Diabetic osteopenia, a known chronic complication of diabetes, has been demonstrated with alterations in the calcium-vitamin D endocrine system. In order to investigate the relationship between the decrease of bone density and the altered mineral metabolism in non-insulin-dependent diabetes mellitus (NIDDM), 104 male clinical-proven NIDDM patients were studied. All patients were examined on metacarpal bone mineral density (m-BMD) by means of computed X-ray densitometry (CXD) methods. The values of the Z-score of m-BMD were significantly lower than those of age-matched controls (P<0.01). There was a positive correlation between m-BMD and serum calcium levels (P<0.01), but a negative correlation was conversely observed between m-BMD and serum intact parathyroid hormone (PTH) (P<0.01), indicating that the negative calcium balance under diabetic conditions could cause the decrease of m-BMD in NIDDM. Interestingly, since a significantly positive correlation was found between circulating levels of calcium and parathyroid hormone-related peptide (PTHrP) (P<0.05) but not PTH, it seems likely that PTHrP plays a more compensatory role on the maintenance of calcium homeostasis than PTH under diabetic conditions. Based on these observations, the CXD method would be useful in measuring the mineral density of cortical bone, and would also be beneficial to investigate underlying pathogenetic mechanism of diabetic osteopenia.

Absorptiometry, Photon↗

PubMed2000

Constitutive trichloroethylene degradation led by tac promoter chromosomally integrated upstream of phenol hydroxylase genes of Ralstonia sp. KN1 and its nucleotide sequence analysis.

Ralstonia sp. KN1-10A is a strain capable of degrading trichloroethylene (TCE) constitutively due to the tac promoter (Ptac) integrated upstream of the phenol hydroxylase genes (phy) in its chromosome. The expression of Ptac was analyzed using luxAB of Vibrio harveyi as a reporter. After determining the nucleotide sequence of phyABCDE required for TCE degradation, a luxAB-encoding fragment was integrated downstream of phyE by homologous recombination in strain KN1-10A, obtaining strain KN1-10A-LX. In the same manner, the luxAB-encoding fragment was integrated into the chromosome of the wild-type strain, KN1. The resultant strain KN1-LX was used to analyze the gene expression caused by phenol induction. The expression induced by Ptac was compared to that by phenol induction. Although the level of luxAB expression led by Ptac was almost equal to that induced by phenol, the TCE degradation rate by the Ptac-carrying KN1-10A-LX was markedly slower than that by the phenol-induced KN1-LX. These results suggest that an important gene for TCE degradation was not transcribed by Ptac in KN1-10A-LX. The nucleotide sequence analysis showed the existence of a small gene, phyZ, upstream of phyA, and Ptac was found to be integrated into the middle of phyZ in KN1-10A-LX. The effect of phyZ on TCE degradation was examined by using recombinant strains expressing phyABCDE with or without phyZ in a plasmid. The coexistence of phyZ markedly accelerated TCE degradation. Through an exhaustive expression analysis, it was demonstrated that the chromosomal integration of Ptac was a very attractive method for high and stable production of phenol hydroxylase for TCE degradation.

Journal Article↗

PubMed2000

Trichloroethylene degradation by Ralstonia sp. KN1-10A constitutively expressing phenol hydroxylase: transformation products, NADH limitation, and product toxicity.

Ralstonia sp. KN1-10A, which was constructed by inserting the tac promoter upstream of the phenol hydroxylase (PH) gene in the chromosomal DNA of the wild-type strain, Ralstonia sp. KN1, is a useful recombinant strain for eliminating trichloroethylene (TCE) from contaminated sites because it exhibits constitutive TCE oxidation activity. During TCE degradation by Ralstonia sp. KN1-10A, noxious chlorinated compounds, such as dichloroacetic acid, trichloroacetic acid, 2,2,2-trichloroethanol, and chloral, were not detected, and more than 95% of chlorine in TCE was released as chloride ions. Among the possible TCE transformation products, only carbon monoxide was detected, and its conversion percentage was 7 mol%. The addition of formate, which Ralstonia sp. KN1-10A could use as an exogenous electron donor, did not enhance the TCE degradation performance, suggesting that NADH depletion did not limit the degradation. The phenol degradation activity of Ralstonia sp. KN1-10A that previously degraded TCE was not markedly lower than that of cells not exposed to TCE, suggesting that Ralstonia sp. KN1-10A was not susceptible to product toxicity associated with TCE degradation. Furthermore, to clarify the mechanisms underlying TCE degradation by PH from Ralstonia sp. KN1, this enzyme was compared with another enzyme, a hybrid aromatic ring dioxygenase exhibiting a high TCE degradation activity in Escherichia coli and Pseudomonas sp. The initial TCE degradation rate of Ralstonia sp. KN1 (pKTP100), which produced PH, was 1 50 lower than that of Ralstonia sp. KN1 (pKTF200), which produced the hybrid aromatic ring dioxygenase. However, because of its lower product toxicity, the strain producing PH could degrade 2.3 times more TCE than that generated by the strain producing the hybrid aromatic ring dioxygenase.

Journal Article↗

PubMed2000

MET-88, a gamma-butyrobetaine hydroxylase inhibitor, improves cardiac SR Ca2+ uptake activity in rats with congestive heart failure following myocardial infarction.

We previously reported that MET-88, 3-(2,2,2-trimethylhydrazinium) propionate, improved left ventricular diastolic dysfunction induced by congestive heart failure (CHF) in rats. The present study was designed to investigate the mechanism by which MET-88 improved the cardiac relaxation impaired in CHF rats. The left coronary artery of the animals was ligated, and the rats were then orally administered vehicle (control), MET-88 at 50 or 100 mg/kg or captopril at 20 mg/kg for 20 days. Myocytes were isolated from the non-infarcted region in the left ventricle, and cell shortening and [Ca2+]i transients were measured with a video-edge detector and by fluorescence analysis, respectively. In CHF control rats, the diastolic phase of cell shortening was prolonged compared with that of the sham-operated (sham) rats. This prolongation was prevented by treatment with MET-88 at 100 mg/kg or captopril at 20 mg/kg. CHF control rats also showed an increase in the decay time of [Ca2+]i transients compared with sham rats. MET-88 at 100 mg/kg and captopril at 20 mg/kg attenuated the increase in decay time of [Ca2+]i transients. Ca2+ uptake activity of the sarcoplasmic reticulum (SR) isolated from the non-infarcted region in the left ventricle was measured, and Lineweaver-Burk plot analysis of the activity was performed. CHF control rats revealed a decrease in the Vmax for SR Ca2+ uptake activity without alteration in Kd. MET-88 at 100 mg/kg significantly prevented the decrease in Vmax, but had no effect on Kd. Also, treatment with MET-88 at 100 mg/kg improved myocardial high-energy phosphate levels impaired in CHF rats. These results suggest that one of the mechanisms by which MET-88 improved cardiac relaxation in CHF rats is based on the amelioration of [Ca2+]i transients through increase of SR Ca2+ uptake activity.

Adenine Nucleotides↗

PubMed2000

The insulinotropic mechanism of the novel hypoglycaemic agent JTT-608: direct enhancement of Ca(2+) efficacy and increase of Ca(2+) influx by phosphodiesterase inhibition.

We examined the effects of the novel hypoglycaemic agent JTT-608 [trans-4-(4-methylcyclohexyl)-4-oxobutyric acid] on insulin secretion using rat pancreatic islets, and analysed the mechanism of its effect. JTT-608 augmented 8.3 mM glucose-induced insulin secretion dose-dependently, and there was a stimulatory effect of 100 microM JTT-608 at both moderate and high concentrations (8.3, 11. 1 and 16.7 mM) of glucose, but not at low concentrations (3.3 and 5. 5 mM). In perifusion experiments, both phases of insulin release were enhanced, and the effect was eliminated 10 min after withdrawal of the agent. In the presence of 200 microM diazoxide and a depolarizing concentration (30 mM) of K(+), there was an augmentation of insulin secretion by 100 microM JTT-608, not only under high levels of glucose but also under low levels, and the effects were abolished by 10 microM nitrendipine. JTT-608 augmented insulin secretion from electrically permeabilized islets in the presence of stimulatory concentrations (0.3 and 1.0 microM) of Ca(2+), and the intracellular Ca(2+) concentration ([Ca(2+)](i)) response under 16.7 mM glucose, 200 microM diazoxide, and 30 mM K(+) was also increased. The cyclic AMP content in the islets was increased by 100 microM JTT-608, and an additive effect to 1 microM forskolin was observed, but not to 50 microM 3-isobutyl-1-methylxanthine (IBMX). JTT-608 inhibited phosphodiesterase (PDE) activity dose-dependently. We conclude that JTT-608 augments insulin secretion by enhancing Ca(2+) efficacy and by increasing Ca(2+) influx. This appears to be a result of the increased intracellular cyclic AMP concentration due to PDE inhibition.

Animals↗

PubMed2000

Evaluation of iNOS-dependent and independent mechanisms of the microvascular permeability change induced by lipopolysaccharide.

1. Subcutaneous injection of lipopolysaccharide (LPS) increases plasma leakage in mouse skin. Pretreatment with LPS conditions mice tolerant to the LPS-induced plasma leakage. Nitric oxide (NO) has been suggested to be involved in these LPS effects. A specific role of inducible NO synthase (iNOS) was investigated in the LPS-induced plasma leakage using iNOS deficient mice. 2. Plasma leakage in mouse skin was measured by the local accumulation of Pontamine sky blue at the site of subcutaneous injection of LPS (Sal. typhimurium). LPS (100 - 400 microg site(-1)) produced a dose-related increase in dye leakage in both iNOS deficient and wild-type mice with about 40% less dye leakage in iNOS deficient mice. 3. Indomethacin (5 mg kg(-1)), N-[-2-cyclohexyloxy]-4-nitrophenyl methanesulphonamide (NS-398) (1 mg kg(-1)), diphenhydramine (10 mg kg(-1)) and anti-TNF-alpha antibody (dilution 1 : 400, 10 ml kg(-1)) inhibited the LPS-induced dye leakage in both iNOS deficient and wild-type mice, whereas N(G)-nitro-L-arginine methyl ester (L-NAME) (10 mg kg(-1)) or aminoguanidine (10 mg kg(-1)) inhibited that in wild-type but not in iNOS deficient mice. 4. Pretreatment with LPS (0.15 mg kg(-1) i.p.) 4 h before decreased the LPS-induced dye leakage in wild-type but not in iNOS deficient mice. LPS pretreatment increased serum corticosterone levels in both mice, while it increased the serum nitrate/nitrite levels in wild-type but not in iNOS deficient mice. 5. These studies indicate that an increase in vascular permeability induced by LPS is mediated by NO produced by iNOS, eicosanoids, histamine and TNF-alpha. The tolerance against LPS-induced vascular permeability change may be mediated by iNOS induction but not by an increased release of endogenous corticosteroids.

Animals↗

PubMed2000

Role of inflammatory mediators in lipid A analogue (ONO-4007)-induced vascular permeability change in mouse skin.

1. Endotoxin shock is accompanied by an increase in peripheral vascular permeability. It has been postulated that most biological activities of LPS are derived from lipid A moiety. Here we examined the effect of lipid A analogue ONO-4007 in increasing vascular permeability and the possible mediators in mouse skin by a dye leakage method. 2. Subcutaneous injection of ONO-4007 (1 - 2 mg site(-1)) induced a dose-dependent increase in vascular permeability which was evident after 120 min. 3. ONO-4007-induced dye leakage was significantly attenuated by pretreatments with anti-tumour necrosis factor-alpha (TNF-alpha) and anti-interleukin-1alpha (IL-1alpha) antibodies, but not with indomethacin (5 mg kg(-1)) or diphenhydramine (10 mg kg(-1)). ONO-4007-induced dye leakage was significantly inhibited by a pretreatment with N(G)-nitro-L-arginine methyl ester (L-NAME) (10 mg kg(-1)) but not with aminoguanidine (50 mg kg(-1)). In inducible nitric oxide synthase (iNOS)-deficient mice, ONO-4007 significantly increased the dye leakage, while ONO-4007 dilated rat thoracic aortic rings pre-contracted with phenylephrine, and the L-NAME pretreatment inhibited the dilation. 4. Thus, TNF-alpha, IL-1alpha and constitutive NOSs-derived nitric oxide but not prostaglandins or histamine play a role in ONO-4007-induced increase in vascular permeability. Although ONO-4007 mimics LPS in increasing vascular permeability, mechanisms of permeability change elicited by ONO-4007 were not identical to those of LPS.

Animals↗

PubMed2000

Prediction of organ-confined disease by prostate-specific antigen nadir after neoadjuvant therapy.

BACKGROUND: It is not clear whether or not serum prostate-specific antigen (PSA) levels after androgen deprivation prior to radical prostatectomy (neoadjuvant therapy) have any value in the prediction of the final pathologic stage. METHODS: We conducted a study on 49 patients who underwent retropubic radical prostatectomy following neoadjuvant therapy for clinical stage T1c, T2, and T3a prostate cancer. We evaluated progression-free survival based on the PSA failure rate and the predictive value of the PSA nadir after neoadjuvant therapy and other clinical factors to determine the most important predictor of organ confinement. RESULTS: Of the 49 patients, 30 had organ-confined disease. Of 31 patients without adjuvant therapy after surgery, the PSA failure-free rates at 2 years were 81.6 and 34.3% in the subset of organ-confined disease and non-organ-confined disease, respectively (P= 0.0031). Of the 18 patients with adjuvant androgen deprivation therapy after surgery, the PSA failure-free rate at 2 years was 100% and 59.7% in patients with organ-confined disease and non-organ-confined disease, respectively. Baseline PSA (P=0.037), PSA nadir (P<0.0001) and PSA density (P=0.003) significantly correlated with organ confinement. Multivariate logistic regression analysis revealed that the PSA nadir was the only independent predictor of organ confinement (P = 0.044). CONCLUSIONS: There was a trend that the patients with non organ-confined disease had a higher probability of PSA failure than did the patients with organ-confined disease. The PSA nadir after neoadjuvant therapy was the strongest predictor of organ confinement. The predictive value of the serum PSA nadir should be validated in well-designed larger population-based studies.

Aged↗

PubMed2000

Possible role of elevation of glutathione in the acquisition of enhanced proliferation of mouse splenocytes exposed to small-dose gamma-rays.

PURPOSE: To examine the relation between the induction of an increased glutathione level and the elevated proliferative response of mouse splenocytes by a small dose of gamma-rays. MATERIALS AND METHODS: Male ICR strain mice, 7 weeks of age, were divided into irradiated and non-irradiated control groups. Irradiation was done with gamma-rays from a 137Cs source at a dose of 50 cGy (1.11 Gy/min). Glutathione content in the splenocytes was measured using a modified spectrophotometric technique. Concanavalin A (Con A)-induced proliferative response of the splenocytes after whole-body gamma-ray irradiation was estimated from the 3H-thymidine incorporation into the cells. RESULTS: The glutathione level in mouse splenocytes increased 2 h after whole-body y-ray irradiation at 50cGy, peaked at 4h and thereafter decreased almost to the zero-time level by 12-h postirradiation. A significant enhancement of Con A-induced proliferation was observed in the splenocytes obtained from the whole-body-irradiated animals between 2h and 6h post-irradiation. Glutathione exogenously added to splenocytes obtained from normal mice enhanced the Con A-induced proliferation of splenocytes in a dose-dependent manner. This enhancement was completely blocked by buthionine sulfoximine, a specific inhibitor of the de novo pathway of glutathione synthesis. CONCLUSIONS: The induction of endogenous glutathione immediately after low-dose gamma-ray irradiation is at least partially responsible for the enhancement of immune function, and may throw light on the mechanisms of carcinostatic effects induced by low dose ionizing radiation.

Animals↗

PubMed2000

Nitrotyrosine formation and its role in various pathological conditions.

The formation of peroxynitrite and nitrotyrosine was examined in a variety of in vitro and in vivo animal models and its relation to cell or tissue damage was examined. polymorphonuclear leukocyte (PMN)-induced injury to cardiac myocytes endothelial cells, activated PMN produced peroxynitrite. Peroxynitrite appears to be responsible for the injury but it was not a major mediator of endothelial cell injury. In the experiment of ischemia-reperfusion injury of the rat brain nitrotyrosine was formed in the peri-infarct and core-of infarct regions. The degradation curve of nitrotyrosine revealed that its t(1/2) was about 2.2 hours. In the radiation-induced lung injury of rats, nitrotyrosine was also formed but it was not the sole mechanism for the injury. Levels of nitrotyrosine correlated with the severity of myocardial dysfunction in the canine model of cytokine-induced cardiac injury. Inhibition of NO generation abolished the formation of peroxynitrite and nitrotyrosine in all experiments. In conclusion; although nitrotyrosine is formed in a variety of pathological conditions where the generation of NO is increased, its presence does not always correlate with the severity of injury.

Animals↗

PubMed2000

Monitoring sialyl lewis x (CD15s) on peripheral lymphocytes for the diagnosis of acute rejection.

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Acute Disease↗

PubMed2000

Insulin is a dominant suppressor of sterol 12 alpha-hydroxylase P450 (CYP8B) expression in rat liver: possible role of insulin in circadian rhythm of CYP8B.

Sterol 12 alpha-hydroxylase (CYP8B) is a key enzyme for regulating the cholic acid/chenodeoxycholic acid ratio in bile acid biosynthesis. The hepatic CYP8B level was elevated in streptozotocin-induced diabetic rats, and the elevated CYP8B was suppressed by insulin administration [Ishida, H. et al. (1999) J. Biochem. 126, 19-25]. The streptozotocin-induced elevation of hepatic CYP8B mRNA concomitantly responded to the decrement of the serum insulin level. The CYP8B mRNA level in the cultivated rat hepatoma H4TG cells was strongly suppressed by insulin, although it was affected by dibutyryl cAMP or thyroxine to lesser extents. These observations demonstrate that CYP8B expression is dominantly regulated by the direct action of insulin on hepatocytes. A marked circadian rhythm (maximum at 13:00-16:00 and minimum at 1:00) was observed both on the mRNA level and the activity of CYP8B. This rhythm was shifted from that of cholesterol 7 alpha-hydroxylase, a rate-limiting enzyme of bile acid biosynthesis, showing a maximum at 22:00 and a minimum at 10:00, and this shift might oscillate the cholic acid/chenodeoxycholic acid ratio, which is increased in the late afternoon and decreased at midnight. The rhythm of CYP8B was the inverse of the circadian variation of serum insulin level and was similar to the circadian rhythm of glucose 6-phosphatase. These facts and the potent suppressive effect of insulin on CYP8B indicate that the oscillation of the serum insulin may be a factor in producing the circadian rhythm of CYP8B.

Animals↗

PubMed2000

Studies on the specificity and sensitivity of the influenza C virus binding assay for 9-O-acetylated sialic acids and its application to human melanomas.

The sensitivity and specificity of two influenza C virus assays, solid-phase and overlay assays, were investigated using naturally occurring 9-O-acetylated GD(3), rat serum glycoproteins containing 60% of N-acetyl-9-O-acetylneuraminic acid, and synthetically O-acetylated sialylated compounds. The sensitivity of the solid-phase assay was higher for glycoproteins containing N-acetyl-9-O-acetylneuraminic acid than for gangliosides, and also differed for various 9-O-acetylated gangliosides. The overlay assay was less sensitive for all glycoconjugates tested. For virus recognition the presentation of the sialic acid within the molecule and the structure of the sialic acid are essential. Investigation of gangliosides from human melanomas and normal skin with the influenza C virus assay showed an increase of O-acetylation of sialic acids in most tumour samples and the occurrence of several O-acetylated gangliosides.

Acetylation↗

PubMed2000

Insulin is the essential factor maintaining the constitutive expression of hepatic sterol 14-demethylase P450 (CYP51).

The role of serum insulin in regulating the expression level of hepatic sterol 14-demethylase P450 (CYP51) was examined. Administration of streptozotocin, which destroys pancreatic beta-cells, caused reduction of CYP51 mRNA level in rats in parallel with the loss of serum insulin. Streptozotocin treatment also reduced the CYP51 activity. The decreased mRNA level and activity of the streptozotocin-treated rats were restored to the normal level within 24 h by repeated administration of insulin. CYP51 level of normal rats was insensitive to the circadian variation of serum insulin and insulin administration, and no significant difference was observed between the hepatic CYP51 activities of Sprague-Dawley and Wistar lean rats, although the serum insulin concentration of the latter was higher than the former. These facts indicate that the expression of hepatic CYP51 is maintained by serum insulin, and its lowest physiological level is sufficient for supporting the expression of CYP51. The responses of CYP51 expression to streptozotocin and insulin treatments were closely similar to those of the sterol regulatory element binding protein (SREBP)-1c expression [Shimomura et al. (1999) Proc. Nat. Acad. Sci. USA 96, 13656-13661]. Based on this fact, the possible contribution of SREBP-1c to the insulin-dependent expression of hepatic CYP51 gene was also discussed.

Animals↗