A non-capacitative pathway activated by arachidonic acid is the major Ca2+ entry mechanism in rat A7r5 smooth muscle cells stimulated with low concentrations of vasopressin.
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1. Depletion of the Ca2+ stores of A7r5 cells stimulated Ca2+, though not Sr2+, entry. Vasopressin (AVP) or platelet-derived growth factor (PDGF) stimulated Sr2+ entry. The cells therefore express a capacitative pathway activated by empty stores and a non-capacitative pathway stimulated by receptors; only the former is permeable to Mn2+ and only the latter to Sr2+. 2. Neither empty stores nor inositol 1,4,5-trisphosphate (InsP3) binding to its receptors are required for activation of the non-capacitative pathway, because microinjection of cells with heparin prevented PDGF-evoked Ca2+ mobilization but not Sr2+ entry. 3. Low concentrations of Gd3+ irreversibly blocked capacitative Ca2+ entry without affecting AVP-evoked Sr2+ entry. After inhibition of the capacitative pathway with Gd3+, AVP evoked a substantial increase in cytosolic [Ca2+], confirming that the non-capacitative pathway can evoke a significant increase in cytosolic [Ca2+]. 4. Arachidonic acid mimicked the effect of AVP on Sr2+ entry without stimulating Mn2+ entry; the Sr2+ entry was inhibited by 100 microM Gd3+, but not by 1 microM Gd3+ which completely inhibited capacitative Ca2+ entry. The effects of arachidonic acid did not require its metabolism. 5. AVP-evoked Sr2+ entry was unaffected by isotetrandrine, an inhibitor of G protein-coupled phospholipase A2. U73122, an inhibitor of phosphoinositidase C, inhibited AVP-evoked formation of inositol phosphates and Sr2+ entry. The effects of phorbol esters and Ro31-8220 (a protein kinase C inhibitor) established that protein kinase C did not mediate the effects of AVP on the non-capacitative pathway. An inhibitor of diacylglycerol lipase, RHC-80267, inhibited AVP-evoked Sr2+ entry without affecting capacitative Ca2+ entry or release of Ca2+ stores. 6. Selective inhibition of capacitative Ca2+ entry with Gd3+ revealed that the non-capacitative pathway is the major route for the Ca2+ entry evoked by low AVP concentrations. 7. We conclude that in A7r5 cells, the Ca2+ entry evoked by low concentrations of AVP is mediated largely by a non-capacitative pathway directly regulated by arachidonic acid produced by the sequential activities of phosphoinositidase C and diacylglycerol lipase. (+info)
Effects of supplementation of organic and inorganic combinations of copper, cobalt, manganese, and zinc above nutrient requirement levels on postpartum two-year-old cows.
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The objective of this study was to determine whether a combination of Cu, Co, Mn, and Zn in an organic or inorganic form fed at higher than nutrient recommendations for 2-yr-old cows from calving to breeding would affect pregnancy rate, calving date, calf performance, and cow liver and serum mineral concentrations. Crossbred 2-yr-old cows were used after calving in 1994 (n = 127) and 1995 (n = 109). Cows were blocked by calving date to one of three treatments: 1) no supplemental minerals (CTL), 2) organic minerals (ORG), or 3) inorganic minerals (ING). Minerals were fed for the same daily intake for both organic and inorganic treatments: Cu (125 mg), Co (25 mg), Mn (200 mg), and Zn (360 mg). Cows were individually fed a mineral-protein supplement with grass hay from calving (February-March) to before breeding (May 15). Hay intakes were calculated using chromium oxide boluses to determine fecal output. Fecal excretion of minerals was calculated following trace element analysis of feces. Liver biopsies were obtained before calving, after calving (start of supplementation), at the end of supplementation, and in midsummer. Over 2 yr, more cows did not become pregnant (P < .01) in ORG (11/78) and ING (11/78) treatments than in CTL (0/80) treatments. A treatment x year interaction was found for day of conception. Cows in the ORG group conceived later (P < .01) than cows in the ING or CTL groups in 1994. In 1995, there was no difference (P > .10) in day of conception among groups. Liver Zn and Mn concentrations were not different (P > .10) and Cu concentrations increased (P < .01) for the ORG and ING groups. Cows in the ORG and ING groups had higher (P < .01) concentrations of Cu, Mn, and Zn in the feces than the CTL cows. Trace elements in the feces did not differ for ORG and ING groups. Results indicate that combinations of Cu, Co, Mn, and Zn fed at higher levels than are required reduced reproductive performance. (+info)
The mode of action of contractile effects induced by external calcium and its related bivalent cations in the KCl-depolarized rat uterus.
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The cumulative log dose-response curve (DRC) isometrically produced by CaCl2 on depolarized uteri of rats in Ca2+-free KCl Ringer's solution (pH 7.8) showed a plateau-shaped type, when responses were plotted as a percent of the maximal tension of 50 micrometer acetylcholine, whereas those produced by BaCl2 or SrCl2 were a simple sigmoid type with the maximal response near the height of the plateau induced by Ca2+. The plateau phase of Ca2+ was inhibited competitively by Mg2+ (0.5--50 mM) and non-competitively by Mn2+ (30 micrometer--1 mM), whereas the maximal contraction of Ca2+ was not inhibited by either ion. Dantrolene (0.1 mM) inhibited both the plateau and the maximal contraction of Ca 2+ and retained the plateau-shaped type. EGTA (2mM) potentiated the plateau to the maximal response level and changed the DRC induced by Ca2+ to the simple sigmoid type. From these results, the plateau of DRC induced by Ca2+ seems to be mainly due to actions of external Ca2+ on the calcium binding of internal surface of cell membrane, suggesting that the internal surface is the site of the bivalent cations tested. (+info)
Improved Mg2+-based reverse transcriptase assay for detection of primate retroviruses.
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The reverse transcriptase (RT) assay is a simple, relatively inexpensive, widely used assay that can detect all retroviruses (known and novel retroviruses as well as infectious and defective retroviruses) on the basis of the divalent cation requirement of their RT enzyme, i.e., Mg2+ or Mn2+. Descriptions of various RT assays have been published; however, they cannot be directly applied to the analysis of biological products or clinical samples without further standardization to determine the lower limit of virus detection (sensitivity), assay variability (reproducibility), or ability to detect different retroviruses (specificity). We describe the detection of type E and type D primate retroviruses, which may be pathogenic for humans, by a new 32P-based, Mg2+-containing RT assay. The results show that the sensitivity of detection is <3.2 50% tissue culture infective doses (TCID50s) for human immunodeficiency virus type 1 (HIV-1) and <1 TCID50 for simian immunodeficiency virus isolated from a rhesus macaque (SIVmac). Analysis of recombinant HIV-1 RT enzyme indicated that 10(-5) U, which is equivalent to 4.25 x 10(4) virions, could be detected. Additionally, genetically distinct type D retroviruses such as simian AIDS retrovirus and squirrel monkey retrovirus were also detected in the assay with similar sensitivities. Thus, the improved RT assay can be used to detect genetically divergent Mg2+-dependent retroviruses of human and simian origin that can infect human cells and that therefore pose a potential health risk to humans. (+info)
Effects of stair-step nutrition and trace mineral supplementation on attainment of puberty in beef heifers of three sire breeds.
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A study was conducted to evaluate the influence of nutrition and sire breed on age at puberty and first lactation milk yield in crossbred beef heifers. After weaning, 208 heifers sired by Hereford, Limousin, or Piedmontese bulls were assigned to either a control (CG) or stair-step gain (SSG) dietary regimen plus a mineral supplement with or without Cu, Zn, and Mn top-dressed onto the feed. Heifers on the SSG regimen were fed a diet intended to supply energy to support gains at a rate of 120% of the CG diet for 55 d and then were switched to a diet formulated to produce an ADG at 70% of the rate of the CG diet for 84 d. They then switched back to the 120% diet for the last 30 d before breeding. Total weight gain and overall rate of gain did not differ among dietary treatments. Hereford- and Limousin-sired heifers gained at similar rates, and Piedmontese-sired heifers gained an average of .10 kg/d slower than the other two sire breed groups. During one period, Piedmontese-sired heifers on the CG diet gained .19 kg/d faster ( P < . 01) when supplemented with mineral than when not. During that same period, there was no influence of mineral supplementation on weight gains for Hereford- or Piedmontese-sired heifers on the high SSG diet, but Limousin-sired heifers tended (P = .07) to gain faster (1.00 vs .85 kg/d) when supplemented with Cu, Zn, and Mn than when not. Piedmontese-sired heifers reached puberty at the earliest age (P = .03), followed by Hereford- and then Limousin-sired heifers. There were no treatment effects on milk yield at an average of 70 d of lactation. However, at approximately 120 d of lactation, Piedmontese-sired heifers were producing less milk (P < .05) than Limousin- but not Hereford-sired heifers. Hereford-sired heifers had lower (P < .05) plasma Cu concentrations than Piedmontese-sired heifers. There were no treatment effects on plasma Zn concentrations. Heifers sired by bulls of breeds that differ in potential muscularity differed in growth, reproduction, milk yield, and plasma mineral concentrations, but dietary treatments resulted in little to no differences in these variables. (+info)
DNA hairpin opening mediated by the RAG1 and RAG2 proteins.
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The lymphoid cell-specific proteins RAG1 and RAG2 initiate V(D)J recombination by cleaving DNA adjacent to recombination signals, generating blunt signal ends and covalently sealed, hairpin coding ends. A critical next step in the reaction is opening of the hairpins, but the factor(s) responsible has not been identified and had been thought to be a ubiquitous component(s) of the DNA repair machinery. Here we demonstrate that RAG1 and RAG2 possess an intrinsic single-stranded nuclease activity capable of nicking hairpin coding ends at or near the hairpin tip. In Mn2+, a synthetic hairpin is nicked 5 nucleotides (nt) 5' of the hairpin tip, with more distant sites of nicking suppressed by HMG2. In Mg2+, hairpins generated by V(D)J cleavage are nicked whereas synthetic hairpins are not. Cleavage-generated hairpins are nicked at the tip and predominantly 1 to 2 nt 5' of the tip. RAG1 and RAG2 may therefore be responsible for initiating the processing of coding ends and for the generation of P nucleotides during V(D)J recombination. (+info)
Concentration of manganese in the tissues of cycling and anestrous ewes.
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A wide range of variation in concentration of manganese was found in the blood of 51 cycling ewes although no significant differences were evident (P less than 0.05) between days 4, 11 and 15 (day 0 = day of estrus) of the estrous cycle. In addition, no differences (P less than 0.05) were found in the manganese concentrations of various soft tissues of 15 ewes, killed on day 4 (five animals), day 11 (five animals) or during anestrus (five animals). The highest concentration of manganese was present in the liver, pancreas and kidney cortex, tissues which are rich in mitochondria. Among the tissues of the reproductive tract, the corpus luteum showed the highest manganese concentration and the level increased significantly (P less than 0.01) between days 4 and 11 suggesting that this trace element may be closely related to the metabolic and possible functional characteristics of this endocrine gland. Uterine horn and caruncles contained greater manganese concentrations than other components of the reproductive tract. The significance of these findings in relation to the effect of manganese intake on ewe fertility remains to be assessed. (+info)
Cooperative interaction of NF-kappaB and C/EBP binding sites is necessary for manganese superoxide dismutase gene transcription mediated by lipopolysaccharide and interferon-gamma.
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Expression of the manganese superoxide dismutase (Mn-SOD) is induced by pro-inflammatory cytokines. We investigated the cis-acting elements within a tumor necrosis factor-responsive element (TNFRE) which was identified in the second intron of the murine Mn-SOD gene. Site-directed mutagenesis, reporter plasmid transfection studies and electrophoretic mobility shift assays demonstrated that inducible transcription factors enhanced the transcriptional activity of the Mn-SOD gene through the TNFRE. The cooperation between proteins binding to the newly identified NF-kappaB and C/EBP sites led to synergistic gene transcription. This report provides the first evidence that cooperation between two distinct cis-acting elements may be required for induction of Mn-SOD gene expression mediated by lipopolysaccharide and interferon-gamma. (+info)