Purification and characterization of 3-hydroxymethylglutaryl-coenzyme A reductase of Schistosoma mansoni: regulation of parasite enzyme activity differs from mammalian host

The enzyme 3-hydroxymethylglutaryl-CoA (HMG-CoA) reductase plays a critical role in regulating the production of cholesterol, dolichols, and ubiquinones in mammals. The inhibition of this enzyme in Schistosoma mansoni is accompanied by a cessation of egg production by the female parasite and a reduced ability of the parasite to properly glycoslyate their proteins. Furthermore, we recently demonstrated that mevinolin, if given continuously over a period of 10-14 days, is a potent antischistosomal drug. In this paper, we describe the properties of purified HMG-CoA reductase from S. mansoni. Using affinity chromatography, we were able to obtain a 417-fold purification of the enzyme which had Km values similar to the rat enzyme for HMG-CoA and NADPH. The Ki value for mevinolin, a potent and selective inhibitor of the rat reductase (Ki = 0.6 nM), was significantly higher (Ki = 46 nM) for the schistosome enzyme. SDS-PAGE and HPLC of the purified enzyme resulted in the appearance of a single protein, which had a molecular weight (66,000) in the range reported for the rat enzyme. Parasite reductase activity, unlike that of its host, did not display a circadian rhythm. Furthermore, agents which elevate (cholestyramine) or decrease (cholesterol) mammalian reductase activity had no effect on the parasite enzyme. Our results suggest that the mechanism which regulates production of the parasite's enzyme may differ from its mammalian host.     

与胎儿珠蛋白基因持续表达有关的(A_γδβ)°—地贫纯合子及杂合子的分子鉴别

我们分子鉴别了一个缺失型中国(A_γδβ)°-地贫家系。先证者为这一缺失的纯合子,具有中度贫血症状。家系的另五个成员均为这一缺失的杂合子,其胎儿血红蛋白(HbF)为16—21％,接近或达到HPFH杂合子的HbF水平,并且几乎不表现贫血症状。限制性内切酶图谱分析证明了β-珠蛋白基因簇内的DNA顺序缺失,缺失的5′端点位于Aγ基因IVSⅡ内,3′端点在β-珠蛋白基因下游区远端,距HPFH-2的3′缺失端点上游区约11kb。缺失的总长度约为80kb。本文讨论了这一缺失导致胎儿血红蛋白在成人中持续活跃表达的可能机制。     

Delayed removal of 125I-labeled very low density lipoprotein from plasma of rats with insulin-deficient diabetes mellitus.

The present studies demonstrate that the removal rate of exogenously labelled 125I-VLDL-protein is prolonged when total serum from insulin-deficient rats combined with isolated 125I-VLDL is injected into normal recipient rats (6.8 +/- 0.7 vs 4.2 +/- 0.4 min; p < 0.01), but not when 125I-VLDL-protein is isolated and injected alone (4.2 +/- 0.8 vs 4.3 +/- 0.8 min). Furthermore, the present studies demonstrate that when isolated 125I-VLDL-protein is recombined with either VLDL-free (d > 1.006 g/ml), or lipoprotein-free serum (d > 1.215 g/ml) from insulin-deficient rats, the defect in removal rate of VLDL-protein observed in total serum is reestablished (125I-VLDL + VLDL-free serum from insulin-deficient rat vs that from normal rat: 7.6 +/- 1.2 vs 4.6 +/- 0.7 min, p < 0.05; and 125I-VLDL + lipoprotein-free serum from insulin-deficient rat vs that from normal rat: 6.4 +/- 0.7 vs 4.1 +/- 0.4 min, p < 0.01). These data suggest that a factor or factors exist in lipoprotein-free serum of insulin-deficient rats which interfere with the normal removal of 125I-VLDL. Since we have previously demonstrated a prolongation in the removal rate of endogenously labeled VLDL-3H-TG, the defect in removal of VLDL from the plasma of insulin-deficient rats appears to include both the lipid and protein moieties of the VLDL particles.     

A molecular thermodynamic approach to predict the secondary structure of homopolypeptides in aqueous systems.

Under physiological conditions, many polypeptide chains spontaneously fold into discrete and tightly packed three-dimensional structures. The folded polypeptide chain conformation is believed to represent a minimum Gibbs energy of the system, governed by the weak interactions that operate between the amino acid residues and between the residues and the solvent. A semiempirical molecular thermodynamic model is proposed to represent the Gibbs energy of folding of aqueous homopolypeptide systems. The model takes into consideration both the entropy contribution and the enthalpy contribution of folding homopolypeptide chains in aqueous solutions. The entropy contribution is derived from the Flory-Huggins expression for the entropy of mixing. It accounts for the entropy loss in folding a random-coiled polypeptide chain into a specific polypeptide conformation. The enthalpy contribution is derived from a molecular segment-based Non-Random Two Liquid (NRTL) local composition model [H. Renon and J. M. Prausnitz (1968) AIChE J., Vol. 14, pp. 135-142; C.-C. Chen and L. B. Evans (1986) AIChE J., Vol. 32, pp. 444-454], which takes into consideration of the residue-residue, residue-solvent, and solvent-solvent binary physical interactions along with the local compositions of amino acid residues in aqueous homopolypeptides. The UNIFAC group contribution method [A. Fredenslund, R. L. Jones, and J. M. Prausnitz (1975) AIChE J., 21, 1086-1099; A. Fredenslund, J. Gmehling, and P. Rasmussen (1977) Vapor-Liquid Equilibrium Using UNIFAC, Elsevier Scientific Publishing Company, Amsterdam], developed originally to estimate the excess Gibbs energy of solutions of small molecules, was used to estimate the NRTL binary interaction parameters. The model yields a hydrophobicity scale for the 20 amino acid side chains, which compares favorably with established scales [Y. Nozaki and C. Tanford (1971) Journal of Biological Chemistry, Vol. 46, pp. 2211-2217; E. B. Leodidis and T. A. Hatton (1990) Journal of Physical Chemistry, Vol. 94, pp. 6411-6420]. In addition, the model generates qualitatively correct thermodynamic constants and it accurately predicts thermodynamically favorable folding of a number of aqueous homopolypeptides from random-coiled states into alpha-helices. The model further facilitates estimation of the Zimm-Bragg helix growth parameter s and the nucleation parameter sigma for amino acid residues [B. H. Zimm and J. K. Bragg (1959) Journal of Chemical Physics, Vol. 31, pp. 526-535]. The calculated values of the two parameters fall into the ranges suggested by Zimm and Bragg.     

Up-regulation of granulocyte-macrophage colony-stimulating factor (GM-CSF) receptors in murine peritoneal exudate macrophages by both GM-CSF and IL-3.

Murine peritoneal exudate macrophages (PEM) display multiple CSF receptors. In this study, the expression of granulocyte-macrophage (GM)-CSF receptors in PEM was studied. PEM displayed over 5000 single type, high affinity GM-CSF receptors/cell with a Kd = 38 to 42 pM and an apparent molecular mass of 86,000 Da. Treatment of PEM with low, but not high, concentrations of recombinant murine (rMu) GM-CSF continuously for 24 h resulted in a marked up-regulation of GM-CSF receptors in PEM. A similar up-regulation of GM-CSF receptors also was detected in PEM cultures treated with rMuIL-3 (1-100 ng/ml) for 24 h or longer, regardless the doses of rMuIL-3 added in this case. Scatchard analysis of equilibrium binding showed that the enhanced binding activities in both cases were due to an increase in total number of GM-CSF receptors rather than changes in receptor affinity. Contrariwise, treatment with recombinant human macrophage-CSF (greater than 100-1000 ng/ml) partially inhibited the expression of GM-CSF receptors in PEM. Removal of rMuGM-CSF from culture medium 24 h after treatment led to a further up-regulation of GM-CSF receptors over a 4 to 24-h period, depending on the doses of initial treatment. On the other hand, removal of rMuIL-3 from culture medium after prolonged treatment did not result in further increase in GM-CSF receptors. The protein synthesis inhibitor cycloheximide abrogated GM-CSF receptor up-regulation induced by both rMuIL-3 and rMuGM-CSF, whereas actinomycin D inhibited only the second (8-24 h) phase of GM-CSF receptor up-regulation induced by exposure to high concentrations rMuGM-CSF (10 ng/ml). These findings suggest that rMuGM-CSF and rMuIL-3 up-regulate GM-CSF receptors in PEM in part through similar or identical metabolic pathways and provide further evidence of a close linkage between IL-3 and GM-CSF receptors.     

Abscisic Acid increases terrestrial plant cell resistance to hydrostatic pressure

Cells of the terrestrial plant species bromegrass (Bromus inermis L.) are not naturally adapted to withstand the hydrostatic pressures encountered in aquatic environments. However, after treatment with the natural plant growth hormone abscisic acid (75 micromolar), bromegrass cells survived a hydrostatic pressure of 101.3 megapascals, approximating the limits of ocean depth (10,860 m). The increased resistance to hydrostatic pressure from 1 to 7 days of abscisic acid treatment paralleled the induced elevation of cell tolerance to freezing stress.     

Quantitative immunoassay of total cellular GAP junction protein connexin32 during liver regeneration using antibodies specific to the COOH-terminus.

A radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA) were used to determine relative concentrations of liver connexin32 (CX32) in rats. The RIA and ELISA utilize synthetic peptides corresponding to regions of the carboxyl-terminus and antibodies raised in rabbits against these peptides. Assuming that affinities of antisera are similar for peptide and native CX32, total cellular CX32 was found to exceed the amount of gap junction protein at the cell surface calculated from morphometric analyses by 1.5-2.0 fold. This finding raises the possibility that some of the protein is present in cytoplasmic compartments or as occult precursors in the plasma membrane. Studies of CX32 content in regenerating rat liver support this conclusion and show a time course of loss and recovery of CX32 that agrees with those reported in studies using other techniques.     

Enzymatic sulfation of gastrin in rat gastric mucosa

An enzyme which catalyzes the transfer of sulfate from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to gastrin (G17) was identified in rat gastric mucosal cells. The enzyme activity was detected in the 105,000xg supernatant fraction. Formation of gastrin sulfate was shown by using 125I-gastrin and non-radioactive PAPS. The product was sensitive to acid hydrolysis, arylsulfatase treatment and removed by gastrin antibody, but not changed by treatments with chondro-4-sulfatase and chondro-6-sulfatase. The product had a molecular weight of 2050 daltons, close to the molecular weight of G17 sulfate, and, therefore, indicating the sulfated product is not APS derived from the degradation of PAPS. The enzyme activity showed a Km value of 5 microM for PAPS and a pH optimum of 6.0. The activity was not detected in the liver preparation.