Transport of magnesium by two isoforms of the Na+-Ca2+ exchanger expressed in CCL39 fibroblasts

Cytoplasmic concentrations of Ca2+ ([Ca2+]i) and Mg2+ ([Mg2+]i) were measured with fluorescent indicators in CCL39 cells, a cell line established from Chinese hamster lung fibroblasts, transfected with complementary deoxyribonucleic acid (cDNA) of the Na+-Ca2+ exchanger isolated either from canine heart (NCX1) or from rat brain (NCX3). Raising extracellular [Mg2+] to 10 mM increased Mg2+ influx and the resultant change in [Mg2+]i (delta[Mg2+]i) was monitored with furaptra under Ca2+-free conditions. In control (vector-transfected) cells, delta[Mg2+]i at 45 min was similar with or without extracellular Na+ (130 mM or 0 mM) and when [Na+]i was raised by 1 mM ouabain treatment. delta[Mg2+]i in NCX1-transfected cells was attenuated significantly in the presence of 130 mM Na+, but became comparable to (or slightly larger than) that in control cells on either removal of extracellular Na+ or treatment with 1 mM ouabain. Cells expressing NCX3 showed an intermediate dependence of delta[Mg2+]i on Na+, probably reflecting a lower degree of expression of the exchanger protein. Extracellular Na+-dependent changes in [Ca2+]i (measured with fura-2 in the presence of extracellular Ca2+ and 10 microM ionomycin, a Ca2+ ionophore) were minimal in control cells, marked in the NCX1-transfected cells and intermediate in the NCX3-transfected cells. These results suggest that the Na+-Ca2+ exchanger (either NCX1 or NCX3) can transport Mg2+ and may play a role in the extrusion of magnesium from cells.     

Cell lines that support replication of a novel herpes simplex virus 1 UL31 deletion mutant can properly target UL34 protein to the nuclear rim in the absence of UL31

Previous results indicated that the herpes simplex virus 1 (HSV-1) U(L)31 gene is necessary and sufficient for localization of the U(L)34 protein exclusively to the nuclear membrane of infected Hep2 cells. In the current studies, a bacterial artificial chromosome containing the entire HSV-1 strain F genome was used to construct a recombinant viral genome in which a gene encoding kanamycin resistance was inserted in place of 262 codons of the 306 codon U(L)31 open reading frame. The deletion virus produced virus titers approximately 10- to 50-fold lower in rabbit skin cells, more than 2000-fold lower in Vero cells, and more than 1500-fold lower in CV1 cells, compared to a virus bearing a restored U(L)31 gene. The replication of the U(L)31 deletion virus was restored on U(L)31-complementing cell lines derived either from rabbit skin cells or CV1 cells. Confocal microscopy indicated that the majority of U(L)34 protein localized aberrantly in the cytoplasm and nucleoplasm of Vero cells and CV1 cells, whereas U(L)34 protein localized at the nuclear membrane in rabbit skin cells, and U(L)31 complementing CV1 cells infected with the U(L)31 deletion virus. We conclude that rabbit skin cells encode a function that allows proper localization of U(L)34 protein to the nuclear membrane. We speculate that this function partially complements that of U(L)31 and may explain why U(L)31 is less critical for replication in rabbit skin cells as opposed to Vero and CV1 cells.     

Effects of sedative odorant inhalation on patients with atopic dermatitis]

Atopic dermatitis (AD) has been clinically well-known to be frequently exacerbated by psychological and physiological stress. In this study, we examined effects of sedative odorant (modified valerian oil) inhalation on patients with AD. We investigated clinical scores, skin physiological parameters and psychological questionnaire (POMS) every 2 weeks. For first 2 weeks, we arranged non-inhalation period. Results for non-inhalation period were compared with these of 2- or 4-week inhalation. As results, sum of skin clinical scores significantly improved after odorant inhalation. Some patients improved for non-inhalation period, too. However, patients that had not improved for non-inhalation period significantly improved after odorant inhalation. Skin conductance and skin dryness/scaling score also improved after odorant inhalation without improving for non-inhalation period. Psychological parameter (POMS) also tended to improve after odorant inhalation. These results suggest that sedative odorants may be useful as a complementary therapy for AD through psychosomatic stress care.     

Cohesin release is required for sister chromatid resolution,but not for condensin-mediated compaction,at the onset of mitosis

The establishment of metaphase chromosomes is an essential prerequisite of sister chromatid separation in anaphase. It involves the coordinated action of cohesin and condensin, protein complexes that mediate cohesion and condensation, respectively. In metazoans, most cohesin dissociates from chromatin at prophase, coincident with association of condensin. Whether loosening of cohesion at the onset of mitosis facilitates the compaction process, resolution of the sister chromatids, or both, remains unknown. We have found that the prophase release of cohesin is completely blocked when two mitotic kinases, aurora B and polo-like kinase (Plx1), are simultaneously depleted from Xenopus egg extracts. Condensin loading onto chromatin is not affected under this condition, and rod-shaped chromosomes are produced that show an apparently normal level of compaction. However, the resolution of sister chromatids within these chromosomes is severely compromised. This is not because of inhibition of topoisomerase II activity that is also required for the resolution process. We propose that aurora B and Plx1 cooperate to destabilize the sister chromatid linkage through distinct mechanisms that may involve phosphorylation of histone H3 and cohesin, respectively. More importantly, our results strongly suggest that cohesin release at the onset of mitosis is essential for sister chromatid resolution but not for condensin-mediated compaction.     

Nucleotide sequence of the gene encoding the matrix protein of mumps virus (Miyahara strain)

The nucleotide sequence of the gene encoding the matrix (M) protein of mumps virus (MuV), Miyahara strain, has been determined from several overlapping cDNA clones. The M protein mRNA is 1248 nucleotides in length, exclusive of the poly(A) tail, and codes for a protein of 375 amino acids (Mr41,556). Comparison of the deduced amino acid sequence of the M protein of the Miyahara strain with that of the SBL-1 strain revealed that the M proteins of both strains are highly conserved. A significantly lower rate of nucleotide differences conducive to amino acid differences in the M gene compared with other genes appeared to indicate the importance of the conserved primary structure of the M protein for its function.Requests for reprints should be addressed to Kiyoshi Tanabayashi, Department of Measles Virus, National Institute of Health, 4-7-1 Gakuen, Musashimurayama, Tokyo 190-12, Japan.     

Iron,coronary artery calcification,and mortality in patients undergoing hemodialysis

ObjectiveA high coronary artery calcification score (CACS) may be associated with high mortality in patients undergoing hemodialysis (HD). Recently, effects of iron on vascular smooth muscle cell calcification have been described. We aimed to investigate the relationships between iron, CACS, and mortality in HD patients.MethodsWe studied 173 consecutive patients who were undergoing maintenance HD. Laboratory data and Agatston’s CACS were obtained at baseline for two groups of patients: those with CACS ≥400 (n = 109) and those with CACS <400 (n = 64). Logistic regression analyses for CACS ≥400 and Cox proportional hazard analyses for mortality were conducted.ResultsThe median (interquartile range) age and duration of dialysis of the participants were 67 (60–75) years and 73 (37–138) months, respectively. Serum iron (Fe) and transferrin saturation (TSAT) levels were significantly lower in participants with CACS ≥400 than in those with CACS <400, although the serum ferritin concentration did not differ between the groups. TSAT ≥21% was significantly associated with CACS ≥400 (odds ratio 0.46, p<0.05). TSAT ≥17%, Fe ≥63 µg/dL, and ferritin ≥200 ng/mL appear to protect against 5-year all-cause mortality in HD patients, independent of conventional risk factors of all-cause mortality (p < 0.05).ConclusionWe have identified associations between iron, CACS, and mortality in HD patients. Lower TSAT was found to be an independent predictor of CACS ≥400, and iron deficiency (low TSAT, iron, or ferritin) was a significant predictor of 5-year all-cause mortality in HD patients.     

Comprehensive genetic screening for vascular Ehlers–Danlos syndrome through an amplification-based next-generation sequencing system

Vascular Ehlers–Danlos syndrome (vEDS) is a hereditary connective tissue disorder (HCTD) characterized by arterial dissection/aneurysm/rupture, sigmoid colon rupture, or uterine rupture. Diagnosis is confirmed by detecting heterozygous variants in COL3A1. This is the largest Asian case series and the first to apply an amplification-based next-generation sequencing through custom panels of causative genes for HCTDs, including a specific method of evaluating copy number variations. Among 429 patients with suspected HCTDs analyzed, 101 were suspected to have vEDS, and 33 of them (32.4%) were found to have COL3A1 variants. Two patients with a clinical diagnosis of Loeys–Dietz syndrome and/or familial thoracic aortic aneurysm and dissection were also found to have COL3A1 variants. Twenty cases (57.1%) had missense variants leading to glycine (Gly) substitutions in the triple helical domain, one (2.9%) had a missense variant leading to non-Gly substitution in this domain, eight (22.9%) had splice site alterations, three (8.6%) had nonsense variants, two (5.7%) had in-frame deletions, and one (2.9%) had a multi-exon deletion, including two deceased patients analyzed with formalin-fixed and paraffin-embedded samples. This is a clinically useful system to detect a wide spectrum of variants from various types of samples.     

Pharmacological effects of concomitant administration of β-adrenoceptor blocker and agonist in normal subjects: characterization by heart rate response to exercise

The effects of a combination regimen of metoprolol and ₁-adrenoceptor agonist denopamine on resting and exercise heart rate have been studied in 10 normal volunteers. Maximal ramp upright bicycle exercise was performed three times at 1-week intervals. Two hours before each exercise test, 5 mg metoprolol plus 20 mg denopamine, 5 mg metoprolol plus a denopamine placebo, or two placebos were orally administered in a double-blind fashion.During exercise after placebo administration, heart rate increased in parallel with the exercise intensity. Compared to the placebo values, resting heart rate was significantly decreased by an average of 10 beats · min^–1 by 5 mg metoprolol, whereas it was not altered by the combination regimen. During exercise, however, both the combination regimen and metoprolol alone showed a significant negative chronotropic effect, decreasing peak exercise heart rate by an average of 14 and 21 beats · min^–1, respectively. Peak oxygen uptake was also significantly decreased by both regimens.We conclude that concomitant administration of 5 mg metoprolol and 20 mg denopamine exerts an effective -adrenoceptor blocking action during exercise but a minimal effect at rest in normal subjects. The combination regimen appears to have a favourable pharmacological profile for -adrenoceptor blocker therapy in patients with chronic heart failure.