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Biomedical subjects

L Zhu

Publications and source records attributed to L Zhu.

At least 379 records · Page 21Linked to original sources
PubMed1995

The pRB-related protein p107 contains two growth suppression domains: independent interactions with E2F and cyclin/cdk complexes.

Unregulated expression of either the retinoblastoma protein (pRB) or the related protein p107 can cause growth arrest of sensitive cells in the G1 phase of the cell cycle. However, growth arrests mediated by p107 and pRB are not identical. Through structure-function and co-expression analyses we have dissected the p107 molecule into two domains that independently are able to block cell cycle progression. One domain corresponds to the sequences needed for interaction with the transcription factor E2F, and the other corresponds to the interaction domain for cyclin A or cyclin E complexes. In cervical carcinoma cell line C33A, which was previously shown to be sensitive to p107 but resistant to pRB growth suppression, only the cyclin binding domain is active as a growth suppressor. Furthermore, we show that these two independent domains are functional in untransformed mouse fibroblasts. Together, these results provide experimental evidence for the presence of two functional domains in p107 and pinpoint an important functional difference between p107 and pRB.

3T3 Cells↗
PubMed1995

DNA duplexes flanked by hybrid duplexes: the solution structure of chimeric junctions in [r(cgcg)d(TATACGCG)]2.

Hybrid duplexes and chimeric duplexes containing hybrid segments linked to pure DNA (or pure RNA) segments are involved in transcription and replication, as well as reverse transcription. A complete understanding of the mechanism of these processes requires detailed information on such duplexes and the junctions between duplexes of differing structure. Using two-dimensional NMR, restrained molecular dynamics and mechanics, and back-calculation refinement against the nuclear Overhauser effect spectra at various mixing times, we have determined the solution structure of the chimeric duplex [r(cgcg)d-(TATACGCG)]2 containing a pure DNA segment in the center of a hybrid duplex. The solution structure differs from the previously determined X-ray structure of the analogous duplex [r(gcg)d(TATACGC)]2, which was found to be A-form throughout [Wang, A.H.-J., et al. (1982) Nature 299, 601-604]. The basic features of the solution structure are (a) the RNA residues are all A-form with C3'-endo sugar conformations, (b) the central DNA segment is B-form, (c) the transition from A-form RNA sugar conformations to B-form DNA sugar conformations involves only the dT5 base step, and (d) although the sugar conformations of the DNA residues A6-G12 are closer to B-form, the basic helical properties of the peripheral RNA.DNA hybrid segments are closer to typical A-form than to B-form.

Base Sequence↗
PubMed1995

Cell cycle control of apoptosis in human leukemic T cells.

Eukaryotic cell cycle is regulated by feedback controls that can arrest the progress of the cell cycle at specific checkpoints. Failure of feedback controls may predispose cells to undergo apoptotic death in response to a variety of stimuli. Transformed cell lines often carry defects in one or more proteins that regulate cell cycle, and therefore can be used as a model to study how cell cycle controls cell death. The present study investigated whether TCR/CD3-induced apoptosis of human leukemic T cells is cell cycle dependent. Studies of Jurkat and Sup-T13, two human leukemic T cell lines, demonstrated that anti-CD3 Ab-driven activation results in a transient, reversible inhibition of DNA synthesis in Jurkat cells, but results in a persistent, irreversible inhibition of DNA synthesis in Sup-T13 cells. Anti-CD3 mAb blocked Jurkat cells at the G1/S interface without inducing cell death. In contrast, anti-CD3 mAb allowed Sup-T13 cells to enter S phase but inhibited progression into G2 phase and triggered cell death. Electron microscopy showed that anti-CD3 mAb-stimulated Sup-T13 cells acquired the characteristic morphology of apoptosis. The nuclear DNA appeared as oligonucleosome-sized fragments by gel electrophoresis, and as an apoptotic peak displaying lower fluorescence than the DNA content of cells in G1 phase by DNA flow microfluorometry. DNA fragmentation correlated with entry into S phase. The DNA synthesis inhibitor aphidicolin arrested Sup-T13 cells at the G1/S interface and prevented the apoptosis. Therefore, DNA synthesis is required for executing the apoptosis program initiated by TCR/CD3 stimulation of Sup-T13 cells. These data are consistent with the hypothesis that the suicide of activated cycling T cells upon encountering autoantigens may be one of the mechanisms for the immune system to prevent autoimmunity.

Apoptosis↗
PubMed1995

Expression of heat-stable antigen on tumor cells provides co-stimulation for tumor-specific T cell proliferation and cytotoxicity in mice.

Heat-stable antigen (HSA/J11d/possibly homologous to CD24), a cell adhesion molecule capable of providing a co-stimulatory signal for T cell proliferation, is expressed on B cells, activated T cells, monocytes, granulocytes, Langerhans cells and thymocytes. Recent studies have demonstrated that co-stimulatory signals provided by cell adhesion molecules such as B7-1 play an essential role in generation of an anti-tumor immune response. To examine whether the co-stimulatory signal provided by HSA can induce an anti-tumor immune response, we have transfected HSA cDNA into the murine melanoma cell line K1735M2, and examined the ability of this transfected cell line to induce tumor-specific T cell responses. The results demonstrate that spleen cells from mice immunized with HSA-transfected K1735M2 cells showed enhanced T cell proliferation in a mixed lymphocyte tumor reaction (MLTR) assay and also demonstrated a significant anti-tumor cytotoxicity to the parent tumor cell (K1735M2). This anti-tumor cytolytic activity could be abrogated by pretreatment of effector cells with anti-mouse CD8 monoclonal antibody and complement. Under similar conditions, spleen cells from C3H mice immunized with vector-transfected K1735M2 cells neither actively proliferate in an MLTR assay, nor did they exert significant cytolytic activity against the respective tumor cells. In summary, our study demonstrated that HSA can provide a co-stimulatory signal for the T cell immune response against tumor cells in a murine model.

Animals↗
PubMed1995

Developmental regulation of troponin I isoform genes in striated muscles of transgenic mice.

The differentiation and diversification of striated muscle is a complex process involving numerous temporal and spatial alterations in the pattern of contractile protein isoform gene expression. In order to gain insight into the regulation of contractile protein isoform changes during skeletal and cardiac muscle formation, the expression of a transgene comprising a chloramphenicol acetyltransferase (CAT) reporter gene linked with sequences from -4200 to +12 of the human slow skeletal troponin I (TnIs) gene, and all three endogenous mouse troponin I (TnI) isoform genes, was investigated in embryonic, neonatal, and postnatal mice. The -4200 TnIsCAT transgene was properly activated in the limb and trunk skeletal muscle primordia and the early embryonic atrium and ventricle of the heart. Along with the endogenous mouse TnIs gene, expression of the CAT transgene began to segregate into the presumptive slow-twitch myofibers at late fetal stages and expression declined in the neonatal and postnatal heart except for the conductive tissues, in which expression persisted into adulthood. However, expression of the CAT transgene during development did not completely follow the endogenous mouse TnIs gene. The expression of the CAT transgene was aberrantly low in the embryonic cardiac outflow tract and the ventricles of the fetal heart. In addition to its expression in striated muscles, the transgene was expressed aberrantly in the primordial axial skeleton. We conclude that the upstream sequences from the human TnIs gene contain sufficient regulatory information to confer appropriate transgene expression during the early differentiation of skeletal muscles and during the establishment of fiber type upon the maturation of myofibers. However, additional regulatory elements are likely to be required for correct temporal and spatial regulation in the heart and somitic mesoderm during development. In vitro DNA transfection of cultured skeletal and cardiac muscle cells identified a cell type-specific enhancer element within the first intron of the TnIs gene whose absence in the transgene may account for the aberrant expression observed in vivo. In addition, we provide the first evidence that the fast-twitch skeletal muscle isoform of troponin I, TnIf, is transiently expressed during early cardiac muscle development.

Animals↗
PubMed1995

An improved NOESY simulation program for partially relaxed spectra: BIRDER.

An improved 2D NOESY simulation program, BIRDER (back-calculation considering incomplete recovery and differential external relaxation), has been developed. BIRDER has the following features: (1) It allows for differential external relaxation rates for protons with different chemical environments, (2) it automatically accounts for incomplete recovery of z magnetization between scans, and (3) it incorporates anisotropic tumbling motional modes for nonspherical molecules. It was found that a lower R factor is obtained if differential external relaxation rates are used in the NOESY spectrum simulation. The method for experimentally determining these external relaxation rates is discussed. In practice, the ability to account for incomplete recovery of z magnetization is very useful in the quantitative analysis of 2D NOESY spectra of spin systems with long longitudinal relaxation times (T1). For example, the T1's of some protons in RNA duplexes are approximately 4-6 s. Thus, a more than 15 s relaxation delay is needed to allow the z magnetization to return to thermal equilibrium between scans, requiring more than a week to obtain a single NOESY spectrum. Unless incomplete recovery of z magnetization is explicitly considered, distance errors may arise when shorter relaxation delays are used. The situation is further complicated in studies of DNA:RNA hybrid or DNA-RNA chimeric duplexes in which the DNA protons relax much faster than the RNA protons. It is shown that BIRDER successfully corrects for the incomplete recovery of z magnetization and makes it possible to save spectrometer time by using shorter relaxation delays without introducing serious errors. The nonsymmetrical nature of NOESY spectra when the spin system is not fully relaxed is discussed.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1995

Donor-specific cytotoxicity induced by allogeneic intestinal epithelial cells in a sponge matrix model.

Small intestinal epithelial cells (IEC) constitutively express MHC class II molecules. However, little is known about the role of IEC in intestinal allograft rejection. The present study examined whether IEC can induce the development of cytotoxic T cells in vivo using a sponge matrix model. IEC isolated from ACI (RT1a) rats were injected into polyurethane sponges implanted i.p. in Lewis (RT1(1)) rats. Sponge grafts with ACI splenocytes or Lewis IEC were used as controls. The sponge grafts were removed and sponge-infiltrating cells (SIC) were harvested on post-operative days (POD) 7, 10, and 14. The phenotype of SIC was determined by FACS analysis and the cell-mediated cytotoxicity was measured using a chromium relapse assay. Non-specific inflammatory cells accumulated in the IEC sponge allografts during the first 10 days. By POD 14, however, 61% of SIC were T lymphocytes and 36% expressed cytotoxic T cell marker (OX-8). The cytotoxicity in IEC sponge allografts was detectable on POD 7 and POD 10, and markedly elevated on POD 14. The cytotoxicity induced by allogeneic splenocytes appeared in the sponge grafts on POD 7, peaked on POD 10, and declined thereafter. The allospecific cytotoxicity induced by IEC was dependent on host macrophages as pretreatment of animals with gadolinium, a rare earth metal that inactivates macrophages, abrogated the induction of cytotoxicity. We conclude that: (1) the migration and maturation of cytotoxic T cells can be induced in vivo by IEC and (2) IEC may contribute to the increased severity of intestinal rejection through interaction with macrophages.

Animals↗
PubMed1995

A Drosophila gene that encodes a member of the protein disulfide isomerase/phospholipase C-alpha family.

Screening of a Drosophila genomic DNA library at reduced stringency hybridization conditions using a rat PLC alpha cDNA probe yielded a gene which encodes a member of the protein disulfide isomerase/PLC alpha family. The gene has been localized to band 74C on the left arm of the third chromosome and has been designated dpdi. Northern analysis shows that the dpdi gene encodes a transcript that is 2.3 kb in length and is present throughout development as well as in both heads and bodies of adults. The deduced dpdi protein is 496 amino acids in length and contains two domains exhibiting high similarity to thioredoxin, two regions that are similar to the hormone binding domain of human estrogen receptor, and a sequence of four amino acids (KDEL) at the C-terminus which has been described by others as being responsible for retention of proteins in the endoplasmic reticulum. Overall, dpdi contains a higher similarity to rat protein disulfide isomerase (53% identical) than to rat PLC alpha (30% identical). However, it is unclear whether dpdi functions in vivo as a PDI or as a PLC, or both. Drosophila, with its well characterized genetics and the ability to generate mutants in a gene that has been cloned, provides an excellent system in which to resolve this issue.

Amino Acid Sequence↗
PubMed1995

Malignant islet cell tumor associated with hypercalcemia.

BACKGROUND: Three cases of islet cell tumors of the pancreas with hypercalcemia were studied, and 16 similar cases have been found in a 25-year review of the English-language literature. The purpose of the study was to review the cause of the hypercalcemia and the clinical characteristics of the tumors. METHODS: Tumor tissue retrieved from paraffin-embedded blocks was studied immunohistochemically for both parathyroid hormone (PTH) and PTH-related protein (PTHrP). PTH was measured in the serum in each patient and the serum PTHrP was measured by immunoassay in one patient. RESULTS: One of our patients had a fatal serum calcium level of 26.4 mg/dl. PTHrP stains were positive in two of our tumors, and one patient had an elevated PTHrP serum level. Serum PTH levels were normal or low in each patient. All three tumors were malignant and extremely vascular. The total group of 19 patients have in common hypercalcemia associated with a normal or low serum PTH level. Although the cause of hypercalcemia has not been proved, the tumors apparently produce PTHrP, because seven of eight tumors stained positive for PTHrP and each of the four patients tested had an elevated PTHrP serum titer. The tumors are extremely vascular, are usually malignant (17 of 18), and become large, but they are compatible with a relatively long patient survival time. CONCLUSIONS: These neuroendocrine tumors associated with hypercalcemia share several characteristics, but a claim that they represent another type of "functioning islet cell tumor" should await a clearer delineation of the cause of the hypercalcemia.

Adenoma, Islet Cell↗
PubMed1995

Structure of a single-cytidine hairpin loop formed by the DNA triplet GCA.

In certain contexts the DNA triplet GGA, when juxtaposed on opposite strands of a DNA duplex, shows the unusual property of pairing with itself in an antiparallel orientation to form the (GGA)2 motif. In this motif the central guanines do not pair but intercalate and stack between sheared G.A pairs. Similar studies with GCA triplets reveal that they do not form analogous paired (GCA)2 motifs but instead strongly promote formation of a hairpin, the structure of which is now reported here. The GCA hairpin loop consists of a single cytidine residue closed by a sheared G.A pair and this structure is discussed in the context of triplet expansions in triplet-repeat diseases.

Base Sequence↗
PubMed1995

The human troponin I slow promoter directs slow fiber-specific expression in transgenic mice.

Troponin I (TnI) is a muscle-specific protein involved in the calcium-mediated contraction of striated muscle. Three TnI isoforms have been identified, each encoded by a separate gene and expressed in specific striated muscles in the adult. The slow isoform gene (TnIs) is transcriptionally regulated during skeletal muscle development such that its expression in the adult is restricted to muscle fibers innervated by a slow nerve. To delineate regions of this gene that are responsive to information imparted by the slow nerve, we generated transgenic mice carrying -4,200 to +12 bp of the human TnIs gene linked to the bacterial chloramphenicol acetyltransferase (CAT) coding region. By Northern blot analysis, we detected transgene transcripts only in muscles containing slow-twitch fibers. CAT histochemical analysis revealed that expression of the transgene is restricted solely to slow-twitch fibers as characterized by type I myosin heavy-chain (MyHC) expression. Using regeneration as a model for neural influenced expression, we show that this gene construct also contains sequences necessary to respond to cues from the central nervous system.

Animals↗
PubMed1995

A model for heat transfer from embedded blood vessels in two-dimensional tissue preparations.

Two-dimensional microvascular tissue preparations have been extensively used to study blood flow in the microcirculation, and, most recently, the mechanism of thermal equilibration between thermally significant countercurrent artery-vein pairs. In this paper, an approximate three-dimensional solution for the heat transfer from a periodic array of blood vessels in a tissue preparation of uniform thickness with surface convection is constructed using a newly derived fundamental solution for a Green's function for this flow geometry. This approximate solution is exact when the ratio K' of the blood to tissue conductivity is unity and a highly accurate approximation when K' not equal to 1. This basic solution is applied to develop a model for the heat transfer from a countercurrent artery-vein pair in an exteriorized rat cremaster muscle preparation. The numerical results provide important new insight into the design of microvascular experiments in which the axial variation of the thermal equilibration in microvessels can be measured for the first time. The solutions also provide new insight into the design of fluted fins and microchips that are convectively cooled by internal pores.

Animals↗
PubMed1995

Differential roles of two tandem E2F sites in repression of the human p107 promoter by retinoblastoma and p107 proteins.

Although many lines of evidence indicate that the cellular protein p107 is closely related to the retinoblastoma protein, the exact function of the p107 gene and its regulation are presently not known. To investigate the molecular mechanism controlling expression of the human p107 gene, a 5' flanking sequence of this gene was isolated and shown to promote high-level expression of a luciferase reporter gene in cycling human 293 and Saos-2 cells. Sequencing and transcription mapping analyses showed that the human p107 promoter is TATA-less and contains a tandem, direct repeat of E2F-binding sites, with the 3' copy overlapping the major transcription initiation site. Deletion analysis of the p107 promoter showed that a promoter DNA fragment containing only the two E2F sites together with the leader sequence could direct relatively efficient expression in 293 cells. Site-directed mutagenesis of these E2F sites revealed that although both sites were important for p107 promoter activity, mutation on the proximal, initiation site copy of the E2F site showed a stronger effect. The human p107 promoter could be repressed by the retinoblastoma protein and its own gene product. Interestingly, the repression was found to be mediated through the 5' copy of the E2F site. These studies demonstrate for the first time differential roles of two tandem E2F sites in promoter regulation.

Base Sequence↗
PubMed1995

Molecular cloning of an amphibian insulin receptor substrate 1-like cDNA and involvement of phosphatidylinositol 3-kinase in insulin-induced Xenopus oocyte maturation.

An insulin receptor substrate 1 (IRS-1)-like cDNA was isolated from a Xenopus ovary cDNA library by low-stringency hybridization using rat IRS-1 cDNA as a probe. The deduced amino acid sequence encoded by this cDNA (termed XIRS-L) is 67% identical (77% similar) to that of rat IRS-1. Significantly, all the insulin-induced tyrosine phosphorylation sites identified in rat IRS-1, including those responsible for binding to the Src homology domains of phosphatidylinositol (PI) 3-kinase, Syp and Grb2, are conserved in XIRS-L. Both mRNA and protein corresponding to the cloned XIRS-L can be detected in immature Xenopus oocytes. Recombinant XIRS-L protein produced in insect cells or a bacterial glutathione S-transferase fusion protein containing the putative PI 3-kinase binding site can be phosphorylated in vitro by purified insulin receptor kinase (IRK) domain, and the IRK-catalyzed phosphorylation renders both proteins capable of binding PI 3-kinase in Xenopus oocyte lysates. Another glutathione S-transferase fusion protein containing the C terminus of XIRS-L and including several putative tyrosine phosphorylation sites is also phosphorylated by IRK in vitro, but it failed to bind PI 3-kinase. Insulin stimulation of immature Xenopus oocytes activates PI 3-kinase in vivo [as indicated by an elevation of PI(3,4)P2 and PI(3,4,5)P3] as well as oocyte maturation (as indicated by germinal vesicle breakdown). Pretreatment of these oocytes with wortmannin inhibited insulin-induced activation of PI 3-kinase in vivo. The same treatment also abolished insulin-induced, but not progesterone-induced, germinal vesicle breakdown. These results (i) identify an IRS-1-like molecule in immature Xenopus oocytes, suggesting that the use of IRS-1-like Scr homology 2 domain-docking proteins in signal transduction is conserved in vertebrates, and (ii) strongly implicate PI 3-kinase as an essential effector of insulin-induced oocyte maturation.

Amino Acid Sequence↗
PubMed1995

Localized distribution of alpha 9 integrin in the cornea and changes in expression during corneal epithelial cell differentiation.

A recently characterized integrin alpha-chain, alpha 9, forms heterodimers with the integrin beta 1-chain and is present in the skin with a distribution similar to that of alpha 2 and alpha 3, other beta 1 integrins. To determine whether alpha 9 is expressed in the stratified squamous epithelium of the cornea, we used immunohistochemical techniques to compare the distribution of alpha 9 in the adult mouse cornea with that of alpha 3. Abundant alpha 9 was expressed in the lateral and basal membranes of the basal cells of the conjunctiva and corneal limbus, but very little alpha 9 was present in the basal cells of the central corneal epithelium. In contrast, alpha 3 was present in the membranes of basal cells of the conjunctiva, limbus, and central cornea. To determine when during postnatal maturation of the corneal epithelium alpha 9 becomes restricted to the limbus, we looked at the distribution of alpha 9 and alpha 3 in the developing mouse eye from birth to eyelid opening. At birth, the basal cells of the cornea and developing limbal region did not express alpha 9, but there was abundant alpha 9 expressed in suprabasal cells between the fused lids and in the basal cells of the skin and conjunctiva. In contrast, alpha 3, integrin was expressed uniformly in the basal cells across the surface of the conjunctiva, limbus, and cornea and was present only in the basal cells of the epithelium between the fused eyelids. In the central cornea, alpha 9 expression increased in basal cells up until Day 10 after birth. After Day 10, alpha 9 expression in the central cornea began to decrease; after the lids were open, alpha 9 expression in the central cornea became restricted to the limbus. In the basal and suprabasal cells between the fused eyelids expression of alpha 9 became increasingly restricted over time to the basal cells. Recent data suggest that alpha 9 beta 1 can interact with tenascin. Our dual labeling confocal microscopy studies indicate that localization of alpha 9 and tenascin are not coordinated in the developing mouse cornea. Many recent studies have shown an important role for beta 1 integrins in mediating epithelial cell differentiation in vitro; in vivo, changes in integrin expression have been found in wound healing, psoriasis, and in basal and squamous cell carcinomas.(ABSTRACT TRUNCATED AT 400 WORDS)

Age Factors↗