Large scale purification of myo-inositol monophosphate phosphatase from porcine brains

myo-Inositol monophosphate phosphatase (IMPase) has been purified 888-fold to apparent homogeneity from procine brains. The purification procedure involves: homogenization, ammonium sulfate fractionation, and a number of ion-exchange and gel-filtration chromatography steps. The purified enzyme exhibited a final specific activity of 932 nmol . min(-1) . mg(-1). The molecular mass of the enzyme was estimated to be 29kDa by SDS poly-acrylamide gel electrophoresis and 58 +/- 5 kDa by HPLC gel filtration in 10mM Tris-HCI, pH 7.4. Kinetic measurements have shown that the apparent K(m) value of the phosphatase for the utilization of inositol-1-phosphate and beta-glycerol phosphate are 3.20 x 10(-4) and 8 x 10(-3) M, respectively. Similar to the same enzyme isolated from bovine brains, the porcine brain enzyme has been shown to be inhibited by lithium. The K(1) was determined to be 6.38 x 10(-4) M and the inhibition is uncompetitive. (c) 1995 John Wiley & Sons, Inc.     

Fluorophore-labeled carbohydrate analysis of immunoglobulin fusion proteins: Correlation of oligosaccharide content with in vivo clearance profile

CTLA4 is a membrane receptor on cytotoxic T cells whose interaction with the B7 counterreceptor on B cells is important in alloantigen responses. Soluble recombinant human and murine CTLA4 were produced using either Chinese hamster ovary or NS-0 cell lines. Expression vectors were constructed containing the gene coding for the extracellular domain of CTLA4 fused to either human lgG1 hinge, CH2, and CH3 domains or murine lgG2a hinge, CH2, and CH3 domain genes. These glycoproteins were produced in hollow-fiber or packed-bed-type bioreactors and purified from conditioned media by protein A affinity chromatography. Batches of purified CTLA4lg were analyzed for size, composition, and isoelectric point (pl) patterns by standard protein methods; oligosaccharide and monosaccharide profiles using several carbohydrate specific techniques; and in vivo clearance profiles using a murine model. Significant differences were observed between lots in their pl, clearance, and crbohydrate profiles. Higher overall pl values correlated with accelerated alpha-phase clearance and changes in oligosaccharide composition as determined by lectin binding analysis and electrophoresis of fluorophore-conjugated carbohydrates. Preparations exhibiting slower clearance profiles had oligosaccharides with higher quantities of N-acetylneuraminic acid and were predominantly of an N-linked biantennary complex-type. Conversely, batches with accelerated clearance profiles had less detectable N-acetylneuraminic acid. Oligosaccharides from murine CTLA4lg produced in NS-0 cells had terminal N-glycolylneuraminic acid but no detectable N-acetylneuraminic acid and had concomitant accelerated clearance. These data suggest that the presence and quantity of N-acetylneuraminic acid is an important component in predicting CTLA4lg plasma clearance rates and that production lots can be analyzed for oligosaccharide heterogeneity and sialic acid content by electrophoresis of fluorophore-conjugated carbohydrates. (c) 1995 John Wiley & Sons, Inc.     

Local positive feedback by calcium in the propagation of intracellular calcium waves.

In many types of eukaryotic cells, the activation of surface receptors leads to the production of inositol 1,4,5-trisphosphate and calcium release from intracellular stores. Calcium release can occur in complex spatial patterns, including waves of release that traverse the cytoplasm. Fluorescence video microscopy was used to view calcium waves in single mouse neuroblastoma cells. The propagation of calcium waves was slowed by buffers that bind calcium quickly, such as BAPTA, but not by a buffer with slower on-rate, EGTA. This shows that a key feedback event in wave propagation is rapid diffusion of calcium occurring locally on a scale of < 1 micron. The length-speed product of wavefronts was used to determine that calcium acting in feedback diffuses at nearly the rate expected for free diffusion in aqueous solution. In cytoplasm, which contains immobile Ca2+ buffers, this rate of diffusion occurs only in the first 0.2 ms after release, within 0.4 micron of a Ca2+ release channel mouth. Calcium diffusion from an open channel to neighboring release sites is, therefore, a rate-determining regenerative step in calcium wave propagation. The theoretical limitations of the wave front analysis are discussed.     

Differential requirement of protein tyrosine kinase and protein kinase C in the generation of IL-2-induced LAK cell and αCD3-induced CD3-AK cell responses

This study examined the role of protein tyrosine kinase (PTK) and protein kinase C (PKC) in the signal transduction pathways for lymphocyte activation through IL-2R to generate LAK cells and through TCR—CD3 to generate CD3-AK cells. Two PTK inhibitors [herbimycin A and genistein (PTK-I)] and two PKC inhibitors [calphositin C and staurosporine (PKC-I)] were used in the experiments. It was found that the primary activation pathway through IL-2R was PTK-dependent; that is, generation of both the IL-2-induced proliferative and the cytotoxic responses was completely abrogated by PTK-I and not by PKC-I. Quite different results were obtained with the αCD3-induced CD3-AK cell response. First, the αCD3-induced proliferation was only partially inhibited by PTK-I or PKC-I alone. Second, generation of CD3-AK cytotoxic response was primarily PKC-dependent; that is, only PKC-I induced significant inhibition. Genistein was found to reduce protein tyrosine phosphorylation in both LAK cells and CD3-AK cells, indicating that CD3-AK cells were also susceptible to PTK-I treatment. Further studies showed that PTK-I and not PKC-I suppressed perforin mRNA expression and N-2-benzyoxycarbonyl-l-lysine thiobeneylester esterase production in LAK cells, and the opposite was true for CD3-AK cells. These results indicate that different pathways were employed in lymphocyte activation through IL-2R and TCR—CD3. The former pathway is primarily PTK-dependent. Activation through TCR—CD3 is a more complex event. Induction of a proliferative response can employ either a PTK- or a PKC-dependent pathway, whereas induction of a cytotoxic response is primarily PKC-dependent. Furthermore, it appears that a PTK-independent pathway exists for the induction of a CD3-AK response and thus suggests that activation of the second messenger PKC may not necessarily be preceded by PTK activation.     

正常人各年龄组染色体着丝粒点(Cd)研究

本文运用本室改良的Cd-NOR银染技术对80例4个年龄组的正常中国人的Cd变化进行了较系统的研究, 结果表明:(1)正常人随年龄增加,Cd消失的频率、Cd变异及Cd-NOR融合频率也相应增加,特别是Ⅲ、Ⅳ组(中、老年组)增加的频率尤为显著;(2)首次对Cd消失的过程提出了独特的观点,即Cd消失首先表现为Cd变小, 随着变小程度的加大,最终导致Cd消失;(3)在本研究中首次观察到单个Cd的现象,作者认为是细胞分裂中期染色体着丝一分为二的延迟现象。各年龄组间单Cd出现频率无统计学差异,同一年龄组中,2号染色体和1号染色体上单Cd出现频率显著高于理论值;(4)随年龄增高,Cd各项观察值的增高在男性与女性间未见明显的差异。 Abstract:The Cd variation of human chromosome in four groups of different age has been investigated.The result shows that the frequencies of Cd disappearing,size variation and Cd-NOR fusion increased with the age rising,especially in the group of aged people.We suggest that the variation of Cd shows the size changes first,and then disappears completely.We also observed some cells in which a few chromosomes shows only a single Cd in centromeric region.Cd variation in different age groups has no significant difference between the male and the female.     

A STAT factor mediates the sexually dimorphic regulation of hepatic cytochrome P450 3A10/lithocholic acid 6 beta-hydroxylase gene expression by growth hormone.

Adult male rodents have a pulsatile profile of growth hormone (GH) release, whereas female rodents have a relatively steady-state pattern with uniform, albeit lower levels of GH. The expression of a number of sexually differentiated hepatic proteins is primarily determined by these plasma GH profiles and only secondarily regulated by gonadal hormones. An important subset of these sexually dimorphic proteins is cytochrome P450s. CYP3A10/6 beta-hydroxylase is a cytochrome P450 that catalyzes the 6 beta-hydroxylation of lithocholic acid. CYP3A10/6 beta-hydroxylase is expressed only in male hamsters; however, mimicking the male GH secretion pattern in females induces expression of the gene to male levels. Using chimeric CYP3A10/6 beta-hydroxylase promoter/luciferase reporter genes transfected into hamster primary hepatocytes, we have shown a GH-mediated induction of promoter activity. A combination of 5'-deletion constructs, heterologous promoter constructs, and specific mutagenesis was used to localize the DNA element involved in the GH-mediated regulation of CYP3A10/6 beta-hydroxylase promoter activity, which resembles a STAT binding site. Footprint and gel shift analyses confirmed that the expression of the protein binding to this site is regulated by GH and that the DNA-protein complex can be partially supershifted by anti-STAT-5 antibodies. This protein is 50% more abundant in male than in female hamster livers, is absent in hypophysectomized female livers, and is restored when hypophysectomized females are injected with GH in a manner that masculinizes female hamsters in terms of CYP3A10/6 beta-hydroxylase expression. The system characterized and described here is ideally suited for dissecting the molecular details governing the sexually dimorphic expression of liver-specific genes.     

Physicochemical properties of alkaline serine proteases in alcohol

The alkaline proteases subtilisin Carlsberg and alcalase possess substantial enzymatic activity even when dissolved in ethanol. The crude enzymes were purified by gel filtration and the main fractions suspended in ethanol to give a translucent suspension. Both the supernatant and the resuspended precipitate after high-speed centrifugation were found to have enzymatic activities. The solubility of subtilisin Carlsberg in anhydrous ethanol was found to be 45.1g/ml and that of alcalase was 48.1g/ml by Coomassie blue dye-binding method using bovine serum albumin as a standard. In the presence of water, the solubility of both enzymes increased with water content. The stability of enzymes incubated in ethanol was assayed by their amidase and transesterase activities using Ala-Ala-Pro-Phe-pNA as substrate in phosphate buffer (pH8.2) and Moz-Leu-OBzl as substrate in anhydrous ethanol, respectively. The soluble enzymes have a half-life of about 36 hr and that of suspended enzymes about 50 hr in the amidase activity assay, whereas the same soluble enzymes have a half-life of about several hours and that of suspended enzymes 1 h by the transesterase activity assay. The stability of both enzymes decreased as water concentration increased. The diastereoselectivity of the enzyme-catalyzed hydrolysis of diastereo pairs of tetrapeptide esters,l-Ala-l-Ala-(d-orl-)Pro-l-Phe-OMe andl-Ala-l-Ala-(d-orl-)Ala-l-Phe-OMe, in phosphate is as high as that of the transesterification of these substrates in ethanol. It is concluded that active sites and selectivity of alkaline serine proteases in anhydrous alcohol are probably very similar to those in aqueous solution in spite of the fact that a lower reactivity is usually associated with the enzymes in nonaqueous solvents.     

Unique and selective mitogenic effects of vanadate on SV40-transformed cells

Vanadate and insulin both function as unique complete mitogens for SV40-transformed 3T3T cells, designated CSV3-1, but not for nontransformed 3T3T cells. The mitogenic effects induced by vanadate and insulin in CSV3-1 cells are mediated by different signaling mechanisms. For example, vanadate does not stimulate the tyrosine phosphorylation of the insulin receptor -subunit nor the 170 kDa insulin receptor substrate-1. Instead, vanadate induces a marked increase in tyrosine phosphorylation of 55 and 64 kDa proteins that is not observed in insulin-stimulated CSV3-1 cells. Perhaps most interestingly, vanadate-induced mitogenesis is associated with the selective induction ofc-jun andjunB expression without significantly inducingc-fos orc-myc. Furthermore, treatment of CSV3-1 cells with genistein abolishes the effects of vanadate on protein tyrosine phosphorylation andc-jun induction. These and related data suggest that modulation of protein tyrosine phosphorylation andc-jun andjunB expression may serve the critical roles in mediating vanadate-induced mitogenesis in SV40-transformed cells.     

A simple method to quantitatively measure polypeptide JHNHα coupling constants from TOCSY or NOESY spectra

A simple linear relationship between the J coupling constant and the linewidth (_1/2) of in-phase NMR peaks has been identified. This relationship permits the rapid and accurate determination of polypeptide J coupling constants from a simple inspection of amide cross peaks in homonuclear ¹H TOCSY or ¹H NOESY spectra. By using the appropriate set of processing parameters we show that J = 0.5(_1/2) – MW/5000 + 1.8 for TOCSY spectra and J = 0.6(_1/2) – MW/5000 – 0.9 for NOESY spectra, where _1/2 is the half-height linewidth in Hz and MW is the molecular weight of the protein in Da. The simplicity of this relationship, combined with the ease with which _1/2 measurements can be made, means that J coupling constants can now be rapidly determined (up to 100 measurements in less than 30 min) without the need for any complex curve-fitting algorithms. Tests on 11 different polypeptides involving more than 650 separate J measurements have shown that this method yields coupling constants with an rmsd error (relative to X-ray data) of less than 0.9 Hz. Furthermore, the correlation coefficient between the predicted NMR coupling constants and those derived from high-resolution X-ray crystal structures is typically better than 0.89. These simple linear relationships have been found to be valid for peptides as small as 1 kDa to proteins as large as 20 kDa. Despite the method's simplicity, these results are comparable to the accuracy and precision of the best techniques published to date.