OCT Activity Research Articles

Renal failure and vitamin D

Secondary hyperparathyroidism (2HPT), a common disorder in patients with chronic renal failure, develops in response to phosphate retention and low serum 1,25-dihydroxyvitamin D(3) [1,25 (OH) (2)D(3), calcitriol] . Replacement therapy with calcitriol or its precursor 1alpha-hydroxyvitamin D(3) [1alpha (OH) D(3), alfacalcidol] often produces hypercalcemia and hyperphosphatemia in these patients. Several vitamin D analogues have been developed that retain the direct suppressive action of 1,25 (OH) (2)D(3) on the parathyroid glands but have less calcemic activity, therapy offering a safer and more effective means of controlling 2HPT. 1,25-D dihydroxy-19-norvitamin D(2) (19-nor D(2)) and 1alpha-hydroxyvitamin D(2) (1alphaOHD(2)) are available in the United States and 1,25-dihydrox-22-oxavitamin D(3) (22-oxacalcitriol, OCT) and 1,25-dihydroxy-26,26,26,27,27,27-hexafluorovitamin D(3) [1,25 (OH)(2)26,27F(6)D(3), falecalcitriol] have been approved for use in Japan. Animal studies have demonstrated that OCT and 19-nor D(2) have a wider therapeutic window for suppression of parathyroid hormone (PTH) because of their lower calcemic activities of OCT has been attributed to its rapid clearance which prevents sustained effects on intestinal calcium absorption and bone resorption, but still allows a prolonged suppression of PTH gene expression and parathyroid cell growth. The calcemic activity of 19-norD(2) diminishes with the duration of treatment by as yet unknown mechanisms. The lower toxicity of 1alphaOHD(2), compared 1alphaOHD(3). has also been noted with chronic, but not acute administration, perhaps due to differential metabolism. The unique actions of falecalcitriol may also result from altered metabolism. A clear understanding of the molecular basis for the selectivity of vitamin D analogues on parathyroid function may allow the design of even more effective analogues.

CDNA cloning of two isoforms of ornithine carbamoyltransferase from Canavalia lineata leaves and the effect of site-directed mutagenesis of the carbamoyl phosphate binding site.

The immunoscreening method was used to isolate cDNAs of 1323 bp (ClOCT1) and 1433 bp (ClOCT2) encoding two ornithine carbamoyltransferases (OCT, EC 2.1.3.3) from the cDNA expression library of Canavalia lineata leaves constructed in a lambdaZAP Express vector. ClOCT1 and ClOCT2 encode 359 and 369 amino acids, respectively. The N-terminals of deduced amino acid sequences of the two cDNAs showed typical features of the transit peptide of chloroplast targeting proteins. The ornithine-binding domain (FMHCLP) and catalytic domain (HPXQ) of ClOCT1 and ClOCT2 and the carbamoyl phosphate (CP)-binding site of ClOCT1 (SMRTR) are identical to OCTs of other plant species, pea and Arabidopsis thaliana. However, the CP-binding site sequence of ClOCT2, SLRTH, has not yet been reported. Both ClOCT1 and ClOCT2 cDNAs were expressed in Escherichia coli BL21 (DE3) by using expression vector pET30a. Recombinant ClOCT1 protein showed 14 times higher ornithine-dependent OCT activity than canaline-dependent OCT activity. In contrast, recombinant ClOCT2 protein showed 13 times higher canaline-dependent OCT activity than ornithine-dependent OCT activity. The two amino acids of the CP-binding site of ClOCT2 (SLRTH) were combinatorially changed to those of the CP-binding site of ClOCT1 (SMRTR) by site-directed mutagenesis. When Leu-118 of ClOCT2 was changed to Met, ornithine-dependent activity was increased significantly. It is assumed that the substrate specificity of ClOCT1 or ClOCT2 proteins partially depends on the amino acid sequence of the CP-binding site.

Suppression of IL-2 and IL-4 gene expression by nodularin through the reduced NF-AT binding activity

Nodularin is a cyclic peptide produced by cyanobacteria. In the present study, the inhibitory effect of nodularin on T lymphocyte functions was demonstrated. Direct addition of nodularin to B6C3F1 mouse splenocyte cultures produced a concentration-dependent inhibition of the lymphoproliferative response to concanavalin A stimulation. Nodularin inhibited PMA plus ionomycin (Io)-induced IL-2 mRNA expression in murine splenocytes and thymocytes as determined by quantitative/competitive RT-PCR. To further characterize the mechanism for the transcriptional regulation of IL-2, the binding activity of transcription factors, NF-AT, AP-1, NF-κB, and Oct, was evaluated by electrophoretic mobility shift assays in mouse splenocytes. Nodularin reduced the NF-AT binding activity in PMA/Io-induced splenocytes, but no significant effect was observed on AP-1, NF-κB, or Oct binding activity. Nodularin also inhibited IL-4 mRNA expression in PMA/Io-stimulated murine splenocytes. These results suggest that T lymphocyte is a possible cellular target of nodularin, and the inhibitory effect of nodularin on T-cell specific transcription factor NF-AT induces T-cell dysfunction, which leads to a diminution in IL-2 and IL-4 gene transcription.

The action of a novel vitamin D3 analogue, OCT, on immunomodulatory function of keratinocytes and lymphocytes.

Topical vitamin D3 has relatively recently been introduced for the treatment of psoriasis. Synthetic vitamin D3 analogues with a high potential for inducing differentiation of cells, but with a low hypercalcemic effect have recently been developed. One such synthetic analogue of 1,25-dihydroxyvitamin D3 (calcitriol), 22-oxacalcitriol (OCT), is a novel agent for the topical treatment of psoriasis. The activity of OCT in vitro was investigated and compared with that of a series of vitamin D3 analogues as to their ability to inhibit murine T lymphocyte proliferation stimulated by con-A, to suppress IL-6 and IL-8 production by keratinocytes stimulated with IL-1alpha and TNFalpha, and to inhibit AP-1- and NFkappaB-dependent reporter gene expression. OCT inhibited the proliferation of lymphocytes and suppressed IL-8 and IL-6 production by keratinocytes to the same extent as the other vitamin D3 analogues. It also inhibited AP-1- and NFkappaB-controlled luciferase activity to the same extent as the other vitamin D3 analogues, which demonstrates its mechanism of action in the suppression of inflammatory processes.

Electrophysiological responses of antennal receptor neurons in female Australian sheep blowflies, Lucilia cuprina, to host odours

Studies were conducted to investigate the electrophysiological responses of receptor neurons on the antennae of female Lucilia cuprina to putative stimulants. Three distinct types of olfactory neurons were identified. Tests with 25 compounds showed 27 out of the 75 neurons examined responded best to racemic 1-octen-3-ol (Oct-best neuron), nine to dimethyldisulphide (DMDS-best neuron) and seven to 2-phenylethanol (PE-best neuron). The remaining cells were not stimulated by any chemical stimulus presented in this study. The olfactory neurons showed low spontaneous activities, 2.2 ~ 2.7 impulses/s, which increased significantly on stimulation with test compounds. Dose-dependent responses were observed with Oct-best neurons. Another type of neuron that had a much higher spontaneous activity (26 impulses/s) was observed. This neuron did not respond to any of the candidate compounds tested but showed a decrease in activity with increased airflow over the antennae and was termed the Airflow-sensitive neuron. The spontaneous activities of Oct-, DMDS-, PE-best and Airflow-sensitive neurons were inhibited by butanoic, pentanoic, hexanoic acids, and 3-methylindole.

Expression of ornithine carbamoyltransferase gene in rat hepatoma-derived cell lines, H4-II-E and R-Y121B

The rat R-Y121B cell line is a unique cell line which has ornithine carbamoyltransferase (OCT, EC 2.1.3.3) and can be continuously cultured in a serum-free medium which lacks arginine but is supplemented with ornithine. The OCT gene expression was examined in R-Y121B cells and their parental H4-II-E cells. OCT activity in R-Y121B cells was about one-tenth of that of the adult rat liver while it was not detected at all in H4-II-E cells. Southern hybridization using an OCT cDNA probe containing the entire coding region showed that the OCT gene structure was apparently not different in R-Y121B cells, H4-II-E cells, or rat liver cells. Northern hybridization using the OCT cDNA probe detected a hybridizing signal in R-Y121B cells but not in H4-II-E cells. Reverse transcription-polymerase chain reaction (RT-PCR) showed that an expected size of the OCT cDNA fragment was amplified in R-Y121B cells but not in H4-II-E cells. The amplified fragment was confirmed to be a real rat OCT cDNA by the digestion of this fragment with two restriction enzymes and by the nucleotide sequencing of the fragment. These results indicated that OCT mRNA was present in R-Y121B cells but not in H4-II-E cells. Amino acid analysis showed that arginine was not present in the culture medium but was present in the hydrolysate of R-Y121B cells. The present experiments indicate that transcription, translation, and processing of OCT proceeds normally and the resultant OCT functions in R-Y121B cells, whereas the transcription does not occur in parental H4-II-E cells even though these cells have the normal gene.

Effects of 22-oxacalcitriol on bone metabolism in vitro: comparison with calcitriol—Effects of 22-oxacalcitriol on osteoclast-like cell formation and bone-resorbing activity

22-Oxacalcitriol (OCT), a synthetic vitamin D3 analog, can mimic the ability of calcitriol to differentiate leukemia and skin cells, to enhance the immune response and to suppress parathyroid hormone secretion, but has much less calcemic activity than that of calcitriol. The mechanism of this selective action remains not fully understood, and the actions of OCT on bone metabolism are little known. The present study was, therefore, designed to investigate the effects of OCT and calcitriol on: the proliferation and functions of osteoblastic MC3T3-E1 cells; osteoclast-like cell formation from hemopoietic blast cells in the absence of stromal cells as well as from unfractionated bone cells in the presence of stromal cells; bone resorption; and the proliferation of MC3T3-E1 cells via monocytes. 22-Oxacalcitriol and calcitriol inhibited [3H]thymidine (TdR) incorporation, alkaline phosphatase activity and collagen synthesis of MC3T3-E1 cells to a similar degree. Both OCT (10(-10)-10(-8) mol/l) and calcitriol significantly and similarly stimulated osteoclast-like cell formation from both hemopoietic blast cells and unfractionated bone cells. 22-Oxacalcitriol (10(-10) and 10(-8) mol/l) significantly stimulated bone resorption, although to a slightly lesser degree than did calcitriol. Human monocyte-conditioned medium (CM) significantly stimulated TdR incorporation into MC3T3-E1 cells. On the other hand, CM obtained from monocytes treated with calcitriol (10(-10)-10(-8) mol/l) significantly inhibited TdR incorporation in a dose-related fashion, whereas CM obtained from monocytes treated with OCT (10(-10)-10(-8) mol/l) significantly stimulated TdR incorporation in a dose-related fashion.(ABSTRACT TRUNCATED AT 250 WORDS)

Intracellular multiplication of Legionella pneumophila in HL-60 cells functionally differentiated in response to 22-oxacalcitriol.

The agent 1,25-dihydroxyvitamin D3 (D3) induces the differentiation of HL-60 human leukemia cells into functional monocyte-like cells that can support the intracellular multiplication of Legionella pneumophila. 22-Oxacalcitriol (OCT), a synthetic analogue of D3, exhibits greater differentiation-inducing activity than D3 in WEHI-3 mouse leukemia cells and has been suggested to be clinically more useful because of its lower hypercalcemic activity. The abilities of OCT and D3 to induce the functional differentiation of human leukemia HL-60 cells have now been investigated. OCT induced the differentiation of HL-60 cells into monocyte-like cells to a similar extent as D3. Thus, both OCT and D3 increased (1) the surface expression of CD11b, CD11c, CD14, and CD35; (2) nonspecific esterase staining; and (3) phagocytic activity toward fluorescent beads. HL-60 cells differentiated in response to OCT also supported the intracellular multiplication of L. pneumophila. Activation of both OCT- and D3-treated HL-60 cells with human recombinant interferon-gamma (IFN-gamma) for 24 h before infection markedly inhibited L. pneumophila multiplication. IFN-gamma activation enhanced superoxide anion generation by D3-treated HL-60 cells but not by OCT-treated HL-60 cells, suggesting that the inhibition of L. pneumophila multiplication in IFN-gamma-activated cells is independent of superoxide generation. Finally, D3, but not OCT, markedly stimulated the formation of osteoclast-like multinucleated cells from mouse bone marrow cells, consistent with the lower hypercalcemic activity of OCT.

Functional interaction between different isoforms of the Oct-2 transcription factor expressed in neuronal cells.

The predominant neuronal isoforms of the Oct-2 transcription factor, Oct 2.4 and Oct 2.5, repress the herpes simplex virus immediate-early promoter both in neuronal cells and in fibroblasts normally lacking Oct-2. In contrast, the predominant B lymphocyte form Oct 2.1, which is present at a lower level in neuronal cells, activates the immediate-early promoter in fibroblasts but represses it in neuronal cells. We show here that both Oct 2.4 and Oct 2.5 can functionally interact with Oct 2.1 and convert it from an activator into a repressor. Hence, the cell type-specific activity of Oct 2.1 results from the presence of Oct 2.4 and 2.5 in neuronal cells and their absence in other cell types. The significance of this effect is discussed in terms of the role of Oct-2 in the regulation of viral and cellular gene expression in neuronal cells.

A New Enzymatic Determination of Ornithine Carbamoyltransferase Activity

Abstract A new enzymatic assay method for ornithine carbamoyltransferase, OCT, activity was developed using ornithine:2-oxo-acid aminotransferase, OKAT, and 1-pyrroline-5-carboxylate dehydrogenase, P5CDH. The method is based on the reaction of L-ornithine formation from L-citrulline by OCT. The OCT activity was measured spectrophotometrically at 340 nm and the minimum detectable activity was about 4 units/L of serum. Results obtained by this method and by the chemical method correlated well. This method is superior to the chemical method, as it does not require boiling of the reaction mixture nor removal of interfering substances in the serum.

α-Tocopherol deficiency fails to aggravate toxic liver injury but liver injury causes α-tocopherol retention

The possible aggravation of liver injury by impaired cellular antioxidant function was investigated. A vitamin E-deficient diet (0.5 mg/kg α-tocopherol; control 100 mg/kg) significantly reduced rat liver α-tocopherol concentrations after 4 weeks (1.8 ± 1.7 μg/g; control 34.4 ± 2.4 μg/g, p < 0.001). The effects of copper loading (Cu, 3 g/kg diet); galactosamine (GalN, 0.85 g/kg i.p.); or carbon tetrachloride (CCl4, 10 mmol/kg i.p.) were examined. Serum aspartate transaminase activity was elevated slightly by vitamin E deficiency but not by hepatic copper accumulation. In vitamin E-replete (E+) and vitamin E-deficient (E−) rats, GalN or CCl4 caused a large and comparable elevation in serum AST and OCT activity. This effect on AST was markedly reduced by copper loading in vitamin E replete (E+) rats, but in E(−) rats copper had significantly less protective effect. Copper also diminished the OCT response to GalN in E+, though not E−, rats. A significant rise in total hepatic α-tocopherol content followed administration of GalN or CCl4 in both normocupric and copper-laden E(−) rats. Thus α-tocopherol deficiency (a) was not hepatotoxic per se; (b) failed to potentiate the toxicity of copper, GalN or CCl4; but (c) partially abolished the protection by copper against toxin-induced liver injury. Retention of hepatic α-tocopherol after liver damage may partly explain low serum vitamin E levels seen in clinical liver disease.

Serum and liver enzyme changes in rats after short term exposure to dichlorvos

1. Rat serum and liver ICDH, OCT, GOT and GPT activities are significantly elevated 2–6 hr after i.p. injections of single doses (2.5 mg/kg body wt) of dichlorvos. 2. ICDH, OCT, GOT and GPT activities are significantly increased in serum and liver of rats receiving daily injections (0.25 mg/kg body wt) for 7 days. 3. Although liver SD is significantly elevated in the single and repeated dosage study, serum SD appears unaffected at these levels of dichlorvos toxicity. 4. Changes in serum OCT activity estimations maybe a useful single parameter for screening pesticides for potential hepatotoxic effects in humans and farm animals.

Determination of Serum Ornithine Carbamyl Transferase with an Ammonia Gas-Sensing Electrode

Abstract Serum OCT activity was determined with a potentiometric ammonia gas-sensing electrode at normal and abnormal levels. It is shown that the potentiometric methods offers substantial advantages over previous techniques in terms of sample size, incubation periods, and measurement times. The proposed method correlates well with independent determinations and has a relative error of less than 4.2%.

Relationship between the carbon skeleton length of ketonic solvents and potentiation of chloroform-induced hepatotoxicity in rats

Previous studies have suggested that ketonic solvents potentiate the hepatotoxic action of CHCl 3 in rats. In addition, the relative potentiating capacity of the ketones appeared to be related to the length of their carbon skeleton. To test this hypothesis CHCl 3-induced liver injury was evaluated in male Sprague-Dawley rats pretreated (15 mmol/kg, p.o.) with acetone (Ac), 2-butanone (Bu), 2-pentanone (Pn), 2-hexanone (Hx) or 2-heptanone (Hp). After 18 h, a challenging dose of CHCl 3, (0.50 or 0.75 ml/kg, i.p.) was given. Liver damage was evaluated 24 h after CHCl 3 administration by determining elevations in plasma GPT and OCT activity. Neither Ac, Bu, Pn, Hx, Hp or the CHCl 3 challenging dosages produced marked liver injury when given alone. However, each of the ketones potentiated CHCl 3-induced liver damage. The severity of the potentiated hepatotoxic response was significantly (positively) correlated with the ketone carbon chain length. These observations suggest that carbon skeleton length may play a role in determining the relative potentiating capacity of ketonic solvents.

Serum glucose phosphate isomerase and ornithine carbamoyltransferase activities are increased in women taking contraceptive steroids.

Serum glucosephosphate isomerase (GPI: EC 5.3.1.9) activity is increased in cancer viral hepatitis and myocardial infarction. We describe a here hitherto unknown cause of high serum GPI activity which may have a bearing on the diagnostic value of this enzyme. We measured GPI activity serum with a kit in 10 healthy women of reproductive age 3 6 and 9 months after they started taking an oral contraceptive (OC; Ovulen Searle) and in 20 age- and sex-matched controls. The data were analyzed statistically by use of Students t-test. Mean (and SD) serum GPI activity was 23.4 (6.6) Bodansky units in the control group and 58.4 (16.3) 144.7 (64.1) and 192.9 (101.3) Bodansky units after the use of the OC for 3 6 and 9 months respectively. The difference at each interval was highly significant (P 0.05) from the control value but the 6-month value was significantly higher (P<0.05). Values exceeding the upper limits of normal (mean+2 SD) were observed in 1 of 10 women at 3 months in 4 at 6 months and in none at 9 months. OCT is mainly a hepatic enzyme so this increase could have resulted from hepatic injury caused by OC. If so the decrease in serum OCT activity to near-normal after 9 months of use shows that the hepatic injury is short lived and reverses itself on continued use of the OC. OCs reportedly increase serum aspartate aminotransferase and alanine aminotransferase alkaline phosphatase and isocitrate dehydrogenase. These effects have been attributed to hepatic injury caused by the OCs. However these enzymes are widely distributed in the body so increases in their activities in serum do not provide conclusive evidence of hepatic injury. (full text)

FETAL LIVER BIOPSY FOR PRENATAL DIAGNOSIS OF ORNITHINE CARBAMYL TRANSFERASE DEFICIENCY

Two male fetuses at risk for ornithine carbamyl transferase deficiency (OCTD) were investigated by a fetoscopic technique for fetal liver biopsy. In one OCT activity was absent in the liver and the pregnancy was terminated. Necropsy confirmed the presence of OCTD. In the other fetus, liver OCT activity was normal and a healthy baby was delivered at term.

ASSIMILATION OF NITROGEN IN ROOT NODULES OF ALDER (ALNUS GLUTINOSA)

SummaryCitrulline is shown to be the predominant free amino acid in alder root nodules, while serine also occurs in relatively large amounts. Citrulline and glutamic acid are prominent free amino acids in alder xylem tissue. The activities of N2‐ase, GS, GDH, and OCT in root nodule homogenates are reported. From the Km values of GDH for NH4+ (16mM) and glutamate (0·9 mM) and the concentrations in the nodule of NH4+ (1·5 HIM) and glutamate (0·5 mM) it is concluded that GDH in alder nodules is responsible for the deamination of glutamate. The important function of GDH in the nitrogen metabolism of alder nodules is confirmed by the much higher activity of this enzyme in homogenates of nodules as compared to that in homogenates of root‐tips and leaves.The vesicle clusters, which contain the N2‐ase activity, did not show activity of GS, GDH and OCT. The cytoplasm of the host cells was shown to possess the GS activity, while GDH and OCT probably are localized in the organelles of the host cells.

Establishment of a rat hepatoma cell line which has ornithine carbamoyltransferase activity and grows continuously in arginine-deprived medium.

The epithelial cell line, H4-II-E derived from Reuber hepatoma H35 has no significant activity of ornithine carbamoyltransferase (OCT, EC 2.1.3.3) and is not able to grow in arginine-deprived medium. A multi-step selection procedure is described which selects from HR-II-E populations, cells with OCT activity which can grow in arginine-deficient, ornithine-supplemented media.

Ornithine transcarbamylase (OCT) in urease used as reagent in the oct activity measurement in biological fluids

Ornithine transcarbamylase (OCT) is demonstrated to be present in urease, used as reagent in the OCT activity measurements in order to destroy the interfering substance urea. Some kinetic properties of the OCT in urease have been compared with those of human liver OCT to consider the consequences of the use of urease for OCT studies in biological materials.

HEREDITARY ORNITHINE TRANSCARBAMYLASE DEFICIENCY

The authors report two male cases of liver ornithine carbamyl transferase deficiency. In one the disease occurred at 8 years of age with hyperammoniemic coma leading to death in 48 hours. In the second case, symptoms appeared on the sixth day of life but the outcome was favorable. The child is normal at 15 months. In both cases, there was a residual 6-10% OCT activity. These observations are similar to two other male cases in the literature and are different from the male neonatal fatal form in which the deficiency is virtually total. They underline the genetically heterogeneous nature of OCT deficiencies and the fact that in this X-transmitted trait, hemizygotes can preserve a functional enzymatic activity compatible with life.