Reduction of whole PTH/intact PTH ratio as a predictor of bone metabolism in cinacalcet treatment of hemodialysis patients with secondary hyperparathyroidism.

UNLABELLED: In cinacalcet treatment of hemodialysis (HD) patients with secondary hyperparathyroidism (SHPT), not only intact parathyroid hormone (I-PTH), whole PTH (W-PTH), and bone markers, but also W-PTH/I-PTH ratio as proportion of active PTH(1-84) molecules were decreased. Changes in W-PTH/I-PTH ratio significantly correlated and predicted changes in bone marker. INTRODUCTION: Cinacalcet partly suppresses the secretion of PTH by enhancing PTH(1-84) degradation into N-truncated fragments. The objectives of this study is to investigate the significance of the N-truncated PTH/PTH(1-84) ratio for the prediction of the effect of cinacalcet in HD patients. METHODS: Serum parameters were measured during 12 weeks of oral cinacalcet administration at 25 mg daily in 39 HD patients with SHPT. RESULTS: Serum Ca, Pi, W-PTH, I-PTH, and W-PTH/I-PTH ratio all decreased significantly in a time-dependent manner during cinacalcet administration. Serum tartrate-resistant acid phosphatase (TRAP) 5b reflected these changes more precisely than serum N-telopeptide of type-I collagen. At 1 week, changes in I-PTH and W-PTH correlated significantly with those in serum Pi, but not Ca. Changes in serum Pi (but not Ca) and serum W-PTH also correlated significantly with changes in serum TRAP5b at both 4 and 12 weeks, while changes in serum I-PTH correlated significantly with those in serum TRAP5b only at 12 weeks. Changes in the serum W-PTH/I-PTH ratio correlated significantly with those in serum TRAP5b at both 4 and 12 weeks, and changes in serum W-PTH/I-PTH ratio at 4 weeks showed a tendency for a correlation with changes in serum TRAP5b at 12 weeks. HD patients with a reduced W-PTH/I-PTH ratio after 4 weeks had a significantly greater reduction of TRAP5b over 12 weeks. CONCLUSION: W-PTH and the W-PTH/I-PTH ratio allow estimation of the potency of cinacalcet in enhancement of PTH degradation, and thus no less reliable markers than I-PTH for reflecting cinacalcet-induced bone resorption.

Role of cystine transport in intracellular glutathione level and cisplatin resistance in human ovarian cancer cell lines.

Transport system x(c)(-) is a member of plasma membrane heterodimeric amino-acid transporters and consists of two protein components, xCT and 4F2hc. This system mediates cystine entry coupled with the exodus of intracellular glutamate and regulates the intracellular glutathione (GSH) levels in most mammalian cultured cells. We studied the activity of system x(c)(-) and GSH content in human ovarian cancer cell line (A2780) and its cisplatin (CDDP)-resistant variant (A2780DDP). The rate of cystine uptake was approximately 4.5-fold higher in A2780DDP cells than in A2780 cells and the cystine uptake in A2780DDP cells was mediated by system x(c)(-). Intracellular GSH content was much higher in A2780DDP cells but it fell drastically in the presence of excess glutamate, which inhibited the cystine uptake competitively. xCT and 4F2hc mRNAs were definitely expressed in A2780DDP cells, but far less in A2780 cells. Expression of system x(c)(-) activity by transfection with cDNAs for xCT and 4F2hc made A2780 cells more resistant to CDDP. Similar results on the cystine uptake were obtained in human colonic cancer cell lines. These findings suggest that the system x(c)(-) plays an important role in maintaining the higher levels of GSH and consequently in CDDP resistance in cancer cell lines.

Transient mitral regurgitation following vagal stimulation-induced ventricular arrhythmia in rabbits: a color Doppler echocardiographic study.

BACKGROUND AND AIM OF THE STUDY: Cervical vagal stimulation in rabbits frequently causes systolic murmur with bigeminy due to premature ventricular contractions. The bigeminy disappears in a few minutes, but the systolic murmur persists for a few days. Peculiar lesions of the mitral valves, mitral annulus and papillary muscles, and an increase in left atrial weight, frequently develop in a week. In this study, color Doppler echocardiography was used to examine whether the systolic murmur was due to mitral regurgitation. METHODS: Echocardiographic monitoring was carried out in anesthetized rabbits restrained in the supine position. RESULTS: Doppler echocardiography and phonocardiography showed systolic murmur at 6 h, three days, and at one, two, three and four weeks after vagal stimulation. At 6 h after stimulation, phonocardiography showed systolic click and late systolic murmur; Doppler echocardiography showed marked mitral regurgitation. The systolic murmur and mitral regurgitation were attenuated and the papillary muscle was swollen three days after vagal stimulation. Following stimulation, mitral regurgitation disappeared within one week, and papillary muscle swelling improved after three weeks. CONCLUSION: Doppler echocardiography confirmed that systolic murmur caused by vagal stimulation in rabbits was due to mitral regurgitation.

Molecular cloning and expression of human xCT, the light chain of amino acid transport system xc-.

Transport of system xc- is an exchange agency with high specificity for anionic form of cystine and glutamate. The protein mediating this transport is a disulfide-linked heterodimer of a light chain named xCT and a heavy chain previously known as 4F2hc. We have isolated two cDNAs encoding xCT from the human cDNA library. One clone coded for a protein of 501 amino acids with 12 putative transmembrane domains. When functionally expressed in Xenopus oocytes together with the human 4F2hc, human xCT induced the transport activity whose characteristics are similar to those of system xc-. Another clone seemed to contain a partial human xCT and a long 3' untranslated region. The human xCT gene was localized at chromosome 4q28-31. Analysis of the 5'-flanking region of the human xCT gene revealed several sites for potentially binding of transcriptional factors, including NF-E2 and AP-1. Transport of cystine via system xc- has been known as a regulatory factor for the intracellular glutathione level, and its transport activity is induced in response to the oxygen tension in culture. Northern blot analysis demonstrated that the expression of both xCT and 4F2hc was significantly enhanced by oxygen. The results suggest that oxygen regulates the activity of system xc- by modulating the expression of both xCT and 4F2hc mRNAs.

Antimutagenicity of sweetpotato (Ipomoea batatas) roots.

Antimutagenicity of the water extracts prepared from the storage roots of four varieties of sweetpotato with different flesh colors was investigated using Salmonella typhimurium TA 98. The extract from the whole roots of the purple-colored Ayamurasaki variety effectively decreased the reverse mutation induced not only by Trp-P-1, Trp-P-2, IQ, B[a]P, and 4-NQO but also by dimethyl sulfoxide extracts of grilled beef. Comparison of the inhibitory activity of the extracts from the normal Ayamurasaki and its anthocyanin-deficient mutant one suggested that the anthocyanin pigment in the flesh decreases the mutagenic activity of the mutagens as heterocyclic amines. Two anthocyanin pigments purified from purple-colored sweet-potato, 3-(6,6'-caffeylferulylsophoroside)-5-glucoside of cyanidin (YGM-3) and peonidin (YGM-6) effectively inhibited the reverse mutation induced by heterocyclic amines, Trp-P-1, Trp-P-2, and IQ in the presence of rat liver microsomal activation systems.

Serum levels of 1,25-dihydroxyvitamin D, 24,25-dihydroxyvitamin D, and 25-hydroxyvitamin D in nondialyzed patients with chronic renal failure.

BACKGROUND: In patients with chronic renal failure (CRF), abnormalities in vitamin D metabolism are known to be present, and several factors could contribute to the abnormalities. METHODS: We measured serum levels of three vitamin D metabolites, 1,25(OH)2D, 24, 25(OH)2D and 25(OH)D, and analyzed factors affecting their levels in 76 nondialyzed patients with CRF (serum creatinine> 1.6 and < 9.0 mg/dl), 37 of whom had diabetes mellitus (DM-CRF) and 39 of whom were nondiabetic (nonDM-CRF). RESULTS: Serum levels of 1,25(OH)2D were positively correlated with estimated creatinine clearance (CCr; r = 0.429; P < 0.0001), and levels of 24,25(OH)2D were weakly correlated with CCr (r = 0.252, P < 0.05); no correlation was noted for 25(OH)D. Serum levels of all three vitamin D metabolites were significantly and positively correlated with serum albumin. Although there were no significant differences in age, sex, estimated CCr, calcium and phosphate between DM-CRF and nonDM-CRF, all three vitamin D metabolites were significantly lower in DM-CRF than in nonDM-CRF. To analyze factors influencing vitamin D metabolite levels, we performed multiple regression analyses. Serum 25(OH)D levels were significantly and independently associated with serum albumin, presence of DM and serum phosphate (R2 = 0.599; P < 0.0001). 24,25(OH)2D levels were significantly and strongly associated with 25(OH)D (beta = 0.772; R2 = 0.446; P < 0.0001). Serum 1,25(OH)2D levels were significantly associated only with estimated CCr (R2 = 0. 409; P < 0.0001). CONCLUSIONS: These results suggest that hypoalbuminemia and the presence of DM independently affect serum 25(OH)D levels, probably via diabetic nephropathy and poor nutritional status associated with diabetes, and that 25(OH)D is actively catalyzed to 24,25(OH)2D in CRF, probably largely via extrarenal 24-hydroxylase. Serum levels of 1,25(OH)2D were significantly affected by the degree of renal failure. Thus, this study indicates that patients with CRF, particularly those with DM, should receive supplements containing the active form of vitamin D prior to dialysis.

Molecular cloning of a novel protein containing the association domain of calmodulin-dependent protein kinase II.

The cDNA of a novel protein, which contains the association domain of alpha isoform of calmodulin-dependent protein kinase II (CaM-kinase II alpha), was cloned from rat skeletal muscle. This protein, called alpha KAP, consisted of 200 amino acid residues with a molecular weight of 22,583. alpha KAP has a highly hydrophobic amino-terminal stretch of 25 amino acids which is absent from CaM-kinase II alpha, suggesting that this protein is either a secretory protein or an integral membrane protein. Northern blot analysis with a probe specific for alpha KAP detected three distinct mRNA species of 4.0, 2.4, and 1.5 kb in rat skeletal muscle. The 4.0- and 2.4-kb RNAs were also detected in heart, and at much lower levels in lung, kidney, and testis. Western blot analysis, using antibody raised against a synthetic peptide corresponding to the carboxyl-terminal 15 amino acids, revealed a single band corresponding in mobility to a molecular weight of 21,000 in crude extracts of both rat skeletal muscle and bacteria transformed with the cDNA, suggesting that no significant post-translational modification, such as excision of the amino-terminal hydrophobic segment, occurred. This, together with the fact that alpha KAP was recovered in the high-speed pellet in skeletal muscle, indicated that this protein may be an integral membrane protein.

Evidence for the existence of Ca2+/calmodulin-dependent protein kinase IV kinase isoforms in rat brain.

Calmodulin-dependent protein kinase IV (CaM-kinase IV), which plays crucial roles in the functioning of Ca2+ in the central nervous system and immune system, is markedly activated upon phosphorylation by the action of CaM-kinase IV kinase. Northern and Western blot analyses of CaM-kinase IV kinase showed relatively weak reactions in the rat cerebellum, where the activity of CaM-kinase IV kinase has been demonstrated to exist, indicating that CaM-kinase IV kinase isoforms distinct from the enzyme cloned from the cerebral cortex may exist in the cerebellum. When the crude extracts of rat cerebral cortex, brain stem, and cerebellum were immunotitrated with antibody against the cloned enzyme, only approximately 46, 56, and 25% of the enzyme activity of the respective extracts were immunoprecipitated. Thus, at least two distinct isoforms of CaM-kinase IV kinase appear to exist in the brain.

cDNA cloning and expression of human calmodulin-dependent protein kinase IV.

Calmodulin-dependent protein kinase IV (CaM-kinase IV) is a Ca(2+)-responsive multifunctional protein kinase which occurs abundantly in the brain and thymus. A human cDNA clone encoding CaM-kinase IV was isolated from a Jurkat cell cDNA library and its nucleotide sequence was determined. The cDNA sequence encoded a protein consisting of 473 amino acids with a molecular weight of 51,925. The nucleotide sequence for the coding region and the deduced amino acid sequence showed 81 and 80% identities with those of the rat enzyme, respectively. Western blot analysis, using a polyclonal antibody raised against the recombinant human CaM-kinase IV, which was expressed in Escherichia coli, revealed two bands corresponding in mobility to molecular weights of 60,000 and 61,000, respectively, in a Jurkat cell extract. The antibody also cross-reacted with both isoforms of CaM-kinase IV from rat cerebellum, the apparent molecular weights being 62,000 and 64,000, respectively.

Requirement of brain extract for the activity of brain calmodulin-dependent protein kinase IV expressed in Escherichia coli.

Calmodulin-dependent protein kinase IV (CaM-kinase IV) is a Ca(2+)-responsive multifunctional protein kinase which occurs abundantly in the brain. When cDNA for rat brain CaM-kinase IV was expressed in Escherichia coli, the enzyme was produced in a good yield, but it did not show significant activity. The inactive recombinant CaM-kinase IV was phosphorylated and became highly active on incubation with a rat brain extract in the presence of both Ca2+/calmodulin and ATP/Mg2+. The recombinant CaM-kinase IV-activating activity in brain was one to two orders of magnitude higher than that in the other tissues examined. These observations suggest that CaM-kinase IV may undergo a posttranslational modification, probably Ca2+/calmodulin-dependent phosphorylation by CaM-kinase IV kinase, before exhibiting activity in the central nervous system.

Partial protection of 1 alpha-hydroxyvitamin D3 against the development of diabetes induced by multiple low-dose streptozotocin injection in CD-1 mice.

1 alpha-Hydroxyvitamin D3 (1 alpha-OH-D3), a precursor of active vitamin D3, 1 alpha,25-dihydroxyvitamin D3, was tested in CD-1 mice for its in vivo effect against the development of diabetes induced by administering multiple low doses of streptozotocin (STZ). Daily intraperitoneal (IP) injections of 35 mg/kg body weight of STZ administered for 5 consecutive days to mice from 7 weeks of age induced a delayed-onset hyperglycemia, insulitis, and beta-cell degranulation in 26 of 28 mice. Only 12 of 29 mice developed diabetes when treated with simultaneous daily IP injections of 1 alpha-OH-D3 for 14 consecutive days, with diabetes defined as a plasma glucose level greater than 200 mg/dL. A daily dose of 0.3 micrograms/kg 1 alpha-OH-D3 also protected against the development of hyperglycemia in five of 13 mice, whereas 0.2 micrograms/kg 1 alpha-OH-D3 was ineffective, indicating a dose-related effect. Histological study showed that, among the 1 alpha-OH-D3-treated mice, the pancreatic islets of euglycemic mice showed neither massive islet infiltration nor beta-cell degranulation, whereas those of the hyperglycemic mice showed insulitis. However, when diabetes was chemically induced with a single high dose of STZ, the simultaneous administration of 1 alpha-OH-D3 to mice failed to protect against the development of hyperglycemia; all five mice so treated developed hyperglycemia. Their pancreatic islets did not show insulitis. Therefore, it is suggested that 1 alpha-OH-D3 may protect against the development of diabetes following administration of multiple low doses of STZ, probably via an immune mechanism.

Effects of uremic serum on 1,25-dihydroxyvitamin D3-induced differentiation of a human promyelocytic leukemia cell line, HL-60.

The mechanism by which resistance to 1,25 dihydroxyvitamin D3 (1,25-(OH)2D3) occurs in patients with chronic renal failure was studied. This agent induces differentiation and 1,25-(OH)2D3-24-hydroxylase activity in the mitochondria of the human promyelocytic leukemia cell line, HL-60, via a steroid-hormone receptor mechanism. HL-60 cells were cultured in RPMI 1640 medium supplemented with 10% normal or uremic serum. Treatment of these cells with 10(-8)M 1,25-(OH)2D3 for 5 days in a medium containing 10% uremic serum from 4 patients with chronic renal failure resulted in a maturation of the cells amounting to 30.3 +/- 18.7% (mean +/- SD) and 32.5 +/- 11.2%, as obtained by NBT reduction assay and NSE assay, respectively. These values were significantly lower than those obtained with 10% serum from 3 normal controls (66.6 +/- 12.8%, 58.3 +/- 10.9%, p less than 0.02). The treatment of HL-60 cells with 1,25-(OH)2D3 in a mixture of 5% normal plus 5% uremic serum caused cell differentiation to an extent similar to that in 10% uremic serum, which suggests the presence of a substance(s) having 1,25-(OH)2D3-inhibitory activity in the uremic serum. Exposure of HL-60 cells to uremic serum significantly impaired their responsiveness to 1,25-(OH)2D3 as assessed by the induction of the cell's ability to hydroxylate the C-24 position of 1,25-(OH)2[3H]D3. The mechanism by which uremic serum confers an impaired cellular response to 1,25-(OH)2D3 seemed to be due, in part, to a decrease in 1,25-(OH)2D3 receptor levels. A significant positive correlation was observed between intracellular cAMP levels and 1,25-(OH)2D3-induced HL-60 cell maturation. In summary, the mechanism by which uremic serum confers 1,25-(OH)2D3 resistance upon HL-60 cells seemed to be due to the presence of 1,25-(OH)2D3-inhibitory activity in uremic serum, which may modulate cellular responsiveness to 1,25-(OH)2D3 by such mechanisms as reducing 1,25-(OH)2D3 receptor levels in the cells, in part through alteration in cAMP metabolism.

An experimental meningitis with Streptococcus pneumoniae in rats: therapeutic effect of aspoxicillin and its penetration into CSF.

An experimental meningitis model was produced in rats with Streptococcus pneumoniae Type III and used to evaluate the therapeutic effect of aspoxicillin in comparison with piperacillin, mezlocillin and ampicillin. At the same time, their bactericidal activities and pharmacokinetics in cerebrospinal fluid (CSF) were determined. The experimental meningitis model was prepared by intracisternal inoculation of the organism. The rats in this model began to die 2 days after the infection and all died within 5 days. Histological examination also revealed that this model was a fatal pneumococcal meningitis model in rats. The concentrations of these penicillins in CSF of the infected rats determined by the high performance liquid chromatography method were significantly increased by bacterial infection. Of these drugs, ASPC gave the highest penetration into the infected CSF and the longest persistency in CSF. In a comparison of the bactericidal activity of these penicillins in this model, aspoxicillin at a dose of 20 mg/kg inhibited the regrowth of bacteria in CSF 24 h after administration, but the other three penicillins did not. To conclude, in this new experimental meningitis model in rats, aspoxicillin showed an excellent therapeutic effect due to its stronger bactericidal action and favourable pharmacokinetic properties.

Innervation of hypothalamic and limbic areas by the cholinergic, the GABA-ergic and the catecholaminergic nerve fibers; a quantitative analysis.

By measuring activities of enzymes involved in synthesis of acetylcholine, gamma-aminobutyric acid (GABA), dopamine or noradrenaline, extent of innervation of cholinergic, GABA-ergic, dopaminergic and noradrenergic fibers were analysed in discrete brain areas and subareas of the baboon and the rabbit with a special reference to the hypothalamus and the preoptic ares. In the latter area, cholinergic and dopaminergic innervation increases medio-lateraly on the one hand, and noradrenergic innervation reversed on the other hand. GABA-ergic innervation is almost evenly distributed in this structure. By placing electrical lesions in the lateral hypothalamus, an analysis was made of inputs of these innervations.