Metabolism of 32-hydroxy-24,25-dihydrolanosterol by purified cytochrome P-45014DM from yeast. Evidence for contribution of the cytochrome to whole process of lanosterol 14 alpha-demethylation

Metabolism of 32-hydroxy-24,25-dihydrolanosterol (lanost-8-ene-3 beta,32-diol), a posturated intermediate of the 14 alpha-demethylation (removal of C-32) of 24,25-dihydrolanosterol (lanost-8-en-3 beta-ol), by a reconstituted system consisting of yeast cytochrome P-450 which catalyzes lanosterol 14 alpha-demethylation (cytochrome P-45014DM) (Yoshida, Y., and Aoyama, Y. (1984) J. Biol. Chem. 259, 1655-1660 and Aoyama, Y., Yoshida, Y., and Sato, R. (1984) J. Biol. Chem. 259, 1661-1666) and NADPH-cytochrome P-450 reductase was studied. The reconstituted system converted both 32-hydroxy-24,25-dihydrolanosterol and 24,25-dihydrolanosterol to 4,4-dimethyl-5 alpha-cholesta-8,14-dien-3 beta-ol, the 14 alpha-demethylated product of the latter. The metabolism of these compounds was inhibited by a low concentration of ketoconazole which is a potent cytochrome P-45014DM inhibitor. Affinity of cytochrome P-45014DM for 32-hydroxy-24,25-dihydrolanosterol was about 20 times higher than for 24,25-dihydrolanosterol and the cytochrome metabolized the former about 4 times faster than the latter under the experimental conditions. Spectral analysis suggested that the 32-hydroxyl group of 32-hydroxy-24,25-dihydrolanosterol interacted with the heme iron of the oxidized cytochrome and this interaction might support the high affinity of this compound for the cytochrome. These lines of evidence indicate that 32-hydroxy-24,25-dihydrolanosterol is the intermediate of the 14 alpha-demethylation of 24,25-dihydrolanosterol by cytochrome P-45014DM. It is also clear that the cytochrome catalyzes further metabolism of the 32-hydroxylated intermediate to the 14 alpha-demethylated product with higher efficiency than the 32-hydroxylation of the substrate. Cytochrome P-45014DM is thus classified as lanosterol C14-C32 lyase.     

Thymic stroma-derived T cell growth factor (TSTGF). I. Functional distinction of TSTGF from interleukins 2 and 4 and its preferential growth-promoting effect on helper T cell clones

A recently established thymic stroma-derived cell line (TSCL) supported the growth of the interleukin (IL) 2-dependent, antigen-specific helper T cell (Th) clone, 9-16, without requirement for IL-2 and antigen, and such growth was substituted by a factor produced into cultures by this established TSCL. This substance, thymic stroma-derived T cell-growth factor (TSTGF), was capable of inducing the proliferation of various Th clones including 9-16 Th clone, but not of cytotoxic T cell clones. TSTGF-induced growth promotion was obtained in a dose-dependent fashion and in maintaining antigen specificity of Th clones. The culture supernatant from the TSCL did not contain detectable level of IL-1, IL-2, IL-3, IL-4, or interferon activity. The proliferation of 9-16 Th clone was stimulated by recombinant IL-2 and IL-4 as well as TSTGF, but not by IL-1, IL-3, or interferons. However, the proliferation of this Th clone by IL-2 or IL-4 was almost completely inhibited by anti-IL-2 receptor or anti-IL-4 monoclonal antibody, respectively, whereas TSTGF-induced growth of 9-16 Th clones was not affected by either type of antibody, demonstrating that TSTGF is functionally distinct from IL-2 and IL-4. In addition, TSTGF activity was also obtained from the culture supernatant of the primary thymic explant, which was freshly prepared. These results indicate that the primary thymic explant as well as an established TSCL produce factors capable of promoting the growth of helper but not cytotoxic type of T cells in the absence of T cell growth factors thus far defined.     

Murine monoclonal antibodies to the myelin-associated glycoprotein (MAG) recognize Leu-7-reactive molecules on human mononuclear cells

It is known that the antibody to human myelin-associated glycoprotein (MAG) reacts with a subset of human mononuclear cells (MNC) mediating a natural killer (NK) activity. The properties of the target molecule of the anti-MAG antibody, however, have not yet been elucidated. Three (GC-J4, MC-P2, and MC-P4) of five murine monoclonal antibodies (mAb) to MAG bound to human MNC. Moreover, MC-P2 and MC-P4 inhibited the binding of 125I-labeled anti-Leu-7 to MNC in a dose-dependent fashion. Conversely, anti-Leu-7 inhibited the binding of MC-P2 and MC-P4 to MNC, but did not inhibit the binding of GC-J4. Therefore, it is possible that MC-P2 and MC-P4 bind directly to or close to the Leu-7 epitope, and that GC-J4 binds to the epitope which is distinct from the Leu-7 epitope. The electrophoretic patterns of immunoprecipitates with GC-J4, MC-P2 and anti-Leu-7 from detergent lysates of surface-labeled human MNC were very similar. The target molecules of anti-Leu-7 and anti-MAG mAb have apparent m.w. of 205, 170, 150, 135, 110, 85, 65, and 55 kDa. All of the molecules precipitated by these mAb are monomeric or noncovalently associated proteins, because the electrophoretic mobilities of the proteins remained unchanged whether the samples were reduced or not. MC-P4 may have a higher affinity for the 65 kDa molecule than the other mAb, and precipitates the 58 kDa molecule as well. Therefore, the fine antigenic specificity of MC-P4 is slightly different from those of anti-Leu-7 or MC-P2. The implication of these results is that mAb, whose specificity is directed to the carbohydrate part of human MAG, reacts with the Leu-7 reactive molecules on human MNC, and that at least two epitopes detected by anti-MAG mAb coexist on the surface molecules with various apparent m.w.     

Uncoupling of Rat Cerebral Cortical α2-Adrenoceptors from GTP-Binding Proteins by N-Ethylmaleimide

Pretreatment of membranes from rat cerebral cortex with N-ethylmaleimide (NEM) decreased [3H]-clonidine binding in a concentration-dependent manner. The Bmax values of high-affinity sites for [3H]clonidine were reduced by 50 microM NEM treatment. Treatment with 500 microM NEM diminished the sum of Bmax of both high- and low-affinity components. GTP, Na+, and Mn2+ exerted little effect on [3H]clonidine binding in NEM-treated membranes. The addition of purified GTP-binding proteins caused an increase in the binding to the membranes pretreated with 50 microM NEM, but did not increase [3H]-clonidine binding in membranes treated with 500 microM NEM. In contrast, NEM pretreatment inhibited islet activating protein (IAP)-catalyzed ADP ribosylation of membrane-bound (41,000-dalton) and purified (39,000/41,000-dalton) GTP-binding proteins. From these results, it is suggested that two or three categories of essential sulfhydryl groups are involved in the coupling between agonist, alpha 2-adrenoceptor, and GTP-binding protein. One is a highly sensitive site to NEM (a concentration range of 1-50 microM), which is probably a cysteine residue, IAP-catalyzed ADP-ribosylating site on the alpha-subunit of GTP-binding protein. Other sites have low sensitivity to NEM (a concentration range of 0.1-1 mM), and are the binding domain of agonist and/or the coupling domain of GTP-binding protein on the alpha 2-adrenoceptor. In addition, Ki-ras p21 protein may lack the capacity to couple with the alpha 2-adrenoceptor.     

Optimum conditions for electric pulse-mediated gene transfer to mammalian cells in suspension

A pulse-generating machine which delivers exponentially decaying pulses over broad range of pulse lengths was used to determine the optimum pulse conditions for gene transfer to FM3A cells. In the transformation of tk- cells with pTK1, a single pulse of 100-2000 microseconds gave a high transformation frequency at 1.5-6 kV/cm and room temperature, the highest transformation frequency obtained being 3 X 10(-3). As the suspension buffer for cells exposed to the pulse, Saline G was better than PBS(-) for obtaining a large number of transformants because it ensured high cell viability.     

Depolarization induced by injection of cyclic nucleotides into frog taste cell

In order to identify the intracellular transmitter involved in the taste transduction process, cyclic nucleotides were iontophoretically injected into the frog taste cells while membrane potentials were recorded intracellularly. Injection of either cyclic GMP or cyclic AMP induced a depolarization response of about 5 mV in the taste cells, but injection of Cl- had no effect. The rate of a repolarization after the depolarization elicited by cyclic GMP was larger than that after cyclic AMP. The possible role of cyclic nucleotide in the taste transduction was discussed.     

N-ethylmaleimide inhibits Ca2+ influx induced by collagen or arachidonate on rabbit platelets

N-Ethylmaleimide dose dependently inhibited platelet aggregation induced by collagen or arachidonate but did not inhibit the aggregation by thrombin or ionophore A23187 within the concentrations tested. [3H]Arachidonate release from membrane phospholipids of the collagen-stimulated platelets was inhibited by N-ethylmaleimide in parallel with the inhibition of aggregation, but not in response to A23187. N-Ethylmaleimide prevented 45Ca2+ influx into platelet cells from outer medium induced by collagen, and also inhibited the increase in the concentration of cytoplasmic free Ca2+, which probably results from Ca2+ influx, as monitored by quin2 fluorescence, under stimulation with arachidonate. The concentration of N-ethylmaleimide giving a complete inhibition of Ca2+ influx was consistent with that required to inhibit collagen- or arachidonate-induced aggregation. Prostaglandin metabolism from arachidonate to thromboxane A2 was not disturbed by N-ethylmaleimide, while phosphatidate formation induced by arachidonate was slightly inhibited by it at concentrations at which aggregation was completely inhibited. These data suggest that N-ethylmaleimide preferentially suppresses increase in cytoplasmic free Ca2+ which is linked to thromboxane A2-receptor occupation in collagen- or arachidonate-stimulated platelets, probably due to blockage of Ca2+ influx through Ca2+-channel protein, thereby inhibiting aggregation induced by these agonists.     

Effect of disuse on the ultrastructure of the achilles tendon in rats

We examined the influence exerted, through disuse of the hindlimb, on the collagen fibres of the achilles tendon in rats. With disuse the body mass decreased by 28%, and the mass of soleus muscle decreased by 20%. A decrease in the surface area and diameter was observed in the experimental group when compared to the control group. A histogram of the collagen fibres showed a decrease of the thick fibres in the experimental group. The maximum surface area of collagen fibres in the experimental group was seen to be only 43% of that of the control group. These results showed a decrease in the thickness of the collagen fibres of the achilles tendon through disuse. This seemed to suggest that resistance to tension is decreased by disuse.     

IS421, a new insertion sequence in Escherichia coli

The nucleotide sequence of a new insertion sequence (IS) in Escherichia coli, IS421, was determined. It is 1340 bp long and contains inverted repeats of 22 bp at its termini. It is flanked by 13 bp direct repeats apparently generated upon insertion. There are two ORFs longer than 200 bp in IS421. One can encode a polypeptide of 371 amino acids (aa) and the other, which is on the other strand, can encode a polypeptide of 102 aa. The C-terminal part of the 371 aa polypeptide shows some homology to that of transposases encoded in some other known IS elements. The copy number of IS421 in chromosomal DNA was 4 for E. coli K-12 and B, and 5 for E. coli C, as determined by the Southern hybridization of restriction fragments.     

Cell swelling induced by the permeant molecules urea or glycerol induces immediate high amplitude thyrotropin and prolactin secretion by perifused adenohypophyseal cells

The permeant molecules, urea and glycerol, evoked a prompt secretory burst of TSH and PRL when added to the extracellular medium of acutely dispersed anterior pituitary cells. Secretion of both hormones was proportional to the concentration of urea or glycerol between 26 and 104 mM (r greater than 0.89, P less than 0.001). Equivalent concentrations of the impermeant molecule, mannitol, did not induce secretion. The acute TSH and PRL secretory responses to TRH, hyposmolarity, and permeant molecules were qualitatively indistinguishable. These data support our hypothesis that cell swelling and resultant plasmalemma expansion is a potent inducer of hormone secretion. Since the secretory response to permeant molecules was not reduced in a Ca2+-free medium containing 0.1 mM EGTA, an increase in Ca2+ transport across the plasmalemma to raise cytosol Ca2+ concentration does not appear involved.