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Biomedical subjects

J Zhu

Publications and source records attributed to J Zhu.

At least 577 records · Page 32Linked to original sources
PubMed1996

Three-dimensional reconstruction of the Escherichia coli 30 S ribosomal subunit in ice.

Three-dimensional (3D) reconstructions of both the heat-activated and non-activated 30 S subunit of the Escherichia coli 70 S ribosome were obtained from a frozen hydrated specimen preparation at 1/37 A-1 resolution. Well-characterized features that can be identified in both reconstructions are the head, the base, the platform and the cleft formed between the head and the platform. Comparisons between the 3D maps of 30 S subunits at 0 degree C, heat-activated at 37 degrees C, and the 30 S subunit portion identified in the cryo-3D map of 70 S ribosome reveal conformational changes the subunit probably undergoes during inactive-active transition and upon association with the 50 S subunit. These comparisons also allow us to localize the sites of association of 30 S and 50 S subunits.

Escherichia coli↗
PubMed1996

Chronic nicotine treatment delays the developmental increase in brain muscarinic receptors in rat neonate.

Developmental increase in the muscarinic receptors ([3H]quinuclidinyl benzylate binding sites) of rat neonate brain (cerebral cortex and cerebellum) was significantly inhibited by chronic nicotine treatment of the dams during pregnancy and lactation. However, development of the nicotinic receptors ([3H]cytisine binding sites) was not inhibited and rather was up-regulated in the cerebral cortex and brainstem by the nicotine treatment. Such inhibition and up-regulation were not seen in nicotine-withdrawn rats after birth. These results suggest that nicotine treatment during lactation may cause a remarkable delay in development of muscarinic neurotransmission in rat neonates.

Aging↗
PubMed1996

Insulin signaling in mice expressing reduced levels of Syp.

Syp is a protein tyrosine phosphatase implicated in insulin and growth factor signaling. To evaluate the role of syp in insulin's regulation of plasma glucose, we generated knockout mice. Homozygous knockout mice die prior to day 10.5 of embryonic development. Hemizygous mice express half the levels of syp protein compared with their wild type littermates but do not display any gross morphological changes. Total body weight (age 2-10 weeks) and plasma insulin and glucose levels both in fasting and glucose-challenged states were comparable in the wild type and the hemizygous mice. No differences were observed in insulin-induced glucose uptake in soleus muscle and epididymal fat; insulin inhibition of lipolysis was also similar. We injected insulin into the portal vein of the mice to examine upstream events of the insulin signaling cascade. Tyrosine phosphorylation of insulin receptor and insulin receptor substrate-1 (IRS-1) from hemizygous tissue was similar to that of wild type tissue. Association of the p85 subunit of phosphatidylinositol 3-kinase to IRS-1 increased an average of 2-fold in both groups. We did not observe an increase of IRS-1/syp association after insulin administration, but we did note a significant basal association in both wild type and hemizygous tissue. Our results do not support a major role for syp in the acute in vivo metabolic actions of insulin.

Animals↗
PubMed1996

Determination of the amino acid residue involved in [3H]beta-funaltrexamine covalent binding in the cloned rat mu-opioid receptor.

We previously demonstrated that [3H]beta-funaltrexamine ([3H]beta-FNA) labeled the rat mu opioid receptor expressed in Chinese hamster ovary cells with high specificity, and [3H]beta-FNA-labeled receptors migrated as one broad band with a mass of 80 kDa. In this study, we determined the region and then the amino acid residue of the mu receptor involved in the covalent binding of [3H]beta-FNA. [3H]beta-FNA-labeled receptors were solubilized and purified to approximately 10% purity by immunoaffinity chromatography with antibodies against a C-terminal domain peptide. The site of covalent bond formation was determined to be within Ala206-Met243 by CNBr cleavage of partially purified labeled mu receptors and determinations of sizes of labeled receptor fragments. The amino acid residue of beta-FNA covalent incorporation was then determined by site-directed mutagenesis studies within this region. Mutation of Lys233 to Ala, Arg, His, and Leu completely eliminated covalent binding of [3H]beta-FNA, although these mutants bound beta-FNA with high affinity. Mutations of other amino acid residues did not affect covalent binding of [3H]beta-FNA. These results indicate that [3H]beta-FNA binds covalently to Lys233. Since [3H]beta-FNA is a rigid molecule, the information will be very useful for molecular modeling of interaction between morphinans and the mu receptor.

Amino Acid Sequence↗
PubMed1996

In vitro studies on cellular and molecular mechanisms of arsenic trioxide (As2O3) in the treatment of acute promyelocytic leukemia: As2O3 induces NB4 cell apoptosis with downregulation of Bcl-2 expression and modulation of PML-RAR alpha/PML proteins.

It has been shown recently in China that arsenic trioxide (As2O3) is a very effective treatment for acute promyelocytic leukemia (APL). APL patients resistant to all-trans retinoic acid (ATRA) and conventional chemotherapy can still respond to AS2O3. In this study, we addressed the possible cellular and molecular mechanisms of this treatment by using NB4 cells as a model. The results show that: (1) As2O3 triggers relatively specific NB4 cell apoptosis at micromolar concentration, as proved by morphology, histogramic related nuclear DNA contents, and DNA gel eletrophoresis. (2) As2O3 does not influence bax, bcl-x, c-myc, and p53 gene expression, but downregulates bcl-2 gene expression at both mRNA and protein levels. (3) As2O3 induces a significant modulation of the PML staining pattern in NB4 cells and HL-60 cells. The micropunctates characteristic of PML-RAR alpha in NB4 cells dissappear after treatment with As2O3, whereas a diffuse PML staining occurs in the perinuclear cytoplasmic region. In addition, a low percentage of untreated NB4 cells exhibits an accumulation of PML positive particles in a compartment of cytoplasm. The percentage of these cells can be significantly increased after As2O3 treatment. A similar PML staining pattern is observed in apoptotic cells. (4) ATRA pretreatment does not influence As2O3-induced apoptosis. These results suggest that induction of cell apoptosis can be one of the mechanisms of the therapeutic effect of As2O3. Moreover, this apoptosis induction occurs independently of the retinoid pathway and may be mediated, at least partly, through the modulation of bcl-2, as well as PML-RAR alpha and/ or PML proteins.

Apoptosis↗
PubMed1996

Distinguishing antemortem from postmortem injuries by LTB4 quantification.

Leukotriene B4 (LTB4) in skin samples from seven forensic cases was detected by HPLC to distinguish their antemortem or postmortem origin. In total, there were thirteen antemortem and seven postmortem specimens. The results showed that LTB4 was found in all antemortem wound specimens which were either fresh, or refrigerated or fixed in formalin for less than 10 days. In contrast, LTB4 could not be detected in postmortem wound specimens. These results suggested that detecting the content of LTB4 is a useful method for distinguishing antemortem from postmortem injuries.

Adult↗
PubMed1996

ATP and SH3 binding sites in the protein kinase of the large subunit of herpes simplex virus type 2 of ribonucleotide reductase (ICP10).

The large subunit of herpes simplex virus type 2 ribonucleotide reductase (ICP10) is a multifunctional protein. It consists of a ribonucleotide reductase and a serine/threonine protein kinase (PK) domain, which has three proline-rich motifs consistent with SH3-binding sites at positions 140, 149, and 396. We used site-directed mutagenesis to identify amino acids required for kinase activity and interaction with signaling proteins. Mutation of Lys176 or Lys259 reduced PK activity (5-8-fold) and binding of the 14C-labeled ATP analog rho-fluorosulfonylbenzoyl 5'-adenosine (FSBA) but did not abrogate them. Enzymatic activity and FSBA binding were abrogated by mutation of both Lys residues, suggesting that either one can bind ATP. Mutation of Glu209 (PK catalytic motif III) virtually abrogated kinase activity in the presence of Mg2+ or Mn2+ ions, suggesting that Glu209 functions in ion-dependent PK activity. ICP10 bound the adaptor protein Grb2 in vitro. Mutation of the ICP10 proline-rich motifs at positions 396 and 149 reduced Grb2 binding 20- and 2-fold, respectively. Binding was abrogated by mutation of both motifs. Grb2 binding to wild type ICP10 was competed by a peptide for the Grb2 C-terminal SH3 motif, indicating that it involves the Grb2 C-terminal SH3.

Adaptor Proteins, Signal Transducing↗
PubMed1996

RIG-E, a human homolog of the murine Ly-6 family, is induced by retinoic acid during the differentiation of acute promyelocytic leukemia cell.

In vivo all-trans-retinoic acid (ATRA), a differentiation inducer, is capable of causing clinical remission in about 90% of patients with acute promyelocytic leukemia (APL). The molecular basis for the differentiation of APL cells after treatment with ATRA remains obscure and may involve genes other than the known retinoid nuclear transcription factors. We report here the ATRA-induced gene expression in a cell line (NB4) derived from a patient with APL. By differential display-PCR, we isolated and characterized a novel gene (RIG-E) whose expression is up-regulated by ATRA. The gene is 4.0 kb long, consisting of four exons and three introns, and is localized on human chromosome region 8q24. The deduced amino acid sequence predicts a cell surface protein containing 20 amino acids at the N-terminal end corresponding to a signal peptide and an extracellular sequence containing 111 amino acids. The RIG-E coded protein shares some homology with CD59 and with a number of growth factor receptors. It shares high sequence homology with the murine LY-6 multigene family, whose members are small cysteine-rich proteins differentially expressed in several hematopoietic cell lines and appear to function in signal transduction. It seems that so far RIG-E is the closest human homolog of the LY-6 family. Expression of RIG-E is not restricted to myeloid differentiation, because it is also present in thymocytes and in a number of other tissues at different levels.

Amino Acid Sequence↗
PubMed1996

Activated abl oncogenes and apoptosis: differing responses of transformed myeloid progenitor cell lines.

Activation of the c-abl protooncogene occurs during the generation of both the Abelson murine leukemia virus and the bcrabl fusion gene. To further dissect the biological properties of these proteins, we studied their effect on apoptosis. Using dimethyl sulfoxide (DMSO) to induce apoptosis in the murine myeloid progenitor cell line 32Dcl3, we examined the effect of expression of both v-abl and bcrabl transgenes on apoptosis. v-abl expressing 32Dcl3 cells are sensitive to apoptosis induction, similar to parental 32Dcl3 cells. In contrast, bcrabl expression 32Dcl3 cells are protected from the apoptotic stimulus resulting from DMSO exposure. Analyzing the expression patterns for Bcl-2 and Bax, two proteins known to modulate the apoptotic response, we found a downregulation of Bcl-2 and enhanced expression of Bax in 32Dcl3 cells. In 32Dcl3/v-Abl cells, Bcl-2 expression remained constant while Bax was upregulated, whereas in 32Dcl3 cells expressing bcrabl, there was continuous expression of Bcl-2 at a level greater than observed in v-abl transformed cells. Taken together, our data demonstrate that although both activated abl gene products promote overlapping effects of some biological responses (i.e., factor-independent proliferation) they diverge in their effect on apoptotic signaling pathways.

Animals↗
PubMed1996

Amino-terminal protein-protein interaction motif (POZ-domain) is responsible for activities of the promyelocytic leukemia zinc finger-retinoic acid receptor-alpha fusion protein.

Promyelocytic leukemia zinc finger-retinoic acid receptor a (PLZF-RARalpha), a fusion receptor generated as a result of a variant t(11;17) chromosomal translocation that occurs in a small subset of acute promyelocytic leukemia (APL) patients, has been shown to display a dominant-negative effect against the wild-type RARalpha/retinoid X receptor alpha (RXRalpha). We now show that its N-terminal region (called the POZ-domain), which mediates protein-protein interaction as well as specific nuclear localization of the wild-type PLZF and chimeric PLZF-RARalpha proteins, is primarily responsible for this activity. To further investigate the mechanisms of PLZF-RARalpha action, we have also studied its ligand-receptor, protein-protein, and protein-DNA interaction properties and compared them with those of the promyelocytic leukemia gene (PML)-RARalpha, which is expressed in the majority of APLs as a result of t(15;17) translocation. PLZF-RARalpha and PML-RARalpha have essentially the same ligand-binding affinities and can bind in vitro to retinoic acid response elements (RAREs) as homodimers or heterodimers with RXRalpha. PLZF-RARalpha homodimerization and heterodimerization with RXRalpha were primarily mediated by the POZ-domain and RARalpha sequence, respectively. Despite having identical RARalpha sequences, PLZF-RARalpha and PML-RARalpha homodimers recognized with different affinities distinct RAREs. Furthermore, PLZF-RARalpha could heterodimerize in vitro with the wild-type PLZF, suggesting that it may play a role in leukemogenesis by antagonizing actions of not only the retinoid receptors but also the wild-type PLZF and possibly other POZ-domain-containing regulators. These different protein-protein interactions and the target gene specificities of PLZF-RARalpha and PML-RARalpha may underlie, at least in part, the apparent resistance of APL with t(11;17) to differentiation effects of all-trans-retinoic acid.

Animals↗
PubMed1996

The region in the mu opioid receptor conferring selectivity for sufentanil over the delta receptor is different from that over the kappa receptor.

We determined the binding domains of sufentanil and lofentanil in the mu opioid receptor by comparing their binding affinities to seven mu/delta and six mu/kappa chimeric receptors with those to mu, delta and kappa opioid receptors. TMHs 6 and 7 and the e3 loop of the mu opioid receptor were important for selective binding of sufentanil and lofentanil to the mu over the kappa receptor. TMHs 1-3 and the e1 loop of the mu opioid receptor conferred binding selectivity for sufentanil over the delta receptor. Thus, the region that conferred binding selectivity for sufentanil differs, depending on chimeras used. In addition, the interaction TMHs 1-3 and TMHs 6-7 was crucial for the high affinity binding of these two ligands. These two regions are likely to contain sites of interaction with the ligands or to confer conformations specific to the mu receptor.

Animals↗
PubMed1996

Establishment of a human acute promyelocytic leukemia-ascites model in SCID mice.

Acute promyelocytic leukemia (APL) is an interesting model for cancer research because of the presence of the specific PML-RARalpha fusion gene associated with the clinical response to retinoic acid differentiation therapy. To better understand and improve differentiation induction with retinoic acid, we have established a human APL-ascites model in SCID mice using the NB4 human APL cell line. NB4 (1 x 10(6) cells) were transplanted into the peritoneum (IP) of SCID mice for 1 month. NB4 ascites cells (A-NB4) appeared, which were then engrafted in SCID mice periodically for 18 passages at an interval of 3 to 4 weeks with a 100% success rate of tumor induction. The mean survival times of SCID mice transplanted with 1 x 10(6) A-NB4 cells was 21.6 +/- 2.3 days. Analysis of the biologic characteristics of ninth passage NB4 ascitic cells was performed and they were found to have the morphologic, immunologic, cytogenetic, and molecular features of cultured NB4 cells. Furthermore, A-NB4 cells were capable of differentiating when treated with all-trans retinoic acid (ATRA), as manifested by enhanced NBT reduction and CD11b expression. In vivo treatment with ATRA in SCID mice for 4 days also increased NBT reduction by A-NB4 cells. ATRA treatment significantly prolonged survival time in the group after transplantation (28.1 +/- 6.8 to 29.1 +/- 8.4 days) compared with the control (P < .001). Furthermore, treatment with adriamycin, an effective chemotherapeutic drug in APL, had a strong growth suppressive effect on A-NB4 cells. These results demonstrate that this SCID-APL (NB4 ascites cells) model is a useful preclinical system for evaluating new or known drugs in the treatment of APL.

Animals↗
PubMed1996

In vitro transcription of the rat insulin-like growth factor-I gene.

Although the liver is the major source of circulating insulin-like growth factor-I (IGF-I), relatively little is known about the regulation of IGF-I gene transcription in this tissue. Since transcripts are initiated largely in exon 1, we established an in vitro transcription system to evaluate activation of transcription via the major exon 1 initiation site. Transcription of a G-free cassette reporter was directed by rat IGF-I genomic fragments, and the adenovirus major late promoter was used as an internal control. Tissue specificity was demonstrated by a 60-90% decrease in transcripts with spleen extracts as compared with liver. 54 base pairs (bp) of upstream sequence were sufficient to direct IGF-I gene transcription, and activity increased 5-fold with 300 bp of upstream sequence. DNase I footprinting revealed four protected regions between -300 and -60 bp; binding was confirmed by gel shift analysis, and tissue specificity was demonstrated by reduced shifts with spleen extracts. The necessity of transcription factor binding to such sites was established by competition analysis, which revealed a specific decrease in IGF-I transcription in the presence of a competing fragment. Use of this in vitro transcription system should permit analysis of the function of individual transcription factors involved in regulation of IGF-I gene expression.

Animals↗
PubMed1996

Interaction between the Grb10 SH2 domain and the insulin receptor carboxyl terminus.

Grb10 is a member of a recently identified family of adapter proteins that are thought to play a role in receptor tyrosine kinase-mediated signal transduction. We identified and isolated the Grb10 SH2 domain based on its interaction with the intracellular domain of the insulin receptor beta-subunit using the yeast two-hybrid system. The interaction was specific for the insulin receptor and the insulin-like growth factor-1 receptor, and it required a catalytically active receptor kinase domain and an intact Grb10 SH2 domain. Glutathione S-transferase fusion proteins containing the Grb10 SH2 domain associated in an insulin-dependent manner with insulin receptors from cell lysates and with purified insulin receptors. Co-precipitation experiments revealed the association of cellular Grb10 with hormone-stimulated insulin receptors in cell extracts. The Grb10 SH2 domain did not bind to an insulin receptor lacking 43 amino acids at the carboxyl terminus, and it exhibited highest affinity for a phosphopeptide containing Tyr(P)-1322. Unlike p85 and Syp, which also bind to Tyr(P)-1322, Grb10 was not found to associate with insulin receptor substrate-1. These results suggest that Grb10 is a novel insulin receptor interactive protein and provide direct evidence for an insulin receptor substrate-1-independent function of the insulin receptor carboxyl terminus in protein binding.

3T3 Cells↗
PubMed1996

Microsatellite instability analysis of primary human brain tumors.

Microsatellite instability, as shown by the presence of additional alleles or shifts of electrophoretic mobility at simple sequence tandem repeat loci, has been demonstrated in hereditary and sporadic colorectal tumors and many other tumor types. To study microsatellite instability in human brain tumors, we examined a total of 144 sporadic neoplasms. These included 33 astrocytic tumors, 23 oligodendrogliomas, six gangliogliomas, 41 meningiomas, 10 vestibular schwannomas and 31 pituitary adenomas. Di-, tri- and tetranucleotide repeat microsatellite markers localized on chromosome 4 and 9, X, 13 and 22, respectively, were used to assess whether instability was a significant aspect of their abnormal chromosomal pattern. Instability of microsatellite markers was detected in four oligodendrogliomas (17.4%), one pituitary adenoma (3.2%), one meningioma (2.4%), one astrocytic tumor (3.0%) and not at all in gangliogliomas and schwannomas. Therefore, our results suggest that the microsatellite instability which occurs in colorectal cancers with defective mismatch repair is infrequent in many types of human brain tumors and that the lower level of instability observed in brain tumors may be reflective of other mechanisms of genetic instability.

Base Sequence↗
PubMed1996

Irreversible binding of cis-(+)-3-methylfentanyl isothiocyanate to the delta opioid receptor and determination of its binding domain.

Binding of cis-(+)-3-methylfentanyl isothiocyanate (SUPERFIT) to cloned opioid receptors stably expressed in Chinese hamster ovary cells was characterized. SUPERFIT inhibited [3H]diprenorphine binding with much higher affinity for the delta than the mu or kappa receptor. Pretreatment with SUPERFIT followed by extensive washing reduced delta binding with an IC50 value of 7.1 nM, yet it did not affect mu and kappa binding up to 0.1 microns. The reduction in delta binding by SUPERFIT pretreatment was due to a decrease in Bmax with no change in Kd. These results indicate that SUPERFIT is a highly selective delta irreversible ligand. We then determined the region in the delta receptor that confered binding selectivity for SUPERFIT by examining its binding to six mu/delta chimeric receptors. SUPERFIT bound to delta, mu/delta 1 (amino acids mu 1-94/delta 76-372), delta/mu 3 (delta 1-134/mu 154-398), and delta/mu 4 (delta 1-187/mu 207-398) receptors with high affinity but to mu, delta/mu 1 (delta 1-75/mu 95-398), mu/delta 3 (mu 1-153/delta 135-372), and mu/delta 4 (mu 1-206/delta 188-372) receptors with low affinity. Pretreatment with SUPERFIT potently inhibited [3H]diprenorphine binding to delta, mu/delta 1, delta/mu 3, and delta/mu 4 but affected binding to mu, delta/mu 1, mu/delta 3, and mu/delta 4 only at much higher concentrations. Thus, the segment from the beginning of the first intracellular loop to the middle of the third transmembrane helix of the delta receptor is important for selective binding of SUPERFIT.

Amino Acid Sequence↗