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Biomedical subjects

X Zhu

Publications and source records attributed to X Zhu.

At least 289 records · Page 16Linked to original sources
PubMed1999

A novel nuclear export activity in HIV-1 matrix protein required for viral replication.

An important aspect of the pathophysiology of human immunodeficiency virus type-1 (HIV-1) infection is the ability of the virus to replicate in non-dividing cells. HIV-1 matrix (MA), the amino-terminal domain of the Pr55 gag polyprotein (Pr55), bears a nuclear localization signal that promotes localization of the viral preintegration complex to the nucleus of non-dividing cells following virus entry. However, late during infection, MA, as part of Pr55, directs unspliced viral RNA to the plasma membrane, the site of virus assembly. How MA can mediate these two opposing targeting functions is not understood. Here we demonstrate that MA has a previously undescribed nuclear export activity. Although MA lacks the canonical leucine-rich nuclear export signal, nuclear export is mediated through the conserved Crm1p pathway and functions in both mammalian cells and yeast. A mutation that disrupts the MA nuclear export signal (MA-M4) mislocalizes Pr55 and genomic viral RNA to the nucleus, thereby severely impairing viral replication. Furthermore, we show that MA-M4 can act in a dominant-negative fashion to mislocalize genomic viral RNA even in the presence of wild-type MA. We conclude that the MA nuclear export signal is required to counteract the MA nuclear localization signal, thus ensuring the cytoplasmic availability of the components required for virion assembly.

Biological Transport↗
PubMed1999

Tyrosine phosphorylation of a low molecular weight protein induced by RANTES in T-lymphocytes.

The beta-chemokine RANTES, a T-lymphocyte activator, chemoattractant, and inducer of homotypic aggregation, is considered to exert extensive effects on T lymphocytes through either G protein-coupled or protein tyrosine kinase (PTK) signaling pathway. In the present study, we analyzed RANTES-induced signal transduction through PTK as an early event in T-lymphocyte activation. Tyrosine phosphorylation is detected by immunoblots in the human T-cell line H9 after incubation with human recombinant RANTES. The tyrosine phosphorylation of a protein with a molecular mass of about 25 kD is measurable as early as 30 s and maximal at 1-5 min; and is a dose-dependent effect. The phosphorylation response can be abrogated by the tyrosine-kinase inhibitor herbimycin A (HA) but is insensitive to heterotrimeric Galphai protein inhibitor pertussis toxin (Ptx). This phenomenon is also observed in a visible homotypic aggregation response after incubation serum-starved H9 cells with RANTES. The phosphorylation response can not be down-regulated by preincubation with either anti-CC chemokine receptor 5 (CCR5) antibody or HIV-1Bal supernatants. Our results suggest that tyrosine phosphorylation of a protein with molecular mass of about 25 kD via Src-family PTK(s) is an early event in T-lymphocyte activation associated with the homotypic aggregation in response to RANTES.

Benzoquinones↗
PubMed1999

Regulation of p21(cip1) expression by growth factors and the extracellular matrix reveals a role for transient ERK activity in G1 phase.

We have examined the regulation of p21(cip1) by soluble mitogens and cell anchorage as well as the relationship between the expression of p21(cip1) and activation of the ERK subfamily of MAP kinases. We find that p21(cip1) expression in G1 phase can be divided into two discrete phases: an initial induction that requires growth factors and the activation of ERK, and then a subsequent decline that is enhanced by cell anchorage in an ERK-independent manner. In contrast to the induction of cyclin D1, the induction of p21(cip1) is mediated by transient ERK activity. Comparative studies with wild-type and p21(cip1)-null fibroblasts indicate that adhesion-dependent regulation of p21(cip1) is important for proper control of cyclin E-cdk2 activity. These data lead to a model in which mitogens and anchorage act in a parallel fashion to regulate G1 phase expression of p21(cip1). They also show that (a) growth factors and growth factor/extracellular matrix cooperation can have different roles in regulating G1 phase ERK activity and (b) both transient and sustained ERK signals have functionally significant roles in controlling cell cycle progression through G1 phase.

Animals↗
PubMed1999

B lymphocyte selection and age-related changes in VH gene usage in mutant Alicia rabbits.

Young Alicia rabbits use VHa-negative genes, VHx and VHy, in most VDJ genes, and their serum Ig is VHa negative. However, as Alicia rabbits age, VHa2 allotype Ig is produced at high levels. We investigated which VH gene segments are used in the VDJ genes of a2 Ig-secreting hybridomas and of a2 Ig+ B cells from adult Alicia rabbits. We found that 21 of the 25 VDJ genes used the a2-encoding genes, VH4 or VH7; the other four VDJ genes used four unknown VH gene segments. Because VH4 and VH7 are rarely found in VDJ genes of normal or young Alicia rabbits, we investigated the timing of rearrangement of these genes in Alicia rabbits. During fetal development, VH4 was used in 60-80% of nonproductively rearranged VDJ genes, and VHx and VHy together were used in 10-26%. These data indicate that during B lymphopoiesis VH4 is preferentially rearranged. However, the percentage of productive VHx- and VHy-utilizing VDJ genes increased from 38% at day 21 of gestation to 89% at birth (gestation day 31), whereas the percentage of VH4-utilizing VDJ genes remained at 15%. These data suggest that during fetal development, either VH4-utilizing B-lineage cells are selectively eliminated, or B cells with VHx- and VHy-utilizing VDJ genes are selectively expanded, or both. The accumulation of peripheral VH4-utilizing a2 B cells with age indicates that these B cells might be selectively expanded in the periphery. We discuss the possible selection mechanisms that regulate VH gene segment usage in rabbit B cells during lymphopoiesis and in the periphery.

Aging↗
PubMed1999

Cytosolic phospholipase A2 activation is essential for beta 1 and beta 2 integrin-dependent adhesion of human eosinophils.

We examined the role of cytosolic phospholipase A2 (cPLA2) during human eosinophil adherence to ICAM-1- or VCAM-1-coated plates. IL-5-stimulated eosinophils adhered to ICAM-1 through the beta 2 integrin CD11b/CD18, while nonstimulated eosinophils did not. By contrast, nonstimulated eosinophils adhered to VCAM-1 through the beta 1-integrin VLA-4/CD29. Both IL-5-induced adhesion to ICAM-1 and spontaneous adhesion to VCAM-1 corresponded temporally to cPLA2 phosphorylation, which accompanied enhanced catalytic activity of cPLA2. The structurally unrelated cPLA2 inhibitors, arachidonyl trifluoromethylketone and surfactin, significantly inhibited eosinophil adhesion to ICAM-1 and VCAM-1 in a concentration-dependent manner. Inhibition of secretory PLA2, 5-lipoxygenase, or cyclooxygenase did not affect eosinophil adhesion. Addition of arachidonic acid to eosinophils after cPLA2 inhibition with arachidonyl trifluoromethylketone or surfactin did not reverse the blockade of adhesion to ICAM-1 or VCAM-1. However, CV-6209, a receptor-specific antagonist of platelet-activating factor, inhibited all integrin-mediated adhesion. The activated conformation of CD11b as identified by the mAb, CBRM1/5, as well as quantitative surface CD11b expression were up-regulated after IL-5 stimulation. However, cPLA2 inhibition neither prevented CBRM1/5 expression nor blocked surface Mac-1 up-regulation caused by IL-5. Our data suggest that cPLA2 activation and its catalytic product platelet-activating factor play an essential role in regulating beta 1 and beta 2 integrin-dependent adhesion of eosinophils. This blockade occurs even in the presence of up-regulated eosinophil surface integrin.

Arachidonic Acids↗
PubMed1999

pRB is required for interferon-gamma-induction of the MHC class II abeta gene.

pRB is required for IFN-gamma-induction of MHC class II in human tumor cell lines, providing a potential link between tumor suppressors and the immune system. However, other genes, such as cyclin D1, show pRB-dependency only in tumor cells, so by analogy, pRB may not be necessary for cII-regulation in normal cells. Here, we demonstrate that induction of the mouse MHC class II I-A heterodimer is normal in RB+/+ mouse embryonic fibroblasts (MEFs), but deficient in RB-/- MEFs. Inducibility is restored in RB-/- MEFs stably transfected with wild type RB cDNA or infected with an adenovirus expressing pRB. Thus, involvement of pRB in MHC class II expression is conserved in the mouse and is not an aberrant feature of tumorigenic, aneuploid, human tumor cells. Although cII genes are generally induced in a coordinate fashion, suggesting a common mechanism, we found that pRB was specifically required for induction of the Abeta, but not Aalpha or other MHC cII genes including Ebeta, Ii and H2-Malpha. Finally, IFN-gamma-induction of class II transactivator (CIITA), was pRB-independent, suggesting that pRB works downstream of this master-regulator of MHC class II expression.

Animals↗
PubMed1999

Activation of neuronal extracellular receptor kinase (ERK) in Alzheimer disease links oxidative stress to abnormal phosphorylation.

Responses to increased oxidative stress may be the common mechanism responsible for the varied cytopathology of Alzheimer disease (AD). A possible link in support of this hypothesis is that one of the most striking features of AD, the abnormal accumulation of highly phosphorylated tau and neurofilament proteins, may be brought about by extracellular receptor kinase (ERK) whose activation is a common response to oxidative stress. In this study, we demonstrate that activated ERK is specifically increased in the same vulnerable neurons in AD that are the site of oxidative damage and abnormal phosphorylation. These findings suggest that ERK dysregulation, likley resulting from oxidative stress, could play an important role in the increased phosphorylation of cytoskeletal proteins observed in AD.

Adult↗
PubMed1999

Direct cloning of cell differential expression genes with full-length by a new strategy based on the multiple rounds of 'long distance' polymerase chain reaction and magnetic beads mediated subtraction.

The paper described a new cDNA subtractive cloning strategy. This strategy was based on the 'cap-finder' method, 'long distance' polymerase chain reaction (PCR), streptavidin magnetic beads mediated subtraction, and spin column chromatography. When PCR products were resolved on agarose gel after three rounds of subtraction, the 'single gene difference' group displayed a predominantly enriched band, but the 'multiple gene difference' group did not display any apparent difference. Of 200 clones inserted with 0.7-2 kb fragments from the 'multiple gene difference' group, 50% were identified to be new sequences and 35% were known. Of 100 new sequences, 35% contained coding regions and 75% were confirmed by dot-blotting to be differentially expressed by genes of the target cell. The results suggested that this strategy might be very efficient for full-length cloning of differentially expressed genes of the cell.

Cloning, Molecular↗
PubMed1999

FYN-T-FYB-SLP-76 interactions define a T-cell receptor zeta/CD3-mediated tyrosine phosphorylation pathway that up-regulates interleukin 2 transcription in T-cells.

Protein-tyrosine kinases p56(Lck), SYK, and ZAP-70 and downstream adaptors LAT and SLP-76 have been implicated as essential components in T-cell activation. Another lymphoid-specific adaptor FYB/SLAP has also been identified as a predominant binding partner of SLP-76 and the Src kinase FYN-T, although its role in the activation process has been unclear. In this study, we demonstrate that FYN-T selectively phosphorylates FYB providing a template for the recruitment of FYN-T and SLP-76 SH2 domain binding. This interaction is unusual in its distinct cytoplasmic localization and its long term stable kinetics of phosphorylation. Furthermore, we demonstrate for the first time that the co-expression of all three components of the FYN-T-FYB-SLP-76 matrix can synergistically up-regulate T-cell receptor-driven interleukin 2 transcription activity. These findings document the existence of a T-cell receptor-regulated FYN-T-FYB pathway that interfaces with the adaptor SLP-76 and up-regulates lymphokine production in T-cells.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Analysis of human peripheral blood T cells and single-cell-derived T cell clones uncovers extensive clonal CpG island methylation heterogeneity throughout the genome.

Methylation of cytosine residues in CpG dinucleotides is generally associated with silencing of gene expression. DNA methylation, as a somatic event, has the potential of diversifying gene expression in individual cells of the same lineage. There is little quantitative data available concerning the extent of methylation heterogeneity in individual cells across the genome. T cells from the peripheral blood can be grown as single-cell-derived clones and can be analyzed with respect to their DNA methylation patterns by restriction landmark genomic scanning. The use of the methylation-sensitive enzyme NotI to cut and end-label DNA fragments before their separation in two dimensions provides a quantitative assessment of methylation at NotI sites that characteristically occur in CpG islands. We have undertaken quantitative analysis of two-dimensional DNA patterns to determine the extent of methylation heterogeneity at NotI sites between peripheral blood single-cell-derived T cell clones from the same individual. A total of 1,068 NotI-tagged fragments were analyzed. A subset of 156 fragments exhibited marked methylation heterogeneity at NotI sites between clones. Their average intensity among clones correlated with their intensity in uncultured, whole-blood-derived T cells, indicating that the methylation heterogeneity observed in clones was largely attributable to methylation heterogeneity between the individual cells from which the clones were derived. We have cloned one fragment that exhibited variable NotI-site methylation between clones. This fragment contained a novel CpG island for a gene that we mapped to chromosome 4. The methylation status of the NotI site of this fragment correlated with expression of the corresponding gene. Our data suggest extensive diversity in vivo in the methylation and expression profiles of individual T cells at multiple unrelated loci across the genome.

Base Composition↗
PubMed1999

Androgen stimulates mitogen-activated protein kinase in human breast cancer cells.

The mechanisms by which androgens modulate breast cancer cell growth are largely unknown. Using cultured human PMC42 breast cancer cells, we have determined effects of the androgen R1881 on the activity of the mitogen-activated protein kinases extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 kinase. R1881 did not alter JNK and p38 kinase activity, but activated ERK in a dose-dependent manner. Activation was rapid, peaking at 5 min followed by a decline to baseline after 30-60 min, and was accompanied by tyrosine phosphorylation of ERK. The androgen antagonist flutamide elevated ERK to similar levels and DNA synthesis to levels half those seen with R1881; in addition, excess flutamide lowered R1881-stimulated DNA synthesis to levels seen with flutamide alone. These findings suggest (i) that in human PMC42 breast cancer cells R1881 activates ERK through a non-genomic mechanism, (ii) that this non-genomic mechanism is equivalently activated by the androgen antagonist flutamide, and (iii) that androgen/antiandrogen effect on DNA synthesis may involve both genomic and non-genomic mechanisms. These findings may have important implications for the clinical use of such agents in breast cancer.

Androgen Antagonists↗
PubMed1999

Structural analysis of the lymphocyte-specific kinase Lck in complex with non-selective and Src family selective kinase inhibitors.

BACKGROUND: The lymphocyte-specific kinase Lck is a member of the Src family of non-receptor tyrosine kinases. Lck catalyzes the initial phosphorylation of T-cell receptor components that is necessary for signal transduction and T-cell activation. On the basis of both biochemical and genetic studies, Lck is considered an attractive cell-specific target for the design of novel T-cell immunosuppressants. To date, the lack of detailed structural information on the mode of inhibitor binding to Lck has limited the discovery of novel Lck inhibitors. RESULTS: We report here the high-resolution crystal structures of an activated Lck kinase domain in complex with three structurally distinct ATP-competitive inhibitors: AMP-PNP (a non-selective, non-hydrolyzable ATP analog); staurosporine (a potent but non-selective protein kinase inhibitor); and PP2 (a potent Src family selective protein tyrosine kinase inhibitor). Comparison of these structures reveals subtle but important structural changes at the ATP-binding site. Furthermore, PP2 is found to access a deep, hydrophobic pocket near the ATP-binding cleft of the enzyme; this binding pocket is not occupied by either AMP-PNP or staurosporine. CONCLUSIONS: The potency of staurosporine against Lck derives in part from an induced movement of the glycine-rich loop of the enzyme upon binding of this ligand, which maximizes the van der Waals interactions present in the complex. In contrast, PP2 binds tightly and selectively to Lck and other Src family kinases by making additional contacts in a deep, hydrophobic pocket adjacent to the ATP-binding site; the amino acid composition of this pocket is unique to Src family kinases. The structures of these Lck complexes offer useful structural insights as they demonstrate that kinase selectivity can be achieved with small-molecule inhibitors that exploit subtle topological differences among protein kinases.

Adenosine Triphosphate↗
PubMed1999

Enhancement of TBP binding by activators and general transcription factors.

Eukaryotic transcriptional activators function, at least in part, by promoting assembly of the preinitiation complex, which comprises RNA polymerase II and its general transcription factors (GTFs). Activator-mediated stimulation of the assembly of the preinitiation complex has been studied in vitro but has been relatively refractory to in vivo analysis. Here we use a DNA-crosslinking/immunoprecipitation assay to study in living cells the first step in the assembly of the preinitiation complex, the interaction between the TATA-box-binding protein (TBP) and its binding site, the TATA box. Analysis of a variety of endogenous yeast genes, and of a series of activators of differing strength, reveals a general correlation between TBP binding and transcriptional activity. Using mutant yeast strains, we show that Mot1 prevents the binding of TBP to inactive promoters and that activator-mediated stimulation of TBP binding requires additional GTFs, including TFIIB and Srb4. Taken together, our results indicate that TBP binding in vivo is stringently controlled, and that the ability of activators to stimulate this step in the assembly of the preinitiation complex is a highly cooperative process involving multiple transcription factors.

Adenosine Triphosphatases↗
PubMed1999

Brain-derived neurotrophic factor promotes survival and chemoprotection of human neuroblastoma cells.

Brain-derived neurotrophic factor (BDNF) promotes neuronal survival and protection against neuronal damage. We addressed whether BDNF might promote survival and chemoprotection in neuroblastoma (NB) using a drug-sensitive human NB cell line. All-trans-retinoic acid (ATRA) induces a striking phenotypic differentiation of NB1643 cells, and exogenous BDNF treatment promotes survival of these differentiated cells. ATRA induces TRKB expression, and exogenous BDNF stimulates both autophosphorylation of TRKB and induction of the immediate early gene, FOS, in these cells. BDNF mRNA is expressed in NB1643 cells. Because the time course of TRKB induction closely parallels phenotypic differentiation of these cells, it seems probable that ATRA induces differentiation of NB1643 cells by establishing an autocrine loop involving BDNF and TRKB. Exogenous BDNF treatment resulted in a further increase in neurite outgrowth, which again suggests that an autocrine loop is involved in differentiation of NB1643 cells in response to ATRA. We then tested whether BDNF might afford drug resistance in NB and found that BDNF does indeed protect in this NB model against cisplatin, a DNA-damaging agent actually used in the treatment of NB.

Antineoplastic Agents↗
PubMed1999

Conversion of an inactive cardiac dihydropyridine receptor II-III loop segment into forms that activate skeletal ryanodine receptors.

A 25 amino acid segment (Glu666-Pro691) of the II-III loop of the alpha1 subunit of the skeletal dihydropyridine receptor, but not the corresponding cardiac segment (Asp788-Pro814), activates skeletal ryanodine receptors. To identify the structural domains responsible for activation of skeletal ryanodine receptors, we systematically replaced amino acids of the cardiac II-III loop with their skeletal counterparts. A cluster of five basic residues of the skeletal II-III loop (681RKRRK685) was indispensable for activation of skeletal ryanodine receptors. In the cardiac segment, a negatively charged residue (Glu804) appears to diminish the electrostatic potential created by this basic cluster. In addition, Glu800 in the group of negatively charged residues 798EEEEE802 of the cardiac II-III loop may serve to prevent the binding of the activation domain.

Amino Acid Sequence↗