Kinetics and regulation of 25-hydroxycholecalciferol 1 alpha-hydroxylase from cells isolated from human term decidua

Kinetics and regulation of 25-hydroxycholecalciferol 1 alpha-hydroxylase from cells isolated from term human decidua were studied. The production of 1 alpha,25-dihydroxycholecalciferol (calcitriol) was linear with time for up to 6 h and was directly proportional to the number of cells up to 20 X 10(6)/dish at a substrate concentration of 100 nM. Under these conditions the apparent Km was 88 nM and the Vmax 3.0 pmol/10(6) cells. The production of [3H]calcitriol was inhibited by 0.1 nM (P less than 0.01) and 1 nM (P less than 0.005) unlabeled calcitriol. Unlike the kidney enzyme and for reasons that remain unclear, neither inorganic phosphate salts nor parathyroid hormone had any acute effect on the calcitriol production. Further studies are required to delineate the regulatory mechanism of this enzyme.     

Interaction of vasoactive intestinal peptide (VIP) with rat lymphoid cells

Receptors for vasoactive intestinal peptide (VIP) have been characterized in rat lymphoid cells. The interaction of [125I] VIP with blood mononuclear cells was rapid, reversible, specific and saturable. At apparent equilibrium, the binding of [125I] VIP was competitively inhibited by native VIP in the 0.01-100 nM range concentration. The binding data were compatible with the existence of two classes of receptors: a high-affinity class with a Kd = 0.050 +/- 0.009 nM and a low binding capacity (2.60 +/- 0.28 fmol/10(6) cells), and a low-affinity class with a Kd = 142 +/- 80 nM and a high binding capacity (1966 +/- 330 fmol/10(6) cells). Secretin, glucagon, insulin and somatostatin did not show any effect at a concentration as high as 100 nM. With spleen lymphoid cells, stoichiometric studies were performed. The binding data were compatible with the existence of two classes of receptors: a high-affinity class with a Kd = 0.100 +/- 0.033 nM and a low binding capacity (4.60 +/- 1.07 fmol/10(6) cells), and low-affinity class with a Kd = 255 +/- 110 nM and high binding capacity (2915 +/- 1160 fmol/10(6) cells). With thymocytes, no binding was obtained under different conditions.     

Specific binding sites for growth hormone in cultured mouse thymic epithelial cells

Growth Hormones bound specifically to murine Thymic epithelial cells, which represent the major component of thymic micro-environment and can be modulated by pituitary hormones. The Kds found with human growth hormone and bovine growth hormone were 0.14 and 0.27 nM with a Bmax 0.56 and 0.35 fmol/10(6) cells respectively. Competition experiment analysis showed ED50 of 0.24 nM for hGH, 0.46 nM for rGH, 0.71 nM for bGH, 11.8 nM for hPRL and 11.2 nM for oPRL. No specific binding of [125I]-oPRL was observed under the same conditions. Both hPRL and bGH showed a negative regulatory effect on the number of the hGH binding sites when incubated with the culture for three days. The presence of GH receptors on Thymic epithelial cells provides biochemical evidence for the effect of GH on thymic function.     

Simple and rapid determination of thiol compounds by HPLC and fluorescence detection with 1,3,5,7-tetramethyl-8-phenyl-(2-maleimide) difluoroboradiaza-s-indacene

A rapid and simple background-free high-performance liquid chromatographic (HPLC) approach has been developed for simultaneously determining free thiol compounds including coenzyme A (CoA), cysteine (Cys), glutathione (GSH) and N-acetyl-cysteine (NAC) in biological samples by using 1,3,5,7-tetramethyl-8-phenyl-(2-maleimide) difluoroboradiaza-s-indacene (TMPAB-o-M) as fluorogenic reagent. After derivatization under physiological conditions within 6 min, baseline separation was finished in just 6 min using isocratic elution with reversed-phase HPLC and fluorescence detection. Excellent linearity was observed for all analytes over their concentration ranges of 1-500 nM and detection limits ranging 0.13 nM for CoA to 0.25 nM for Cys (S/N=3) were achieved. The utility of the proposed method has been validated by measuring thiol compounds mentioned above in tissue, fluid and cell samples. The results indicated that this approach was well suited for high-throughput quantitative determination of thiols and study of the physiological role of them.     

Evaluation of endothelin receptor populations using endothelin-1 biotinylated at lysine-9 sidechain.

Optimal conditions for biotinylation of endothelin-1 (Et-1) were determined using biotinylating reagents of variable linker arm length and mono- vs dual-biotinylated Et-1. Specific modification of lysine-9 sidechain with NHS-LC-biotin (Et-1[BtK9]) produced a derivative with maximal binding and retention of vascular smooth muscle contractile activity. The Et-1[BtK9] probe bound to Chinese hamster ovary cells transfected with EtA receptor cDNA (CHO[EtR]), but not untransfected cells. Binding to rat vascular smooth muscle cells (VSMC) was detectable at 0.01 nM with maximal binding at 1 nM. Displacement of 1 nM Et-1 [BtK9] binding by Et-1 indicated an IC50 value of 6 +/- 3 nM. Et-1 displaced Et-1[BtK9] binding to VSMC and CHO[EtR] to a greater extent than endothelin-3, indicating predominant expression of EtA receptor sub-type. Thus, biotinylation of Et-1 at the lysine-9 sidechain may be of general use for localization and typing of Et-receptor populations.     

Preferential binding of insulin-like growth factor-II (IGF-II) to a putative alpha 2 beta 2 IGF-II receptor type in C2 myoblasts.

We have studied insulin-like-growth-factor (IGF) binding in two subclones of the C2 myogenic cell line. In the permissive parental subclone, myoblasts differentiate spontaneously into myotubes in medium supplemented with fetal calf serum. Unlike permissive myoblasts, inducible myoblasts require high concentrations of insulin (1.6 microM) or lower concentrations of IGF-I (25 nM) to differentiate, and expression of MyoD1 is not constitutive. IGF receptors were studied in microsomal membranes of proliferating and quiescent myoblasts and myotubes. IGF-II binding was also studied in inducible myoblasts transfected with the MyoD1 cDNA (clone EP5). Both inducible and permissive cells exhibited a single class of binding sites with similar affinity for IGF-I (Kd 0.8-1.2 nM). Affinity cross-linking of [125I]IGF-I to microsomal membranes, under reducing conditions, revealed a binding moiety with an apparent molecular mass of 130 kDa in permissive cells and 140 kDa in inducible cells, which corresponded to the alpha subunit of the IGF-I receptor. In permissive quiescent myoblasts, linear Scatchard plots suggested that [125I]IGF-II bound to a single class of binding sites (Kd 0.6 nM) compatible with binding to the IGF-II/M6P receptor. This was confirmed by affinity cross-linking experiments showing a labeled complex with an apparent molecular mass of 260 kDa and 220 kDa when studied under reducing and non-reducing conditions, respectively. In contrast, competitive inhibition of [125I]IGF-II binding to inducible quiescent myoblasts generated curvilinear Scatchard plots which could be resolved into two single classes of binding sites. One of them corresponded to the IGF-II/M6P receptor (Kd 0.2 nM) as evidenced by cross-linking experiments. The second was the binding site of highest affinity (Kd 0.04 nM) which was less inhibited by IGF-I than by IGF-II and was not inhibited by insulin. It migrated in SDS/PAGE at a position equivalent a molecular mass of 140 kDa, under reducing conditions, and at approximately 300 kDa, under non-reducing conditions. The labeling of this atypical binding moiety was not inhibited by anti(IGF-II/M6P-receptor) immunoglobulin. It was also observed in permissive and inducible myoblasts at proliferating stage. It was absent for permissive quiescent myoblasts and from permissive and inducible myotubes. Forced expression of MyoD1 in inducible cells (EP5 cells) dramatically reduced [125I]IGF-II binding to this atypical receptor. It emerges from these experiments that C2 cells express a putative alpha 2 beta 2 IGF-II receptor structurally related to the insulin/IGF-I receptor family. It is present in myoblasts but not in myotubes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Globular adiponectin activates Akt in cultured myocytes

The serine/threonine kinase Akt plays an important role in insulin-mediated glucose uptake. Adiponectin (Adp) is known to sensitize this process. The purpose of the current study is to investigate if Adp activates Akt independently from insulin; and if Adp synergizes with insulin on Akt phosphorylation in the rat skeletal muscle L6 cells. Differentiated L6 cells were serum-starved and exposed to various concentrations (0-100nM) of recombinant globular Adp (gAdp) and/or insulin for different time periods at 37°C. Phosphorylation of Akt was monitored by Western blot using an antiserum against pSer(473) or pThr(308) Akt. The results demonstrate that gAdp activates Akt in dose- and time-dependent manners. When L6 cells were treated with sub-maximal concentrations of both insulin (10nM) and gAdp (10nM) for 10min neither synergistic nor additive activation of Akt was observed. Similar non-synergistic or non-additive effect of gAdp on insulin-induced Akt activation was also observed in mouse C2C12 myocytes and rat vascular smooth muscle PAC cells. Moreover, pretreatment of the L6 cells with wortmannin (100nM) for 20min significantly reduced gAdp (100nM) induced and insulin (100nM) induced Akt activation by ～80 and ～70%, respectively. These data suggest that adiponectin stimulates Akt activation via the wortmannin sensitive pathway in L6 cells; and that its effects on Akt phosphorylation are not additive to those of insulin.     

The effect of pulsed electromagnetic fields on the physiologic behaviour of a human astrocytoma cell line

We evaluated the effects of 50 Hz pulsed electromagnetic fields (EMFs) with a peak magnetic field of 3 mT on human astrocytoma cells. Our results clearly demonstrate that, after the cells were exposed to EMFs for 24 h, the basal [Ca(2+)](i) levels increased significantly from 124+/-51 nM to 200+/-79 nM. Pretreatment of the cells with 1.2 microM substance P increased the [Ca(2+)](i) to 555+/-278 nM, while EMF exposure caused a significant drop in [Ca(2+)](i) to 327+/-146 nM. The overall effect of EMFs probably depends on the prevailing Ca(2+) conditions of the cells. After exposure, the proliferative responses of both normal and substance P-pretreated cells increased slightly from 1.03 to 1.07 and 1.04 to 1.06, respectively. U-373 MG cells spontaneously released about 10 pg/ml of interleukin-6 which was significantly increased after the addition of substance P. Moreover, immediately after EMF exposure and 24 h thereafter, the interleukin-6 levels were more elevated (about 40%) than in controls. On the whole, our data suggest that, by changing the properties of cell membranes, EMFs can influence Ca(2+) transport processes and hence Ca(2+) homeostasis. The increased levels of interleukin-6 after 24 h of EMF exposure may confirm the complex connection between Ca(2+) levels, substance P and the cytokine network.     

Two high-affinity ligand binding states of uterine estrogen receptor distinguished by modulation of hydrophobic environment

The steroid binding function of soluble (cytosolic) estrogen receptors from calf uteri was evaluated under conditions known to modify the extent of hydrophobic interaction with receptor-associated proteins. Receptor preparations were equilibrated into 6 M urea (+/- 0.4 M KCl) buffers and control buffers (+/- 0.4 M KCl) by chromatography through small columns of Sephadex G-25 or by dialysis at 0-6 degrees C. Equilibrium dissociation constants (Kd) and binding capacities (n) of experimental and control receptor preparations were determined by 13-point Scatchard analyses using concentrations of 17 beta-[3H]estradiol from 0.05 to 10 nM. Nonspecific binding was determined at each concentration by parallel incubations with a 200-fold molar excess of the receptor-specific competitor diethylstilbestrol. The control receptor population was consistently found to be a single class of binding sites with a high affinity for estradiol (Kd = 0.36 +/- 0.09 nM, n = 14) which was unaffected by G-25 chromatography, by dialysis, by dilution, or by the presence of 0.4 M KCl. However, equilibration into 6 M urea induced a discrete (10-fold) reduction in receptor affinity (Kd = 3.45 +/- 0.86 nM, n = 6) to reveal a second, thermodynamically stable, high-affinity binding state. The presence of 0.4 M KCl did not significantly influence the discrete change in receptor affinity induced by urea. However, KCl did help prevent the reduction in binding capacity induced by urea. The effects of urea on both receptor affinity and binding capacity were reversible, suggesting a lack of covalent modification.(ABSTRACT TRUNCATED AT 250 WORDS)     

Relative efficiencies of the bacterial, yeast, and human DNA methyltransferases for the repair of O6-methylguanine and O4-methylthymine. Suggestive evidence for O4-methylthymine repair by eukaryotic methyltransferases

The suicidal inactivation mechanism of DNA repair methyltransferases (MTases) was exploited to measure the relative efficiencies with which the Escherichia coli, human, and Saccharomyces cerevisiae DNA MTases repair O6-methylguanine (O6MeG) and O4-methylthymine (O4MeT), two of the DNA lesions produced by mutagenic and carcinogenic alkylating agents. Using chemically synthesized double-stranded 25-base pair oligodeoxynucleotides containing a single O6MeG or a single O4MeT, the concentration of O6MeG or O4MeT substrate that produced 50% inactivation (IC50) was determined for each of four MTases. The E. coli ogt gene product had a relatively high affinity for the O6MeG substrate (IC50 8.1 nM) but had an even higher affinity for the O4MeT substrate (IC50 3 nM). By contrast, the E. coli Ada MTase displayed a striking preference for O6MeG (IC50 1.25 nM) as compared to O4MeT (IC50 27.5 nM). Both the human and the yeast DNA MTases were efficiently inactivated upon incubation with the O6MeG-containing oligomer (IC50 values of 1.5 and 1.3 nM, respectively). Surprisingly, the human and yeast MTases were also inactivated by the O4MeT-containing oligomer albeit at IC50 values of 29.5 and 44 nM, respectively. This result suggests that O4MeT lesions can be recognized in this substrate by eukaryotic DNA MTases but the exact biochemical mechanism of methyltransferase inactivation remains to be determined.     

Insulin-like growth factor-I feedback regulation of growth hormone and luteinizing hormone secretion in the pig: evidence for a pituitary site of action

Three experiments (EXP) were conducted to determine the role of insulin-like growth factor-I (IGF-I) in the control of growth hormone (GH) and LH secretion. In EXP I, prepuberal gilts, 65 ± 6 kg body weight and 140 days of age received intracerebroventricular (ICV) injections of saline (n = 4), 25 μg (n = 4) or 75 μg (n = 4) IGF-I and jugular blood samples were collected. In EXP II, anterior pituitary cells in culture collected from 150-day-old prepuberal gilts (n = 6) were challenged with 0.1, 10 or 1000 nM [Ala15]-h growth hormone-releasing hormone-(1-29)NH2 (GHRH), or 0.01, 0.1, 1, 10, 30 nM IGF-I individually or in combinations with 1000 nM GHRH. Secreted GH was measured at 4 and 24 h after treatment. In EXP III, anterior pituitary cells in culture collected from 150-day-old barrows (n = 5) were challenged with 10, 100 or 1000 nM gonadotropin-releasing hormone (GnRH) or 0.01, 0.1, 1, 10, 30 nM IGF-I individually or in combinations with 100 nM GnRH. Secreted LH was measured at 4 h after treatment. In EXP I, serum GH and LH concentrations were unaffected by ICV IGF-I treatment. In EXP II, relative to control all doses of GHRH increased (P < 0.01) GH secretion. Only 1, 10, 30 nM IGF-I enhanced (P < 0.02) basal GH secretion at 4 h, whereas by 24 h all doses except for 30 nM IGF-I suppressed (P < 0.02) basal GH secretion compared to control wells. All doses of IGF-I in combination with 1000 nM GHRH increased (P < 0.04) the GH response to GHRH compared to GHRH alone at 4 h, whereas by 24 h all doses of IGF-I suppressed (P < 0.04) the GH response to GHRH. In EXP III, all doses of IGF-I increased (P < 0.01) basal LH levels while the LH response to GnRH was unaffected by IGF-I (P > 0.1). In conclusion, under these experimental conditions the results suggest that the pituitary is the putative site for IGF-I modulation of GH and LH secretion. Further examination of the role of IGF-I on GH and LH secretion is needed to understand the inhibitory and stimulatory action of IGF-I on GH and LH secretion.     

Regulation of protein turnover by recombinant human insulin-like growth factor-I in L6 myotube cultures

Muscle cell culture experiments were conducted to determine the relative regulatory effects of insulin-like growth factors (IGF) on protein turnover. The effects of recombinant (rc) human IGF-I, ovine somatomedin (oSm/oIGF-I), and insulin on rates of protein labeling and degradation in L6 myotube cultures were evaluated. Myotube cultures were treated with growth factors following a 4-h serum-free incubation period. Protein labeling was measured by determining the rate of [3H] leucine incorporation into cell protein. Protein degradation was measured by a pulse-chase procedure using [3H] leucine. The apparent half maximal stimulation of protein labeling (12%, 8%, 7%) occurred at approximately .1 nM rcIGF-I, 1 nM oSm/oIGF-I and 15 nM insulin, respectively. The apparent half maximal inhibition of proteolysis (18%, 15% and 11%) occurred at .4 nM rcIGF-I, .6 nM oSm/oIGF-I and 4 nM insulin, respectively. The magnitude of the response for protein labeling and degradation was greatest for rcIGF-I. The results provide additional evidence that IGFs play a primary role in regulating protein turnover in muscle.     

Induction in primary culture of 'gluconeogenic' and 'glycolytic' hepatocytes resembling periportal and perivenous cells

Adult rat hepatocytes were kept in primary culture for 48 h under different hormonal conditions to induce an enzyme pattern which with respect to carbohydrate metabolism approximated that of periportal and perivenous hepatocytes in vivo. 1. Glucagon-treated cells compared with control cells possessed a lower activity of glucokinase, a 4.5-fold higher activity of phosphoenolpyruvate carboxykinase and unchanged levels of glucose-6-phosphatase, phosphofructokinase, fructose-bisphosphatase and pyruvate kinase; they resembled in a first approximation the periportal cell type and are called for simplicity 'periportal'. Inversely, insulin-treated cells compared with control cells contained a 2.2-fold higher activity of glucokinase, a slightly decreased activity of phosphoenolpyruvate carboxykinase, increased activities of phosphofructokinase and pyruvate kinase and unaltered levels of glucose-6-phosphatase and fructose-bisphosphatase; they resembled perivenous cells and are called simply 'perivenous'. Gluconeogenesis and glycolysis were studied under various substrate and hormone concentrations. 2. Physiological concentrations of glucose (5 mM) and lactate (2 mM) gave about 80% saturation of gluconeogenesis from lactate and less than 15% saturation of glycolysis at a simultaneous 40% inhibition of the glycolytic rate by lactate. 3. Comparison of the two cell types showed that under identical assay conditions (5 mM glucose, 2 mM lactate, 0.5 nM insulin, 0.1 muM dexamethasone) gluconeogenesis was 1.5-fold faster in the 'periportal' cells and glycolysis was 2.4-fold faster in the 'perivenous' cells. 4. Metabolic rates were under short-term hormonal control. Insulin increased glycolysis three fold in both cell types with a half-maximal effect at about 0.4 nM, but did not influence the gluconeogenic rate. Glucagon inhibited glycolysis by 70% with a half-maximal effect at about 0.1 nM. Gluconeogenesis was stimulated by glucagon (half-maximal dose: 0.5 nM) 1.8-fold only in 'periportal' cells containing high phosphoenolpyruvate carboxykinase activity, not in the 'perivenous' cells with a low level of this enzyme. 5. A comparison of the two cell types showed that with maximally stimulating hormone concentrations gluconeogenesis was threefold faster in 'periportal' cells and glycolysis was eightfold faster in 'perivenous' cells. The results support the view that periportal and perivenous hepatocytes in vivo catalyse gluconeogenesis and glycolysis at inverse rates.     

t-[35S]Butylbicyclophosphorothionate Binding Sites in Invertebrate Tissues

Specific high affinity binding of the cage convulsant t-[35S]butylbicyclophosphorothionate (TBPS) was observed in membrane homogenates of housefly heads and crayfish abdominal muscles. [35S]TBPS binding in these two invertebrate tissues was inhibited by biologically active cage convulsants, picrotoxin analogs, and barbiturates. The housefly binding sites were inhibited most potently by several insecticides. Approximately 50% of total binding was displaceable by excess (0.1 mM) nonradioactive TBPS, picrotoxinin, ethyl bicyclophosphate, or dieldrin. Optimal binding assay conditions for housefly homogenates included pH 7.5, 22 degrees C temperature, 0.3 M chloride concentration, and incubation for 60 min; for crayfish homogenates, 4 degrees C temperature and 150-min incubations were optimal. Scatchard plots of equilibrium binding indicated one site in both tissues (KD = 50 nM, Bmax = 250 fmol/mg protein in housefly; KD = 25 nM, Bmax = 100 fmol/mg protein in crayfish). Association kinetics in housefly were consistent with one rate constant (k+1 = 8 X 10(6) M-1 min-1), but dissociation was described better by two rate constants (k-1 = 0.28 min-1 and 0.042 min-1; calculated KD values of 80 nM and 12 nM). Displacement by cage convulsants showed Hill numbers near 0.5, also consistent with two populations of affinity, while displacement by other drugs showed Hill numbers near 1.0. [35S]TBPS binding in insects was most potently inhibited by the insecticides dieldrin (IC50 = 50 nM), aldrin, and lindane (200 nM), in a stereospecific manner, consistent with this binding site being the receptor for biological toxicity. [35S]TBPS binding was also inhibited by relatively high concentrations of some pyrethroid insecticides, such as deltamethrin and cypermethrin (1-2 microM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Cortisol and IGF-1 synergistically up-regulate taurine transport by the rat skeletal muscle cell line, L6

This study was undertaken to evaluate effects of exercise-induced hormones, cortisol, IGF-1, and beta-endorphin, on the regulation of taurine transport activity in rat skeletal myoblasts, L6 cells. Challenge of L6 cells with cortisol (100 nM) for 24 hrs resulted in a 165% increase in taurine transport activity, 220% increase in Vmax of the taurine transporter, and 55% increase in taurine transporter/ beta-actin mRNA level compared with untreated control cells. Neither IGF-1 (1 approximately 100 nM) nor beta-endorphin (1 approximately 20 nM), added in the incubation medium separately for 24 hrs, affected taurine uptake by L6 cells. However, when cells were co-treated with IGF-1 (10 nM) plus cortisol (100 nM), taurine transport activity (37% increase, p < 0.05), Vmax of the transporter (54%, p < 0.05), and taurine transporter/ beta-actin mRNA level were further increased compared to the value for cells treated with cortisol alone. These results suggest that taurine transport by skeletal muscle cells appear to be synergistically up-regulated during a prolonged exercise via elevated levels of cortisol and IGF-1 in muscle.     

Effects of opioid peptides on immunoreactive corticotropin-releasing factor release from the rat hypothalamus in vitro

Effects of opioid peptides on immunoreactive corticotropin-releasing factor (I-CRF) release from the rat hypothalamus were examined using a rat hypothalamic perifusion system and a rat CRF RIA in vitro. beta-Endorphin (0.3 - 30 nM), dynorphin (0.3 - 30 nM) and FK 33-824 (1 - 10 microM) suppressed basal I-CRF release in a dose-dependent fashion. At 2.2 nM concentrations of these peptides, mean percent inhibition was 56% for beta-endorphin; less than 5% for alpha-endorphin; 44% for dynorphin; 23% for leucine-enkephalin; 6% for methionine-enkephalin; less than 5% for FK 33-824; and less than 5% for D-ala2, D-leu5-enkephalin. The inhibitory effects of beta-endorphin and enkephalins were completely blocked by naloxone, but those of dynorphin were only partially blocked. These results suggest that opioid peptides act through opioid receptors and inhibit I-CRF release from the hypothalamus under our conditions. Therefore, endogenious opioid peptides may have a physiological role in the CRF-releasing mechanism of the hypothalamus.     

High-affinity binding of an influenza hemagglutinin-derived peptide to purified HLA-DR

Immunogenic peptides have been shown to bind detergent-solubilized class II (Ia) molecules from mice. In this investigation, we report that highly purified HLA-DR (DR) molecules in detergent solution are capable of binding a synthetic peptide (HAp) derived from the influenza hemagglutinin sequence. Although the presentation of this peptide has been demonstrated only to DR1-restricted Th cells, the association rate constants for the formation of HAp-DR1, -DR5, and -DR8 complexes were essentially identical (ka = 1.1 x 10(2) to 1.6 x 10(2) M-1 s-1). By contrast, the value of the rate constants for the dissociation of preformed HAp-DR1, -DR5, and -DR8 complexes varied nearly threefold (kd = 1.6 x 10(6) to 4.4 x 10(-6) s-1). The value of the equilibrium dissociation constants (KD) derived from these rate constants were 13 nM, 24 nM, and 28 nM, for HAp-DR1, -DR5, and -DR8 complexes, respectively. Scatchard analysis demonstrated that the KD obtained from the rate constants for the HAp-DR1 reaction was in excellent agreement with that obtained under equilibrium conditions. SDS-PAGE confirmed that the HAp-DR complexes were remarkably stable, as HAp remained associated with the DR alpha beta heterodimer after treatment of the complexes with SDS and beta-mercaptoethanol. Steady-state binding studies demonstrated that 18% of all DR1 molecules had bound HAp at equilibrium, whereas only 3.8% of all DR8 molecules had bound HAp under identical conditions. The slight differences in the KD for HAp-DR complexes suggest that differences in the affinity of a peptide for DR alleles alone may not always explain the process of MHC restriction.     

Effect of differently induced plasma norepinephrine increments on norepinephrine sulfate formation

We investigated whether similar increments in venous plasma norepinephrine (NE) concentration caused by exercise and by intravenous NE infusion will elevate plasma norepinephrine sulfate (NES) to similar concentrations. In randomized order venous plasma NE concentration was elevated to similar concentrations by bicycle exercise (BE; 65% VO(2)max) and by intravenous NE infusion at rest (INF; 0.14 microg/min/kg). N = 11 subjects participated in the study. Increments in plasma NE and the area under curve of plasma NE were similar during BE (11.2 +/- 1.3 nM; 411 +/- 23 nM/min; means +/- S.E.) and INF (12.6 +/- 1.9 nM; 429 +/- 27 nM/min). Plasma NES was significantly elevated to similar concentrations with BE (from 5.7 +/- 1.0 to 8.5 +/- 1.3 nM) and with INF (from 5.6 +/- 0.9 to 8.9 +/- 1.0 nM). Plasma NE and NES concentration during control conditions remained unchanged. Heart rate decreased significantly to 43 +/- 1 beats/min with INF and increased significantly to 162 +/- 3 beats/min with BE. Systolic blood pressure increased with both, INF and BE (155 +/- 3 mmHg; 179 +/- 6 mmHg, respectively). Present findings firstly show that intravenously infused NE is sulfoconjugated in humans, indicating that a major part of NE is sulfoconjugated in blood or at sites easily accessible from blood. Secondly, plasma NE may be a useful additional marker for NES release.     

Biochemical and cytochemical studies of preadipocyte differentiation in serum-free culture of porcine stromal-vascular cells: interaction of dexamethasone and growth hormone.

Stromal-vascular cells from adipose tissue of pigs 5-7 days of age were grown in serum for 2-3 days and switched to serum-free (insulin, transferrin and selenium) conditions +/- test hormones for 6-7 days. The interaction of dexamethasone (DEX) and human growth hormone (hGH) was evaluated since glucocorticoids augment and hGH antagonizes the effect of insulin. Low levels (1-10 nM) of DEX with insulin doubled (p less than 0.05) specific activity of glycerol phosphate dehydrogenase (GPDH) and doubled (p less than 0.05) the number of detectable fat cells relative to insulin alone. DEX with insulin enhanced the morphological differentiation of preadipocytes and markedly increased fat cell cluster numbers in the presence of hGH. Furthermore, 1-10 nM of DEX partially blocked (p greater than 0.05) the inhibitory effect of 10 nM hGH on GPDH activity, but 1-100 nM DEX had no effect (p greater than 0.05) on the ability of hGH to compromise lipid deposition. DEX alone (no insulin or hGH) induced the appearance of esterase-reactive but lipid-free cells. Cells with these characteristics were increased in number by DEX in the presence of hGH but were nearly absent in the presence of insulin and DEX. Therefore, transient exposure to GH in vivo may have no permanent effect on adipose tissue development in the continued presence of glucocorticoids.