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Biomedical subjects

X Zhu

Publications and source records attributed to X Zhu.

At least 667 records · Page 37Linked to original sources
PubMed1994

Circulating T cell repertoire complexity in normal individuals and bone marrow recipients analyzed by CDR3 size spectratyping. Correlation with immune status.

The analysis of the T cell repertoires involved in local or systemic immune responses is beginning to play an important role in many clinical situations. These include autoimmunity, response to viral or bacterial superantigens, alloimmunity including allograft rejection, and tumor immunity. Here we analyze circulating T cell repertoires by determining TCR beta-chain gene complexity using a modification of V beta family-specific PCR. This approach, called CDR3 size spectratyping, uses the size heterogeneity of the CDR3 as a further source of specificity in TCR analysis. It has been used here to analyze the complexity and stability of circulating T cell repertoires in normal adults, including bone marrow donors, and bone marrow transplant recipients. Normal spectratypes are both complex and stable. The repertoire complexity of marrow recipients correlates with their state of immune function. Contractions and gaps in repertoires are revealed in individuals suffering from recurrent infections associated with T cell impairment. Spectratype analysis is applicable to other studies of specific repertoire skewing such as may be associated with immunodeficiency or found at sites of immune activity.

Adult↗
PubMed1994

Kinetic characteristics of phosphofructokinase from Bacillus stearothermophilus: MgATP nonallosterically inhibits the enzyme.

The kinetic mechanism of phosphofructokinase from Bacillus sterothermophilus has been investigated using steady-state measurements. The double-reciprocal patterns observed for initial velocity, product inhibition, and mixed alternate substrate studies of the reverse reaction establish that the mechanism involves rapid-equilibrium random binding of substrates and the formation of an abortive complex composed of enzyme, MgADP, and fructose 6-phosphate (E-MgADP-Fru-6P). Initial velocity patterns for the forward reaction show significant nonlinearity and resemble those seen for competitive substrate (MgATP) inhibition of an enzyme that obeys a random mechanism. A mutant BsPFK enzyme (GV212) was used to show that the inhibition is not due to MgATP binding in the effector site. Product and dead-end inhibition studies of the forward reaction are consistent with a random mechanism, after taking into account the effects of substrate inhibition by MgATP. Initial velocity measurements at low MgATP concentration show that the binding of MgATP is not a rapid-equilibrium process; i.e., the rate of catalysis is faster than the rate of substrate binding. It is concluded that the kinetic mechanism of the forward reaction is sequential random, with the rate of MgATP binding slower than the catalytic rate. A model is presented that incorporates these results and proposes that substrate binding proceeds through two alternative pathways, one of which is kinetically disfavored. The observed MgATP substrate inhibition arises from both reaction flux through the disfavored pathway and, to some extent, abortive binding of MgATP in the Fru-6P site.

Adenosine Diphosphate↗
PubMed1994

Interleukin-2-induced tyrosine phosphorylation of Shc proteins correlates with factor-dependent T cell proliferation.

Interleukin-2 (IL-2) is a growth factor involved in the clonal expansion of antigen-activated T lymphocytes. Interaction of IL-2 with its receptor triggers tyrosine phosphorylation of a series of proteins and results in the activation of p21ras. We report here that Shc, an SH2-containing adaptor protein, is tyrosine-phosphorylated following IL-2 stimulation. IL-2-induced tyrosine phosphorylation of Shc was detectable within seconds following IL-2 addition, reaching its highest level by 15 min. Tyrosine phosphorylation of Shc was induced in multiple IL-2-dependent T cell lines and was found to correlate with IL-2-dependent cell proliferation. Tyrosine-phosphorylated Shc was found to be capable of associating with the SH2 domain of Grb2 following IL-2 stimulation. These results indicate that tyrosine phosphorylation of Shc and its association with Grb2 may be important events in IL-2-initiated signal transduction events in T cells.

Adaptor Proteins, Signal Transducing↗
PubMed1994

A covalently trapped folding intermediate of subtilisin E: spontaneous dimerization of a prosubtilisin E Ser49Cys mutant in vivo and its autoprocessing in vitro.

The propeptide of subtilisin E (the N-terminal 77 amino acid extension) is required for the proper folding of the nascent mature protein and is also a potent and specific inhibitor of the active enzyme. Previous studies have demonstrated that the propeptide can renature denatured mature sequence either in cis or in trans and can be considered an intramolecular chaperone, since it is not required for activity of the mature enzyme. In this paper it is shown that a prosubtilisin-S49C mutant can be expressed in Escherichia coli either as a monomer or as a disulfide-linked dimer, (prosubtilisin-S49C)2, depending on the vector selected. Interconversion between (prosubtilisin-S49C)2 and prosubtilisin-S49C could be readily achieved by reduction and oxidation in denaturing solutions, such as guanidine hydrochloride or urea. While the monomer can undergo autoprocessing in vitro under refolding conditions, the dimer is trapped in an intermediate state which could not be processed into active enzyme. Remarkably, the autoprocessing of this trapped intermediate could be induced readily upon reduction by dithiothreitol. This disulfide-linked (prosubtilisin-S49C)2 is fairly stable, but does tend to aggregate when the ionic strength of the solution is reduced below 0.1 M. The disulfide-linked (prosubtilisin-S49C)2 has far- and near-UV CD spectra revealing the presence of both secondary and tertiary structures, respectively, similar to those of the active mature monomer. Hence this autoprocessing-competent state appears to be a "late" folding intermediate, arising after the "molten globule" state formed in the absence of the prosequence, that has no discernible tertiary structure.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1994

Strategies for studying mouse and human immune responses to human papillomavirus type 16.

Cytotoxic T lymphocytes (CTL) are an important protective mechanism in viral infection and can be effective against tumours. We have investigated the tumour-associated E6 and E7 genes of human papillomavirus type 16 as CTL targets. In H-2b mice we have defined epitopes in E6 and E7 which can readily generate CTL in vivo and we have shown that HLA-A2.1 transgenic mice can generate an HLA-A2.1-restricted response. We have been unable to reveal a primed CTL response in humans. These paradoxical findings imply that human papillomavirus may fail to stimulate a systemic CTL response and/or employ strategies for evading or down-regulating such a response.

Amino Acid Sequence↗
PubMed1994

Heterologous systems for expression of mammalian sulfotransferases.

This paper describes the use of both mammalian and bacterial expression systems as tools to study the structural and functional relationships of proteins encoded by cDNAs to both rat and human aryl sulfotransferases. In particular, we describe the use of the mammalian COS cell system for functional expression studies, and the use of Escherichia coli for the expression and purification of a sulfotransferase fusion protein suitable as an antigen for the generation of sulfotransferase antibodies.

Animals↗
PubMed1994

Effects of estrogens on lipoprotein metabolism and cardiovascular disease in women.

Estrogen use is associated with protection from cardiovascular disease in postmenopausal women. This benefit appears to be magnified among women with pre-existing heart disease. The possible bias of intrinsically better health in women using estrogen has not been ruled out in observational studies. Therefore, two double-blind randomized clinical trials are underway in postmenopausal women. One in women with coronary disease is known as HERS (Heart Estrogen-progestin Replacement Study) and another in predominantly healthy women is the WHI (Women's Health Initiative). Several mechanisms of estrogen mediated protection from cardiovascular disease have been identified including increased HDL, lower LDL, lower VLDL-cholesterol/triglyceride ratio, increased clearance of intermediate density lipoprotein (IDL) and LDL via an upregulated LDL receptor, diminished penetration and degradation of LDL in the arterial wall, an inhibition of LDL oxidation by various estrogens and a reversal of inappropriate acetylcholine (EDRF)-mediated vasoconstriction in arteriosclerotic vessels. The predominating mechanism is not known, but estrogen replacement therapy is both likely to be beneficial to female health, pending randomized trials, as well as a tool to understand mechanisms of prevention of coronary artery disease.

Adolescent↗
PubMed1994

Effects of space flight stress on proopiomelanocortin, proenkephalin A, and tachykinin neuropeptidergic systems in the rat posterior pituitary.

beta-endorphin-like immunoreactivity (BE-li), methionine enkephalin-like immunoreactivity (ME-li), and substance P-like immunoreactivity (SP-li) were measured in the posterior pituitary of rats that experienced a 5-day space flight in a Space Shuttle. ME-li and SP-li were both significantly lower compared to the control rats. However, there was no difference in BE-li between flight and control rats. These data suggest that the space flight stress diminished the methionine enkephalin (ME) and substance P (SP) concentrations in the posterior pituitary without affecting the beta-endorphin (BE) concentration. Thus, the proenkephalin A and tachykinin, but not proopiomelanocortin, neuropeptidergic systems in the posterior pituitary may respond to this type of unique stress.

Animals↗
PubMed1994

Recipient-mediated effect of a traditional Chinese herbal medicine, ren-shen-yang-rong-tang (Japanese name: ninjin-youei-to), on hematopoietic recovery following lethal irradiation and syngeneic bone marrow transplantation.

Ren-shen-yang-rong-tang (Japanese name: Ninjin-youei-to, NYT), a traditional Chinese herbal medicine, was evaluated for recipient-mediated effect on hematopoietic recovery in a murine model of syngeneic bone marrow transplantation (BMT). BALB/c recipient mice were preconditioned with a lethal total body irradiation (TBI) at a dose of 6.5 Gy and transplanted with syngeneic bone marrow (BM) cells. NYT treatments, given intraperitoneally (i.p.) once per day for 3 consecutive days in a dose of 0.625 mg, were performed either before or after TBI and BMT to assess any recipient-mediated effect of this compound. NYT pretreatment was as effective as NYT posttreatment in enhancing the total number of colony-forming unit erythroid (CFU-E) and colony-forming unit granulocyte-macrophage (CFU-GM) per marrow and spleen after TBI and BMT. NYT pretreatment caused a significant increase in marrow and splenic CFU-E and CFU-GM numbers over a prolonged period following TBI and BMT, and affected late-stage erythropoiesis (CFU-E) more profoundly than early-stage erythropoiesis (burst-forming unit erythroid, BFU-E). NYT pretreatment significantly accelerate recovery of not only erythrocyte and leukocyte counts but also platelet counts after transplantation with a limited number (1 x 10(5)) of BM cells. The same treatment, however, was significantly less effective in hematopoietic recovery after transplantation with a minimal number (1 x 10(4)) of BM cells, indicating that NYT accelerates recovery of donor-derived rather than recipient-derived cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Effects of 15-deoxyspergualin on proliferative responses and cytokine gene expression in vitro.

15-deoxyspergualin (DSG)-treated BALB/c spleen cells showed increased spontaneous proliferation and increased alloreactive mixed lymphocyte reactions (MLRs) when a 3-h treatment was carried out. However, when spleen cells were treated with DSG for 5 days without washing out DSG, decreased spontaneous proliferation was observed, although alloreactive MLRs against C3H/He and C57BL/6 alloantigens were increased. In contrast, cyclosporin A (CsA) induced markedly decreased alloreactive MLRs. Decreased concanavalin A (Con A)- and pokeweed mitogen (PWM)-induced responses were observed in spleen cells treated with DSG for 3 h; whereas increased phytohemagglutinin (PHA)-induced responses were observed. On the other hand, increased Con A- and PHA-induced responses were observed in spleen cells treated with DSG for 2 days, whereas PWM-induced responses were decreased. CsA-treatment induced markedly decreased mitogen-induced responses. These results suggest that the immunosuppressive mechanism of DSG differs from that of CsA. Reverse transcriptase-polymerase chain reaction (RT-PCR) method showed that interleukin 1 beta (IL-1 beta), IL-2, IL-3, IL-4, IL-5, and leukemia inhibitory factor (LIF) mRNA expression in DSG-treated spleen cells were increased by Con A stimulation, thus indicating that DSG modulates cytokine gene expression and inducing immunosuppressive mechanisms different from CsA.

Animals↗
PubMed1994

Decreased myo-inositol uptake is associated with reduced bradykinin-stimulated phosphatidylinositol synthesis and diacylglycerol content in cultured neuroblastoma cells exposed to L-fucose.

L-Fucose is a potent, competitive inhibitor of myo-inositol transport by cultured mammalian cells. Chronic exposure of neuroblastoma cells to L-fucose causes a concentration-dependent decrease in myo-inositol content, accumulation, and incorporation into phosphoinositides. In these studies, L-fucose supplementation of culture medium was used to assess the effect of decreased myo-inositol metabolism and content on bradykinin-stimulated phosphatidylinositol synthesis and diacylglycerol production. Chronic exposure of cells to 30 mM L-fucose caused a sustained decrease in bradykinin-stimulated, but not basal, 3H-inositol phosphate release and 32P incorporation into phosphatidylinositol in cells incubated in serum-free, unsupplemented medium. In addition, 32P incorporation into phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate was not altered in L-fucose-conditioned cells. Acute exposure of cells to serum-free medium containing 30 mM L-fucose did not affect either basal or bradykinin-stimulated 32P incorporation into phosphatidylinositol. Basal diacylglycerol content was decreased by 20% in cells chronically exposed to 30 mM L-fucose, although analysis of the molecular species profile revealed no compositional change. Bradykinin stimulated diacylglycerol production in neuroblastoma cells by increasing the hydrolysis of both phosphoinositides and phosphatidylcholine. Bradykinin-stimulated production of total diacylglycerol was similar for control and L-fucose-conditioned cells. However, there was a decrease in the bradykinin-induced generation of the 1-stearoyl-2-arachidonoyl diacylglycerol molecular species in the cells chronically exposed to 30 mM L-fucose. This molecular species accounts for about 70% of the composition of phosphoinositides, but only 10% of phosphatidylcholine. The results suggest that a decrease in myo-inositol uptake results in diminished agonist-induced phosphatidylinositol synthesis and phosphoinositide hydrolysis in cultured neuroblastoma cells grown in L-fucose-containing medium.

Animals↗
PubMed1994

Sequences responsible for intracellular localization of beta-actin messenger RNA also affect cell phenotype.

We have characterized the structure and function of RNA sequences that direct beta-cytoplasmic actin mRNA to the cell periphery were mapped to two segments of 3'-untranslated region by expression of LacZ/beta-actin chimeric mRNAs in chicken embryo fibroblasts (CEFs). A 54-nt segment, the "RNA zipcode," and a homologous but less active 43-nt segment each localized beta-galactosidase activity to the leading lamellae. This zipcode contains the full activity, and mutations or deletions within it reduce, but do not eliminate, its activity, indicating that several motifs contribute to the activity. Two of these motifs, when multimerized, can regenerate almost full activity. These sequences are highly conserved in evolution, since the human beta-actin zipcode, positioned identically in the 3'UTR localizes equally well in chicken cells. Complementary phosphorothioate oligonucleotides against the zipcode delocalized endogenous beta-actin mRNA, whereas those complementary to the region just outside the zipcode, or sense oligonucleotides, did not. Actin mRNA or protein levels were unaffected by the antisense treatments, but a dramatic change in lamellipodia structure, and actin stress fiber organization was observed using the same antizipcode oligonucleotides which delocalized the mRNA. Hence, discrete 3'UTR sequences direct beta-actin isoform synthesis to the leading lamellae and affect cell morphology, presumably through the actin cytoskeleton.

Actins↗
PubMed1994

Stress response of senescent T lymphocytes: reduced hsp70 is independent of the proliferative block.

Senescent human T lymphocyte cultures are unable to undergo proliferation, but show no difference from early passage cells in cytotoxic function or surface antigenic profile. A second feature of senescent T cells is the dramatic reduction in hsp70 production in response to heat shock. This decline is associated with a decrease in binding of nuclear extracts to the consensus heat shock element. Interestingly, the progressive decline in the heat shock response of cultured T cells correlates with the percent proliferative life span completed rather than with the actual proliferative activity at the time of heat shock. This suggests that for senescent T cells the reduced ability to respond to heat shock by producing hsp70, although possibly lying at the level of transcriptional control, may nevertheless be unrelated to the reduced DNA synthesis or the diminished proliferative activity also manifested by these cells.

Cell Division↗
PubMed1994

Limitations of predictive motifs revealed by cytotoxic T lymphocyte epitope mapping of the human papilloma virus E7 protein.

Human papilloma virus (HPV) type 16 is found in the majority of cervical cancer patients and the transforming protein E7 is consistently expressed in cancer cells, making it a potential target for immune attack. In this study we have investigated whether E7 gains access to the MHC class I processing pathway and provides cytotoxic T lymphocyte (CTL) stimulating peptide epitopes. CTL were induced in H-2b mice by immunization with recombinant vaccinia virus expressing E7 (Vac-E7). To map CTL recognition, natural peptides were purified from cells expressing either intact or truncated E7 protein. Following peptide separation by HPLC one major CTL epitope was detected and truncated constructs localized this epitope to the C-terminal region. Mapping with synthetic peptides indicated that residues 49-57 (RAHYNIVTF) were recognised by anti-E7 CTL. Synthetic 49-57 peptide was used to induce CTL, which recognized the same HPLC purified natural peptide fractions as anti-E7 CTL. Binding motifs for H-2b class I molecules did not predict residues 49-57 to be a CTL epitope, but instead the sequence 21-28 (DLYCYEQL) which contains a Kb anchor motif. Synthetic 21-28 peptide was found to bind to Kb class I molecules and readily induced CTL, indicating that the T cell repertoire of H-2b mice can recognize this epitope. However, these CTL did not recognize peptides isolated from E7 expressing cells, showing that natural processing did not produce detectable levels of the 21-28 epitope. Together, the data demonstrate that an unexpected E7 peptide can function as a major CTL epitope.

Amino Acid Sequence↗
PubMed1994

Immune response to human papillomavirus type 16 E6 gene in a live vaccinia vector.

Immunization of mice with a recombinant vaccinia virus expressing the human papillomavirus type 16 (HPV-16) E6 gene elicits specific antibody, proliferative and cytotoxic T lymphocyte responses. T and B cell epitopes were mapped by using synthetic peptides. This study provides the background to future investigation aimed at developing prophylactic and therapeutic vaccines against HPV-16 infection and cervical cancer.

Amino Acid Sequence↗