Biomedical subjects

W Gu

Publications and source records attributed to W Gu.

At least 127 records · Page 7Linked to original sources

PubMed1994

Thermostable variants of bovine beta-lactoglobulin.

The thermal stability of bovine beta-lactoglobulin (BLG) has been enhanced by the introduction of an additional disulfide bond. Wild-type BLG has two disulfide bonds, C106-C119 and C66-C160, with a free cysteine at position 121. We have designed, with the aid of molecular modeling calculations, two mutants of a recombinant BLG (rBLG), L104C and A132C. Molecular dynamics simulations were performed at 300K to study the effect of these alterations on the conformation of the protein. These mutants were then created by site-directed mutagenesis and purified from Escherichia coli carrying a tac expression vector using a two-step renaturation method. Formation of disulfide linkages in the correct arrangement, as designed, was confirmed by peptide mapping. In contrast to wild-type rBLG, which polymerizes at temperatures > 65 degrees C, neither of the mutant proteins polymerized. The conformational stability of the L104C and A132C mutant proteins against thermal denaturation has been substantially increased (8-10 degrees C) as compared with wild-type rBLG. Furthermore, the A132C rBLG exhibits an enhanced stability against denaturation by guanidine hydrochloride as compared with the wild-type or L104C rBLG.

Amino Acid Sequence↗

PubMed1994

Inactivation of tumor suppressor genes, p53 and Rb1, in plasma cell dyscrasias.

The role of loss or inactivation of the retinoblastoma (Rb1) and p53 tumor suppressor genes in the pathogenesis of various human malignancies has been well established, yet little is known regarding plasma cell dyscrasias. In the present study, the loss of Rb1 protein expression, and the presence of Rb1 gene rearrangements as well as the presence of p53 somatic mutations (exons 5 through 9) were investigated in a panel of plasma cell dyscrasias, including 15 monoclonal gammopathies of undetermined significance (MGUS), 63 multiple myelomas (MM), and 18 plasma cell leukemias (PCL). In the same panel of cases, we established the frequency of ras oncogene mutations, the main genetic lesion associated with MM. We report that loss of Rb1 protein and p53 mutations are detectable in 34.7 and 9.8% of MM and PCL primary cases; no lesion was found in MGUS. In advanced stage MM, and PCL cases, Rb1 and p53 inactivation, as well as ras mutations were detected. Our findings show that Rb1 and p53 inactivation are associated with aggressive plasma cell dyscrasias, suggesting a role for these lesions in tumor progression rather than initiation.

Base Sequence↗

PubMed1993

Nascent RNA cleavage by arrested RNA polymerase II does not require upstream translocation of the elongation complex on DNA.

Obstacles incurred by RNA polymerase II during primary transcript synthesis have been identified in vivo and in vitro. Transcription past these impediments requires SII, an RNA polymerase II-binding protein. SII also activates a nuclease in arrested elongation complexes and this nascent RNA shortening precedes transcriptional readthrough. Here we show that in the presence of SII and nucleotides, transcript cleavage is detected during SII-dependent elongation but not during SII-independent transcription. Thus, under typical transcription conditions, SII is necessary but insufficient to activate RNA cleavage. RNA cleavage could serve to move RNA polymerase II away from the transcriptional impediment and/or permit RNA polymerase II multiple attempts at RNA elongation. By mapping the positions of the 3'-ends of RNAs and the elongation complex on DNA, we demonstrate that upstream movement of RNA polymerase II is not required for limited RNA shortening (seven to nine nucleotides) and reactivation of an arrested complex. Arrested complexes become elongation competent after removal of no more than nine nucleotides from the nascent RNA's 3'-end. Further cleavage of nascent RNA, however, does result in "backward" translocation of the enzyme. We also show that one round of RNA cleavage is insufficient for full readthrough at an arrest site, consistent with a previously suggested mechanism of SII action.

Animals↗

PubMed1993

Opposite regulation of gene transcription and cell proliferation by c-Myc and Max.

c-Myc and Max are nuclear phosphoproteins capable of forming DNA-binding, homo- and heteropolymeric complexes in vitro and in vivo. Using a transient cotransfection assay involving c-Myc and Max expression vectors and a reporter gene plasmid containing the Myc/Max binding site, we find that Max represses transcription, whereas a significant stimulation is obtained when Max is coexpressed with c-Myc. Analysis of specific mutants indicates that transcriptional activation requires both the c-Myc and the Max dimerization and DNA-binding domains, as well as the c-Myc transactivation function; transcriptional repression by Max requires both DNA binding and dimerization. Analogously, in stably transfected human B-lymphoblastoid cell lines, overexpressed c-Myc and Max synergize to cause malignant transformation, whereas overexpression of Max alone leads to growth inhibition. These results indicate that the c-Myc and Max are transcriptional regulators with the ability to oppositely regulate target-gene expression and cell proliferation, most likely as the result of the opposite effects of heterodimeric c-Myc-Max (positive) versus homodimeric Max (negative) complexes.

Animals↗

PubMed1993

Interaction of myogenic factors and the retinoblastoma protein mediates muscle cell commitment and differentiation.

The experiments reported here document that the tumor suppressor retinoblastoma protein (pRB) plays an important role in the production and maintenance of the terminally differentiated phenotype of muscle cells. We show that pRB inactivation, through either phosphorylation, binding to T antigen, or genetic alteration, inhibits myogenesis. Moreover, inactivation of pRB in terminally differentiated cells allows them to reenter the cell cycle. In addition to its involvement in the myogenic activities of MyoD, pRB is also required for the cell growth-inhibitory activity of this myogenic factor. We also show that pRB and MyoD directly bind to each other, both in vivo and in vitro, through a region that involves the pocket and the basic-helix-loop-helix domains, respectively. All the results obtained are consistent with the proposal that the effects of MyoD on the cell cycle and of pRB on the myogenic pathway result from the direct binding of the two molecules.

Antigens, Polyomavirus Transforming↗

PubMed1993

[Research on anti-idiotypic antibodies to bovine abortus].

Based on the establishment of McAbs to a local Brucella strain S85A, the McAb A7 was selected and tested as a protective one. Using A7 to immunize rabbits, we got high titer anti-idiotypic antibodies which were then proved to possess internal image by competitive inhibiting ELISA and catch blocking ELISA. The anti-idiotypic antibodies were then purified and mixed with glycerin to immunize guinea pigs and mice. The results showed that: immunized guinea pigs and mice had produced certain titer antibodies to Brucella. 79.2% of immunized guinea pigs had been protected from the challenge of 544A 4 months later from primary immunization. The number of T ANAE+ in peripheral blood of immunized mice had increased significantly compared to controls. These results suggest that the anti-idiotypic antibodies not only possess the antigen internal image but have the available immunogenicity stimulate animals producing Ab3 and make them get free from the infection.

Animals↗

PubMed1993

Transcription elongation by RNA polymerase II: mechanism of SII activation.

RNA chain elongation by RNA polymerase is a dynamic process. Techniques that allow the isolation of active elongation complexes have enabled investigators to describe individual steps in the polymerization of RNA chains. This article will describe recent studies of elongation by RNA polymerase II (pol II). At least four types of blockage to chain elongation can be overcome by elongation factor SII: (a) naturally occurring "arrest" sequences, (b) DNA-bound protein, (c) drugs bound in the DNA minor groove, and (d) chain-terminating substrates incorporated into the RNA chain. SII binds to RNA polymerase II and stimulates a ribonuclease activity that shortens nascent transcripts from their 3' ends. This RNA cleavage is required for chain elongation from some template positions. As a result, the pol II elongation complex can repeatedly shorten and reextend the nascent RNA chain in a process we refer to as cleavage-resynthesis. Hence, assembly of large RNAs does not necessarily proceed in a direct manner. The ability to shorten and reextend nascent RNAs means that a transcription impediment through which only half the enzyme molecules can proceed per encounter, can be overcome by 99% of the molecules after six iterations of cleavage-resynthesis. Surprisingly, the boundaries of the elongation complex do not move upstream after RNA cleavage. The physico-chemical alterations in the elongation complex that accompany RNA cleavage and permit renewed chain elongation are not yet understood.

Animals↗

PubMed1992

A protective monoclonal antibody specifically recognizes and alters the catalytic activity of schistosome triose-phosphate isomerase.

mAb M.1 was previously shown to recognize a 28-kDa Ag in all stages of the human helminth parasite, Schistosoma mansoni, and to bind to the surface membranes of newly transformed schistosomula in a transient manner. Here we demonstrate that M.1 passively transfers partial resistance (41-49%) to cercarial challenge in naive mice. Thus, the 28-kDa Ag recognized by M.1 is a putative vaccine candidate. After immunoaffinity purification, tryptic digests of the 28-kDa Ag were prepared and individual peptides were sequenced. Amino terminus sequences of tryptic peptides of the 28-kDa Ag had high (79-87%) sequence homology with the mammalian glycolytic/gluconeogenic enzyme triosephosphate isomerase (TPI). Purified, native 28-kDa Ag from adult parasites was shown to function enzymatically in an analogous manner to yeast and mammalian TPI in the reverse reaction. Addition of M.1 antibody to the enzyme reaction altered the catalytic activity of schistosome TPI. To determine the immunologic cross-reactivity of this vaccine candidate with mammalian TPI, Western blot analysis was performed and demonstrated that M.1 was immunologically specific for the schistosome enzyme.

Amino Acid Sequence↗

PubMed1992

Dependence of reaction rate of 5,5'-dithiobis-(2-nitrobenzoic acid) to free sulfhydryl groups of bovine serum albumin and ovalbumin on the protein conformations.

The effect of protein conformations on the reaction rate of Ellman's reagent, 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB) with sulfhydryl (SH) groups of proteins was examined. The stopped-flow method was applied to follow the reaction of DTNB with SH group of two proteins, bovine serum albumin (BSA) and ovalbumin (OVA), at various concentrations of guanidine hydrochloride and urea. The rates for both the proteins were faster in guanidine than in urea. The rate sharply depended on the protein conformations, which were monitored by changes of helix contents on the basis of the circular dichroism measurements. The reaction rate of DTNB with SH groups of BSA was maximal around 2 M guanidine and 5 M urea. On the other hand, the reaction rate of DTNB with OVA was maximal at 3.5 M guanidine, while it gradually increased with an increase in the urea concentration. The amount of reactive SH group participating in the reaction with DTNB was also estimated by the absorbance change at 412 nm. The magnitudes of absorbance change for the reaction with free SH groups of OVA at low concentrations of the denaturants were appreciably smaller than those for BSA with one free SH group. Most of the four SH groups of OVA might react with DTNB above 5 M guanidine, although only a part of them did even at 9 M urea.

Dithionitrobenzoic Acid↗

PubMed1992

Molecular dynamics simulations of the whey protein beta-lactoglobulin.

Molecular dynamics simulations have been used to model the motions and conformational behavior of the whey protein bovine beta-lactoglobulin. Simulations were performed for the protein by itself and complexed to a single retinol ligand located in a putative interior binding pocket. In the absence of the retinol ligand, the backbone loops around the opening of this interior pocket shifted inward to partially close off this cavity, similar to the shifts observed in previously reported molecular dynamics simulations of the uncomplexed form of the homologous retinol binding protein. The protein complexed with retinol does not exhibit the same conformational shifts. Conformational changes of this type could serve as a recognition signal allowing in vivo discrimination between the free and retinol complexed forms of the beta-lactoglobulin molecule. The unusual bending of the single alpha-helix observed in the simulations of retinol binding protein were not observed in the present calculations.

Animals↗

PubMed1992

Role of nitrogen regulator I (NtrC), the transcriptional activator of glnA in enteric bacteria, in reducing expression of glnA during nitrogen-limited growth.

During nitrogen-limited growth, transcription of glnA, which codes for glutamine synthetase, requires sigma 54-RNA polymerase and the phosphorylated from the nitrogen regulator I (NRI; also called NtrC). In cells in which the lac promoter controlled expression of the gene coding for NRI, increasing the intracellular concentration of NRI lowered the level of glutamine synthetase. The reduction in glutamine synthetase does not appear to result from the NRI-dependent sequestering of any protein that affects transcription of glnA. Our results also suggest that the negative effect of a high concentration of NRI on glnA expression is a major determinant of the level of glutamine synthetase activity in nitrogen-limited cells of a wild-type strain. We propose that the inhibition results from an impairment of the interaction between NRI-phosphate and RNA polymerase that stimulates glnA transcription. We discuss a model that can account for this reduction in glutamine synthetase.

Bacteriophage lambda↗

PubMed1992

Lipopolysaccharide priming potentiates calcium ionophore stimulated human placental prostaglandin E2 release in vitro.

In this study, the effect of bacterial endotoxin (lipopolysaccharide; LPS) and of LPS priming on the in vitro release of PGE2 from human placental explants was investigated. Both LPS and the calcium ionophore A23187 significantly stimulated PGE2 release (P less than 0.05). Simultaneous exposure of placental explants to both LPS and A23187 revealed no additive or synergistic stimulation of PGE2 release. LPS priming of placental tissue significantly increased A23187-stimulated PGE2 release when compared to non-LPS-primed tissues. The addition of exogenous arachidonic acid (the substrate for PGE2 synthesis) also significantly (P less than 0.05) stimulated PGE2 release. There was, however, no significant further stimulation of PGE2 release following LPS priming in arachidonic acid treated explants. These data suggest that LPS not only increases basal PGE2 release in human placentae, but also potentiates agonist-stimulated PGE2 release, possibly by increasing tissue capacity for endogenous arachidonic acid liberation.

Calcimycin↗

PubMed1992

Identification of cis-acting DNA elements involved in the regulation of angiotensinogen gene expression.

Angiotensinogen is the precursor molecule of one of the most potent vasoactive substances, angiotensin-II. Angiotensinogen is normally synthesized in the liver and secreted into the plasma where it is converted into angiotensin-II by the combined proteolytic action of renin and angiotensin converting enzyme. Angiotensinogen levels in the plasma are modulated by a number of pathological and physiological factors. In order to understand the regulation of angiotensinogen gene expression, we have constructed an expression vector in which 688 bp of the 5'-flanking region of the rat angiotensinogen gene were attached to the chloramphenicol acetyl transferase (CAT) coding sequence. We have also obtained 5'-sequential deletion mutants from the rat angiotensinogen promoter attached to the CAT gene, and have identified multiple cis-acting DNA sequences involved in the regulation of angiotensinogen gene expression by transient transfection of these recombinant DNA molecules in human hepatoma cell lines, Hep3B, and HepG2.

Angiotensinogen↗

PubMed1991

Arachidonic acid metabolites in pregnant rat uterus.

Production of prostaglandin E2 (PGE2), F2 alpha (PGF2 alpha) and 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) by pregnant rat uterus were measured in vitro. At mid-pregnancy, myometrium incubated with decidua attached released more prostanoids into the culture medium than when incubated without. As pregnancy progressed to 21 days more prostanoids were detected in the culture medium. However, no significantly increased conversion of exogenous arachidonic acid (AA) by myometrium was found.

Animals↗

PubMed1991

The blood supply to the heart and brain in the growth retarded guinea pig fetus.

Blood flow to the heart and brain of 31 control and 15 growth retarded (IUGR) guinea pig fetuses was measured between 60-64 days of pregnancy by the microsphere technique. The animals were anaesthetized with diazepam and pentobarbitone. Brain weight was reduced by 11% in IUGR fetuses from 2.61 +/- 0.03 to 2.33 +/- 0.05 g and heart weight by 39% from 0.42 +/- 0.01 to 0.25 +/- 0.01 g, compared to a decrease in body weight of 42% from 83.6 +/- 2.3 to 48.2 +/- 2.2 g. The myocardial blood flow of control animals was negatively correlated to arterial O2 content (r = 0.78, P less than 0.001) and arterial pH (r = 0.68, P less than 0.001). Brain blood flow was inversely correlated to arterial O2 content in control fetuses (r = 0.79, P less than 0.001). Eight regions of the brain were examined: cerebral hemispheres, caudate nucleus, hippocampus, thalamus + hypothalamus, cerebellum, pons, and medulla. Regional blood flows were significantly correlated to fetal oxygenation in the controls. Growth retarded fetuses were characterized by poor oxygenation (arterial O2 content less than or equal to 2.5 mM) and were frequently acidaemic (pH less than 7.20). No relation could be demonstrated between the myocardial or cerebral blood flows of IUGR fetuses and arterial O2 content or pH. It is concluded that growth retarded fetuses are unable to maintain O2 delivery to the brain and myocardium by increases in blood flow. Although O2 extraction could be increased to meet the O2 requirements of the heart, IUGR fetuses had a lower rate pressure product, suggesting a decline in myocardial O2 consumption.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1990

Prostaglandin E2 and F2 alpha in mid-pregnant rat uterus and at parturition.

Prostaglandin E2 (PGE2) and F2 alpha (PGF2 alpha) produced by 15 days pregnant rat myometrium, by parturient rat myometrium and myometrium plus endometrium were measured in vitro. The results showed that the PGs produced by parturient myometrium were higher than these obtained during mid-pregnancy. Myometrium with endometrium released more PGs than myometrium alone, and the addition of arachidonic acid (AA) at 10 DM did not show any significant effect. Exogenous progesterone or estradiol-17b at a concentration of 1 Dmol had no effect on parturient uterine PG secretions.

Animals↗

PubMed1990

Effect of indomethacin on materno-fetal amino acid transfer in the dual-perfused human placental cotyledon.

Indomethacin (1, 10 and 100 mumol L-1) was incrementally infused (8 min for each concentration) into the maternal and fetal circulations of dual-perfused human placental lobules, and measurements were made of materno-fetal amino acid transfer (using the non-metabolizable amino acid alpha-aminoisobutyric acid, or AIB) and fetal effluent perfusate prostaglandin (PG) E levels. No significant changes in AIB transfer compared with controls were observed at any of the doses of indomethacin infused. There was, however, a significant dose-dependent reduction compared with controls in PGE in the fetal circulation with the infusion of indomethacin.

Amino Acids↗

PubMed1990

[Effect of blood-activating and stasis-removing drugs on hypercoagulability data in nephrotic syndrome].

UNLABELLED: Platelet aggregation test (PAgT), plasma factor VIII related antigen (VIII R:Ag) and kaolin partial thromboplastic time (KPTT) in 32 nephrotic children were determined. RESULTS: (1) PAgT induced by ADP and adrenaline which included 1 minute and 5 minute aggregation rate in the patients (30. 78 +/- 7.44%, 72.56 +/- 18.09%, 22.16 +/- 9.24%, 67.53 +/- 22.32%) was significantly increased (P less than 0.02-0.001) as compared with the normal control group (30.65 +/- 8.38%, 57.98 +/- 13.60%, 18.14 +/- 7.33%, 55.92 +/- 16.10%) except 1 minute aggregation rate induced by ADP (P greater than 0.05). (2) VIII R:Ag and KPTT in patients (226.97 +/- 50.47%, 32.04 +/- 5.88 sec) were obviously different (P less than 0.001, P less than 0.02) from the normal control group (107.11 +/- 24.55%, 35.42 +/- 5.00 sec). The results suggested that PAgT, VIII R:Ag and KPTT could be used as laboratory data reflecting the hypercoagulable state in nephrotic children. According to their age, sex and the clinical types of nephrotic syndrome, 24 nephrotic children with abnormality of PAgT, VIII R:Ag and KPTT were random divided into group 1 given prednisone only and group 2 given prednisone, blood-activating and stasis-removing drugs. RESULTS: the difference of PAgT, VIII R:Ag and KPTT in two groups between pre- and post-treatment was significant statistically (P less than 0.05-P less than 0.001). The decline in PAgT and VIII R:Ag was faster in group 2 than in group 1 (P less than 0.05-P less than 0.01). The results showed that blood-activating and stasis-removing drugs were likely to improve abnormal PAgT and VIII R:Ag in nephrotic children.

Child↗