The control of gene transcription by vitamin D compounds is initiated by binding to the VDR, which enhances the receptor's ability to heterodimerize to RXR, interact with response elements in target genes and attract components of the transcriptional initiation complex. A number of factors are capable of influencing this process, including (i) the rate of uptake and catabolism of the ligand, (ii) the nature of the conformational change induced by a specific ligand, (iii) the cellular content of the VDR, (iv) post-translational modifications of the VDR and (v) the availability of other transcriptional components. Vitamin D analogues may affect these factors differently to 1,25(OH)2D3 to produce unique biological profiles that can be exploited for therapeutic use. (+info)
An estimation of the requirement for folic acid in gestating sows: the metabolic utilization of folates as a criterion of measurement.
(10/13134)
Sows at their second parity were randomly distributed in five groups of seven animals each to determine the dietary concentration of folic acid that optimizes the metabolic utilization of the vitamin during gestation. The groups differed by dietary supplement of folic acid: 0, 5, 10, 15, or 20 ppm. Sows were fed 2.5 kg of diet each day. The response of serum folates and folate binding capacity to treatments and the excretion of urinary folates after an i.v. injection of folic acid were measured. The total daily excretion of urinary folates was corrected according to the response to one i.v. injection of saline on the day preceding the i.v. injection of folic acid. The decrease of total serum folates throughout gestation was less pronounced in the groups fed 15 and 20 ppm of dietary folic acid (supplement x period interaction, P<.06) than it was in the other three treatments. The proportion of i.v. folic acid not recovered in sow urine (injected - excreted) decreased as the amount of dietary folic acid increased to reach a minimum, which differed according to the period (supplement x period interaction, P<.02); it was 15 ppm during wk 1 of gestation and 10 ppm for the other periods studied. The unrecovered folates increased over a dietary concentration of 15 ppm. These minimum values correspond to the most appropriate feed concentration that covered the whole body utilization (tissue and cell metabolism, catabolism, and storage) of folates by the sows and could be interpreted as a reliable index of the requirement. (+info)
Cloning and expression of a novel member of the low voltage-activated T-type calcium channel family.
(11/13134)
Low voltage-activated Ca2+ channels play important roles in pacing neuronal firing and producing network oscillations, such as those that occur during sleep and epilepsy. Here we describe the cloning and expression of the third member of the T-type family, alpha1I or CavT.3, from rat brain. Northern analysis indicated that it is predominantly expressed in brain. Expression of the cloned channel in either Xenopus oocytes or stably transfected human embryonic kidney-293 cells revealed novel gating properties. We compared these electrophysiological properties to those of the cloned T-type channels alpha1G and alpha1H and to the high voltage-activated channels formed by alpha1Ebeta3. The alpha1I channels opened after small depolarizations of the membrane similar to alpha1G and alpha1H but at more depolarized potentials. The kinetics of activation and inactivation were dramatically slower, which allows the channel to act as a Ca2+ injector. In oocytes, the kinetics were even slower, suggesting that components of the expression system modulate its gating properties. Steady-state inactivation occurred at higher potentials than any of the other T channels, endowing the channel with a substantial window current. The alpha1I channel could still be classified as T-type by virtue of its criss-crossing kinetics, its slow deactivation (tail current), and its small (11 pS) conductance in 110 mM Ba2+ solutions. Based on its brain distribution and novel gating properties, we suggest that alpha1I plays important roles in determining the electroresponsiveness of neurons, and hence, may be a novel drug target. (+info)
Effects of impaired Ca2+ homeostasis on contraction in postinfarction myocytes.
(12/13134)
The significance of altered Ca2+ influx and efflux pathways on contractile abnormalities of myocytes isolated from rat hearts 3 wk after myocardial infarction (MI) was investigated by varying extracellular Ca2+ concentration ([Ca2+]o, 0.6-5.0 mM) and pacing frequency (0.1-5.0 Hz). Myocytes isolated from 3-wk MI hearts were significantly longer than those from sham-treated (Sham) hearts (125 +/- 1 vs. 114 +/- 1 micrometer, P < 0.0001). At high [Ca2+]o and low pacing frequency, conditions that preferentially favored Ca2+ influx over efflux, Sham myocytes shortened to a greater extent than 3-wk MI myocytes. Conversely, under conditions that favored Ca2+ efflux (low [Ca2+]o and high pacing frequency), MI myocytes shortened more than Sham myocytes. At intermediate [Ca2+]o and pacing frequencies, differences in steady-state contraction amplitudes between Sham and MI myocytes were no longer significant. Collectively, the interpretation of these data was that Ca2+ influx and efflux pathways were subnormal in MI myocytes and that they contributed to abnormal cellular contractile behavior. Because Na+/Ca2+ exchange activity, but not whole cell Ca2+ current, was depressed in 3-wk MI rat myocytes, our results on steady-state contraction are consistent with, but not proof of, the hypothesis that depressed Na+/Ca2+ exchange accounted for abnormal contractility in MI myocytes. The effects of depressed Na+/Ca2+ exchange on MI myocyte mechanical activity were further evaluated in relaxation from caffeine-induced contractures. Because Ca2+ uptake by sarcoplasmic reticulum was inhibited by caffeine and with the assumption that intracellular Na+ and membrane potential were similar between Sham and MI myocytes, myocyte relaxation from caffeine-induced contracture can be taken as an estimate of Ca2+ extrusion by Na+/Ca2+ exchange. In MI myocytes, in which Na+/Ca2+ exchange activity was depressed, the half time of relaxation (1.54 +/- 0.14 s) was significantly (P < 0.02) prolonged compared with that measured in Sham myocytes (1.10 +/- 0.10 s). (+info)
Contributions of mitochondria to animal physiology: from homeostatic sensor to calcium signalling and cell death.
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Over recent years, it has become clear that mitochondria play a central role in many key aspects of animal physiology and pathophysiology. Their central and ubiquitous task is clearly the production of ATP. Nevertheless, they also play subtle roles in glucose homeostasis, acting as the sensor for substrate supply in the transduction pathway that promotes insulin secretion by the pancreatic -cell and that modulates the excitability of the hypothalamic glucose-sensitive neurons involved in appetite control. Mitochondria may also act as sensors of availability of oxygen, the other major mitochondrial substrate, in the regulation of respiration. Mitochondria take up calcium, and the high opacity mitochondrial calcium uptake pathway provides a mechanism that couples energy demand to increased ATP production through the calcium-dependent upregulation of mitochondrial enzyme activity. Mitochondrial calcium accumulation may also have a substantial impact on the spatiotemporal dynamics of cellular calcium signals, with subtle differences of detail in different cell types. Recent work has also revealed the centrality of mitochondrial dysfunction as an irreversible step in the pathway to both necrotic and apoptotic cell death. This review looks at recent developments in these rapidly evolving areas of cell physiology in an attempt to draw together disparate areas of research into a common theme. (+info)
Structural and functional consequences of antigenic modulation of red blood cells with methoxypoly(ethylene glycol).
(14/13134)
We previously showed that the covalent modification of the red blood cell (RBC) surface with methoxypoly(ethylene glycol) [mPEG; MW approximately 5 kD] could significantly attenuate the immunologic recognition of surface antigens. However, to make these antigenically silent RBC a clinically viable option, the mPEG-modified RBC must maintain normal cellular structure and functions. To this end, mPEG-derivatization was found to have no significant detrimental effects on RBC structure or function at concentrations that effectively blocked antigenic recognition of a variety of RBC antigens. Importantly, RBC lysis, morphology, and hemoglobin oxidation state were unaffected by mPEG-modification. Furthermore, as shown by functional studies of Band 3, a major site of modification, PEG-binding does not affect protein function, as evidenced by normal SO4- flux. Similarly, Na+ and K+ homeostasis were unaffected. The functional aspects of the mPEG-modified RBC were also maintained, as evidenced by normal oxygen binding and cellular deformability. Perhaps most importantly, mPEG-derivatized mouse RBC showed normal in vivo survival ( approximately 50 days) with no sensitization after repeated transfusions. These data further support the hypothesis that the covalent attachment of nonimmunogenic materials (eg, mPEG) to intact RBC may have significant application in transfusion medicine, especially for the chronically transfused and/or allosensitized patient. (+info)
Shrinkage-induced activation of Na+/H+ exchange in rat renal mesangial cells.
(15/13134)
Using the pH-sensitive dye 2', 7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF), we examined the effect of hyperosmolar solutions, which presumably caused cell shrinkage, on intracellular pH (pHi) regulation in mesangial cells (single cells or populations) cultured from the rat kidney. The calibration of BCECF is identical in shrunken and unshrunken mesangial cells if the extracellular K+ concentration ([K+]) is adjusted to match the predicted intracellular [K+]. For pHi values between approximately 6.7 and approximately 7.4, the intrinsic buffering power in shrunken cells (600 mosmol/kgH2O) is threefold larger than in unshrunken cells (approximately 300 mosmol/kgH2O). In the nominal absence of CO2/HCO-3, exposing cell populations to a HEPES-buffered solution supplemented with approximately 300 mM mannitol (600 mosmol/kgH2O) causes steady-state pHi to increase by approximately 0.4. The pHi increase is due to activation of Na+/H+ exchange because, in single cells, it is blocked in the absence of external Na+ or in the presence of 50 microM ethylisopropylamiloride (EIPA). Preincubating cells in a Cl--free solution for at least 14 min inhibits the shrinkage-induced pHi increase by 80%. We calculated the pHi dependence of the Na+/H+ exchange rate in cell populations under normosmolar and hyperosmolar conditions by summing 1) the pHi dependence of the total acid-extrusion rate and 2) the pHi dependence of the EIPA-insensitive acid-loading rate. Shrinkage alkali shifts the pHi dependence of Na+/H+ exchange by approximately 0.7 pH units. (+info)
A surrogate measure of whole body leucine transport across the cell membrane.
(16/13134)
Based on a mass-balance model, a surrogate measure of the whole body leucine transport into and out of cells under steady-state conditions was calculated as u/DeltaTTR, where u is the infusion rate of (stable label) leucine tracer and DeltaTTR is the difference between the tracer-to-tracee ratio of extracellular and intracellular leucine. The approach was evaluated in ten healthy subjects [8 males and 2 females; age, 31 +/- 9 (SD) yr; body mass index, 24.0 +/- 1.6 kg/m2] who received a primed (7.58 micromol/kg) constant intravenous infusion (7.58 micromol. kg-1. h-1) of L-[1-13C]leucine over 180 min (7 subjects) or 240 min (3 subjects). Five subjects were studied on two occasions >/=1 wk apart to assess reproducibility. Blood samples taken during the last 30 min of the leucine infusion were used to determine plasma leucine concentration (129 +/- 35 micromol/l), TTR of leucine (9.0 +/- 1.5%), and TTR of alpha-ketoisocaproic acid (6.7 +/- 0.8%). The latter TTR was taken as the measure of the free intracellular leucine TTR. The whole body inward and outward transport was 6.66 +/- 3.82 micromol. kg-1. min-1; the rate of leucine appearance due to proteolysis was 1.93 +/- 0.24 micromol. kg-1. min-1. A positive linear relationship between the inward transport and plasma leucine was observed (P < 0.01), indicating the presence of the mass effect of leucine on its own transport. The transport was highly variable between subjects (between-subject coefficient of variation 57%) but reproducible (within-subject coefficient of variation 17%). We conclude that reproducible estimates of whole body transport of leucine across the cell membrane can be obtained under steady-state conditions with existing experimental and analytical procedures. (+info)