomeric excess (%ee) of (R)-1-phenylethyl acetate reached approximately 90 and 95%ee, respectively. The whole-cell biocatalyst for optical resolution of chiral compounds produced in this study maintained its activity over 25 batch-reaction cycles.

Self-renewal and differentiation of a basic fibroblast growth factor-dependent multipotent hematopoietic cell line derived from embryonic stem cells.

Despite the accumulation of informat on on the origin of hematopoietic stem cells, it is still unclear how these cells are generated in ontogeny. Isolation of cell lines equivalent to early embryonic hematopoietic progenitor cells can be helpful. A multipotent hematopoietic progenitor cell line, A-6, was isolated from H-1 embryonic stem (ES) cells. The self-renewal of A-6 cells was supported by basic-fibroblast growth factor (b-FGF) and their differentiation into definitive erythroid cells, granulocytes and macrophages was induced after co-culture with ST-2 stromal cells. A-6 cells were positive for the surface markers of hematopoietic stem cell, c-kit, CD31, CD34, Flt3/Flk2, PgP-1, and HSA, but were negative for that of the differentiated cells. Reverse transcription-polymerase chain reaction analysis showed that A-6 cells produced mRNA from SCL/tal-1 and GATA-2 genes. Among various cytokines examined, on y stem cell factor (SCF) and Flt3/Flk2 ligand (FL) supported the proliferation of A-6 cells instead of b-FGF. The FL, as well as b-FGF, supported the self-renewal of A-6 cells, whereas SCF induced differentiation into myeloid cells. A-6 cells will be useful for the characterization of hematopoietic progenitor cells derived from ES cells and provide a model system to realize the control mechanisms between self-renewal and different ation of hematopoietic stem cells.

Upregulation of the elongation factor-1alpha gene by p53 in association with death of an erythroleukemic cell line.

Genes upregulated by p53 were screened using an erythroleukemic cell line (1-2-3) that expresses only the temperature-sensitive p53 by the mRNA differential display method. One of the upregulated genes was identified as the elongation factor-1alpha (EF-1alpha) gene, an essential component of the eukaryotic translation apparatus. Three p53-responsive elements were found in the mouse EF-1alpha gene and in the corresponding human, rat, and frog genes. These elements conferred the capacity for induction by p53. EF-1alpha is also a microtubule-severing protein. Upon the temperature-shift, the cells developed the morphology and the localization of alpha-tubulin similar to those of the cells treated with vincristine, a drug that affects microtubules. The microtubule-severing associated with upregulation of EF-1alpha by p53 may be a cause of the cell death.

Up-regulation of cell cycle-associated genes by p53 in apoptosis of an erythroleukemic cell line.

A murine erythroleukemic cell line (1-2-3) which expresses only the temperature-sensitive mutant p53 gene (Ala-to-Val substitution at codon 135) was established. When these cells were cultured at 32 degrees C, the growth rate was reduced significantly and DNA fragmentation, a typical character of apoptosis, was observed. In this process, p53 migrated from cytoplasm to nucleus and protein complexes binding to the p53-responsive element were detected in nuclear extracts of the cells cultured at 32 degrees C by gel-shift assay and transactivation from the p53-responsive element was detected. The expression of the p21 (waf1/cip1/sdi1), cyclin G and gadd45 genes was increased (about 3 to 4 fold that at 37 degrees C), when the cells were cultured at 32 degrees C. However, the expression of the bax gene was increased slightly (about 1.5 fold that at 37 degrees C) and no significant change was detected in expression of the mdm2 gene. No change in the amount of Fas antigen was observed by flow cytometric analysis. Transcripts of the bcl-2 and fasl gene were not detected in the cells both at 37 degrees C and 32 degrees C. These results suggest that up-regulation of the genes associated with the cell cycle and/or DNA replication, such as p21, cyclinG and gadd45 rather than bax, fas, fasl and bcl-2 may be important for induction of apoptosis of this erythroleukemic cell line by p53.

Genomic imprinting of the human serotonin-receptor (HTR2) gene involved in development of retinoblastoma.

Epidemiological and genetic studies of retinoblastoma (RB) suggested that imprinted genes might be genetically linked to the RB gene. In this study, we found that the human serotonin-receptor, HTR2, gene, which had been mapped nearby the RB gene on chromosome 13, was expressed only in human fibroblasts with a maternal allele and not in cells without a maternal allele. The 5' genomic region of the human HTR2 gene was cloned by PCR-mediated method. Only the 5' region of the gene was methylated in cells with the maternal gene, and it was not methylated in cells without the maternal gene. A polymorphism of PvuII site of the gene was also found and useful for the segregation analysis in a family of a RB patient and for analysis of loss of heterozygosity on chromosome 13 in tumor and its parental origin. These results suggest that the human HTR2 gene might be affected by genomic imprinting and that exclusive expression of the maternal HTR2 gene may be associated with the delayed occurrence of RB, which had lost the maternal chromosome 13.

A C3H strain-specific allele (alpha va126) of the murine alpha-globin gene newly detected by UT-PAGE and RT-PCR-SSCP analysis.

An extra band. distinct from the well-characterized globin chains (alpha, beta-maj, beta-min, beta-s), was detected in an adult erythrocyte sample of the C3H strain by urea triton polyacrylamide gel electrophoresis (UT-PAGE) analysis. The extra band was recognized by an antibody against the alpha-globin chain by Western blot analysis. Reverse transcription, polymerase chain reaction and single strand conformation polymorphism (RT-PCR-SSCP) analysis and direct sequencing analysis of cDNA of the alpha-globin gene revealed a nucleotide substitution (GGA to GTA) corresponding to an amino acid substitution (Gly to Val) at codon 26 in the alpha-globin gene only in the erythrocyte sample of the C3H strain. Polypeptides generated by in vitro translation from the alpha-globin gene with the nucleotide substitution at codon 26 (alpha Val26) had the same mobility as that of the extra band of the C3H strain in UT-PAGE. These results suggest that the substitution GGA (Gly) to GTA (Val) at codon 26 of the murine alpha-globin gene may directly affect the mobility of alpha-globin in UT-PAGE and the base substitution may be a C3H strain-specific polymorphism.

A follistatin-like gene, mac25, may act as a growth suppressor of osteosarcoma cells.

mac25, a retinoic acid-inducible gene that is expressed at high levels in senescent epithelial cells, was initially cloned as a gene that is differentially expressed in meningioma. Although the homology of its product with members of family of insulin-like growth factor-binding proteins was suggested, the product also exhibits strong homology to follistatin, an activin-binding protein. However, a domain corresponding to the carboxyl terminus of follistatin is not found in mac25. The carboxyl-terminally truncated form of follistatin, generated by alternative splicing, has stronger activin-binding activity than the complete form. This result suggests that mac25 might act as an activated follistatin. Clonal growth of a p53-deficient osteosarcoma cell line was strongly inhibited when the murine mac25 gene, as well as the p53 gene, was introduced. Resembling activins that belong to the transforming growth factor-beta (TGF-beta) superfamily, mac25 and p53 might associate with similar but distinct targets, namely cyclin-dependent kinase inhibitors. However, there is no evidence for compensation of p53 function by mac25 in the development of p53-deficient mice, as judged from the pattern of expression of mac25 in mice. mac25 might act as a tumor suppressor, modulating signaling of the TGF-beta family, as does alpha-inhibin.

No significant changes in the expression of the fas,fasl,bcl-2 and bax genes in apoptosis of an erythroleukemic cell line by p53.

A murine erythroleukemic cell line (1-2-3) which expresses only the temperature-sensitive mutant p53 gene (Ala-to-Val substitution at codon 135) was established. When these cells were cultured at 32 degrees C, the growth rate was reduced significantly and DNA fragmentation, a typical character of apoptosis, was observed. In this process, p53 migrated from cytoplasm to nucleus and protein complexes binding to the p53-responsive element were detected in nuclear extracts of the cells cultured at 32 degrees C by gel-shift assay and transactivation from the p53-responsive element was detected. The expression of the p21 (waf1/cip1/sdi1), cyclin G and gadd45 genes was increased (about 3- to 4-fold that at 37 degrees C), when the cells were cultured at 32 degrees C. However, no significant change was detected in expression of the mdm2, bax and fas genes. No change in the amount of Fas antigen was observed by flow cytometric analysis. Transcripts of the bcl-2 and fas1 gene were not detected in the cells either at 37 degrees C or 32 degrees C. These results suggest that other gene(s) than bax, fas, fas1 and bcl-2 may be important for induction of apoptosis of this erythroleukemic cell line by p53.

Repression of p53-dependent sequence-specific transactivation by MEF2c.

Lambda ZAP cDNA library constructed from spleen of a p53-deficient mouse was screened by South-Western technique using Fragment A, a DNA sequence that p53 specifically binds to, as a probe. One (clone 2) of six clones isolated was identical to MEF2c, a MADS-family transcription factor. Transcripts of the mef2c gene was also detected in spleen where the expression has not been reported so far. Isolated clones except clone 2 had a growth-suppression activity on p53-deficient osteosarcoma cell line (Saos II). Clone 2 repressed the transactivation from Fragment A by p53, suggesting that MEF2c may act as a negative regulator of p53-responsive element.

[A case of successful treatment of chronic sternal osteomyelitis and mediastinitis after ascending aorta and hemiarch reconstruction].

Mediastinitis is one of severe and fatal complications after cardiac surgery, especially in the presence of a prosthetic graft. A 70-year-old male, who was suffered from chronic sternal osteomyelitis and mediastinitis after ascending aorta and hemiarch reconstruction was reported. He developed high fever and leucocytosis on the 16th postoperative day. Wound culture was positive for Grampositive organisms. He was diagnosed as having sternal osteomyelitis and mediastinitis and treated by sternal bone debridement and irrigation with dilute povidone iodine solution with no effects. Total excision of the infected sternum, irrigation with non-diluted solution of povidone iodine and omental transfer were performed successfully. The literature regarding omental transfer for mediastinitis and infected prosthetic grafts was briefly reviewed.

A mouse erythroleukemia cell line possessing friend spleen focus-forming virus gp55 transgene and temperature-sensitive mutant p53 gene.

Two different erythroleukemia cell lines have been established from the splenic lesions of transgenic mice possessing the Friend spleen focus-forming virus (F-SFFV) gp55 gene. One showed a near-diploid karyotype and a temperature-sensitive (ts) p53 mutation, and the other, a hyper-triploid karyotype with double p53 mutations found by single-strand conformation polymorphism (SSCP) analysis. The cell lines both retained No.11 chromosomes on which p53 genes are localized. Another p53 allele in the cell line with the ts-p53 mutation appeared intact in the SSCP analysis of the genomic exon 5. The cells with the ts-mutant p53 gene showed no apparent change with temperature shift in their growth or dimethylsulfoxide-induced differentiation, although the wild-type p53 gene on the other allele was not expressing. This ts-p53Val-135 gene made p53-deficient fibroblasts anchorage-independent at 37 degrees C but not at 32 degrees C. This non-virus-producing, mouse erythroleukemia cell line will be useful for the study of mutated p53 function during the induction of erythrodifferentiation or apoptotic change.

Parthenogenetic activation of oocytes in c-mos-deficient mice.

In Xenopus the c-mos proto-oncogene product (Mos) is essential for the initiation of oocyte maturation, for the progression from meiosis I to meiosis II and for the second meiotic metaphase arrest, acting as an essential component of the cytostatic factor CSF. Its function in mouse oocytes is unclear, however, as is the biological significance of c-mos mRNA expression in testes and several somatic tissues. We have generated c-mos-deficient mice by gene targeting in embryonic stem cells. These mice grew at the same rate as their wild-type counterparts and reproduction was normal in the males, but the fertility of the females was very low. The c-mos-deficient female mice developed ovarian teratomas at a high frequency. Oocytes from these females matured to the second meiotic metaphase both in vivo and in vitro, but were activated without fertilization. The results indicate that in mice Mos plays a role in the second meiotic metaphase arrest, but does not seem to be essential for the initiation of oocyte maturation, spermatogenesis or somatic cell cycle.

[A case of a right subclavian arterial aneurysm associated with the aortic arch anomaly in childhood].

Aneurysms in the subclavian arteries are extremely rare in childhood. A 7-year-old boy was admitted with hoarseness and abnormal shadow in the chest film. Radiologic studies demonstrated a right subclavian arterial aneurysm associated with the aortic arch anomaly. Proximal and distal ligation of the subclavian arterial aneurysm and reconstruction of blood flow by an aorto-subclavian bypass using a 5 mm GORE-TEX prosthetic graft were performed through a standard median sternotomy incision with extension to the right supraclavicular space. The postoperative course was uneventful and he was discharged in 3 weeks. The literature of the subclavian arterial aneurysm was briefly reviewed.