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Biomedical subjects

X Hu

Publications and source records attributed to X Hu.

At least 361 records · Page 20Linked to original sources
PubMed1996

Specific replacement of consecutive AGG codons results in high-level expression of human cardiac troponin T in Escherichia coli.

The adult isoform of human cardiac troponin T (TnT) contains 288 amino acids, 14 of which (4.9%) are encoded by the rarely used arginine codons (12 AGG, 2 AGA) in Escherichia coli genes. To generate sufficient quantity of TnT protein for antibody production, we cloned the corresponding cDNA and expressed it in E. coli. A low-level expression of TnT that comprised only about 1% of total cell protein was initially observed with the use of the native cDNA. The existence of two pairs of consecutive AGG codons AGG(165) AGG(166) and AGG(215) AGG(216) in the cDNA was suspected to be the main cause for this low-level expression. These two pairs of consecutive AGG codons were successively replaced with the major synonymous codon CGT by site-directed mutagenesis. As suspected, a 10-fold increase in TnT expression was obtained when one pair of the rare arginine codons was replaced and a 40-fold increase was achieved when both pairs of the rare codons were replaced. Our finding demonstrates the importance of consecutive rare codons in the suppression of high-level expression of heterologous proteins in E. coli and suggests that in order to maximize protein expression, a similar approach may be taken with other genes which contain consecutive rare codons.

Adult↗
PubMed1996

Evidence for site selection during synaptogenesis: the surface distribution of synaptic sites in photoreceptor terminals of the files Musca and Drosophila.

1. Photoreceptor terminals in the flies Musca domestica and Drosophila melanogaster have been reconstructed in three dimensions from serial EM to reveal the surface distributions of afferent tetrad synapses. 2. The terminals are cylindrical and surround two target cells; they have synaptic sites distributed along their length and around their circumference, except for a strip along the face that lies furthest away from the target cells. 3. Over their inner faces, the terminals have presynaptic sites that are distributed evenly. 4. The distribution of sites in maps plotted from reconstructed membrane surfaces was examined by quadrat analyses. The frequency of sites per quadrat division was not Poissonian, i.e. was non-random. Thus, some form of site selection must exist during synaptogenesis. 5. The sites were shown by variance ratio analysis to be regular (evenly dispersed, not clustered). This suggests that some form of interaction exists, so as to reduce the probability that a synapse will form close to an already existing synaptic site. 6. Distances between nearest-neighbour pairs of synapses had a closest minimum spacing of about 0.8 micron in Musca that was violated by about 5% of pairs, whereas the corresponding distances were about 0.2 micron shorter in Drosophila, which had 13% of pairs situated closer together than 0.8 micron. 7. During synaptogenesis, either initially in the pupa or later in the adult, the probability that a synapse will form is therefore effectively zero within these distances from an existing synaptic site, perhaps through an inhibitory influence exerted by the latter. The nearest-neighbour distances are normally distributed. 8. Unlike the distribution of presynaptic sites, the distribution of postsynaptic sites over the surfaces of the dendrites of the target cells is not even. Although not studied in detail, the corresponding nearest-neighbour distances are much smaller, as little as 0.1 micron. Thus the wider spacing seen between sites over the receptor terminals is a function of the presynaptic cells, and not of their postsynaptic partners, and implies the existence of interactions between synaptic sites.

Animals↗
PubMed1996

Experimental study on the early efficacy of excimer laser with adjunctive balloon angioplasty in dog's femoral artery.

The effect of domestically-manufactured excimer laser with adjunctive balloon angioplasty in achieving revascularization and reduction of residual stenosis was assessed. 20 femoral arteries with thrombosis and occlusion from 12 dogs were subject to angiography. At first excimer laser angioplasty was done followed by balloon angioplasty. The diameter and residual stenosis of revascularized vessel were measured. The result showed that 17 out of 20 vessels (85%) were revascularized. The diameter of revascularized vessel by excimer laser were 1.22 +/- 0.14 mm, while residual stenoses were 54% +/- 5%. After adjunctive balloon angioplasty the diameter and residual stenoses were 2.04 +/- 0.16 mm and 20% +/- 7% respectively (P < 0.05 and P < 0.01). Complication in form of vasoperforation occurred in 3/20 vessels (15%). It is concluded that China- manufactured excimer laser angioplasty is effective when used for revascularization. While the reduction of narrowing and residual stenoses was enhanced after adjunction of balloon angioplasty. This method can be employed in treating peripheral occlusive disease effectively and safely.

Angioplasty, Balloon, Laser-Assisted↗
PubMed1996

Study on the relationship between cytogenetics and phenotypic effect in Turner's syndrome.

The cytogenetics and clinical stigmata in 5 cases of Turner's syndrome were studied. Three of them were non-mosaic 1(Xq) and two with partial monosome of a X chromosome short are (Xp21), whose DNA replication patterns of inactive X chromosome were analyzed by RBG technique. Results showed that differences between the replication patterns in cases of X chromosome deletion (Xp21) and normal females existed; that the behavior of abnormal X expressed nonrandom inactivation. It was suggested that the phenotype may be closely related with both X chromosome replication pattern and its inactivation behavior, which might be useful in genetic counselling.

Adolescent↗
PubMed1996

The presence of membrane-bound stem cell factor on highly immature nonmetachromatic mast cells in the peripheral blood of a patient with aggressive systemic mastocytosis.

BACKGROUND: Systemic mastocytosis is characterized by mast cell infiltration of bone marrow and tissues in the absence of identified circulating bone marrow-derived progenitors. A 58-year-old man was first seen with aggressive systemic mastocytosis manifested by urticaria pigmentosa, hepatosplenomegaly, generalized bone lesions, anemia, thrombocytopenia, monoclonal gammopathy, and increased urine histamine levels. OBJECTIVES AND METHODS: A rapidly progressive anemia and thrombocytopenia dictated a splenectomy. We sought to identify the mast cell progenitors in the peripheral blood and to provide evidence of their maturation in tissues with immunohistochemical and ultrastructural analyses. RESULTS: The peripheral blood contained 1% to 3% nonmetachromatic mononuclear cells with eccentric nuclei that expressed the mast cell proteases, tryptase and carboxypeptidase A, along with c-kit, stem cell factor (SCF), and high-affinity IgE receptor (Fc epsilon RI), but not chymase. Similar mononuclear cells colocalized in the spleen and lymph nodes with mature, metachromatic mast cells that expressed tryptase, chymase, carboxypeptidase A, c-kit, SCF, and Fc epsilon RI. Electron microscopy disclosed, at each site, a mature mast cell population with electron-dense, scroll-poor granules. CONCLUSIONS: The peripheral blood of a patient with aggressive systemic mastocytosis contained immature mononuclear cells of the mast cell lineage that express c-kit, SCF, tryptase, carboxypeptidase A, and Fc epsilon RI. These cells were also found in the skin, spleen, and lymph nodes where they presumably expand, differentiate, and mature, assuming the mast cell phenotype for those tissues characterized by metachromasia, expression of a full range of mast cell-related secretory granule proteases, and ultrastructural appearance. The presence of SCF on the surface membrane of the circulating, highly immature mast cells suggests an autocrine regulation of the c-kit-SCF interaction.

Cell Differentiation↗
PubMed1996

FGF-1-dependent proliferative and migratory responses are impaired in senescent human umbilical vein endothelial cells and correlate with the inability to signal tyrosine phosphorylation of fibroblast growth factor receptor-1 substrates.

Senescent cells do not proliferate in response to exogenous growth factors, yet the number and affinity of growth factor receptors on the cell surface appear to be similar to presenescent cell populations. To determine whether a defect in receptor signaling exists, we analyzed human umbilical vein endothelial cells (HUVEC) since HUVEC growth is absolutely dependent upon the presence of FGF. We report that in both presenescent and senescent HUVEC populations, FGF-1 induces the expression of cell cycle-specific genes, suggesting that functional FGF receptor (FGFR) may exist on the surface of these cells. However, the tyrosine phosphorylation of FGFR-1 substrates, Src and cortactin, is impaired in senescent HUVEC, and only the presenescent cell populations exhibit a FGF-1-dependent Src tyrosine kinase activity. Moreover, we demonstrate that senescent HUVEC are unable to migrate in response to FGF-1, and these data correlate with an altered organization of focal adhesion sites. These data suggest that the induction of gene expression is insufficient to promote a proliferative or migratory phenotype in senescent HUVEC and that the attenuation of the FGFR-1 signal transduction pathway may be involved in the inability of senescent HUVEC to proliferate and/or migrate.

Base Sequence↗
PubMed1996

Blunt cardiac injuries in children: a postmortem study.

We reviewed the records of the Chief Coroner for all pediatric (< 16 years of age) trauma fatalities in Ontario (pediatric population of 2 million) for the period January 1, 1988 through December 31, 1990. Forty-one (14.5%) of 282 patients for which complete autopsy data were available had sustained cardiac injuries. Nineteen patients (46%) died at the scene of the accident, 15 patients (37%) died in an emergency department, and seven patients (17%) died during hospitalization. Rupture of a cardiac chamber occurred in 16 cases; it was the main cause of death in eight cases and a contributing factor in the remainder. Cardiac contusion without chamber rupture was present in 25 cases, but in none of the cases was it the cause of death. Brain injury was the cause of death in 16 (64%) of the cases of cardiac contusion. Cardiac injuries are more common among children who die from blunt trauma than previous reports have suggested. However, because these injuries are often rapidly fatal, many patients die before they reach a hospital. With improvements in emergency medical services and the resulting reduction in transit time, more patients may reach trauma centers alive. A high index of suspicion and rapid diagnosis and treatment of these injuries can save the lives of some of these patients.

Adolescent↗
PubMed1996

Siderophore-Mediated Aluminum Uptake by Bacillus megaterium ATCC 19213.

The bacterium Bacillus megaterium ATCC 19213 is known to produce two hydroxamate siderophores, schizokinen and N-deoxyschizokinen, under iron-limited conditions. In addition to their high affinity for ferric ions, these siderophores chelate aluminum. Aluminum was absorbed by B. megaterium ATCC 19213 through the siderophore transport receptor, providing an extra pathway for aluminum accumulation into iron-deficient bacteria. At low concentrations of the metal, siderophore-mediated uptake was the dominant process for aluminum accumulation. At high concentrations of aluminum, passive transport dominated and siderophore production slowed the passive transport of aluminum into the cell. Siderophore production was affected by the aluminum content in the media. High concentrations of aluminum increased production of siderophores in iron-limited cultures, and this production continued into stationary phase. Aluminum did not stimulate siderophore production in iron-replete cultures. The production of siderophores markedly affected aluminum uptake. This has direct implications on the toxicity of heavy metals under iron-deficient conditions.

Journal Article↗
PubMed1996

De novo synthesis of the early transcription factor 70-kilodalton subunit is required for morphogenesis of vaccinia virions.

Vaccinia virus early transcription factor (VETF) is a heterodimeric protein that is packaged in virus particles for expression of early genes during the next round of infection. To investigate additional roles of VETF, we constructed a conditionally lethal recombinant vaccinia virus in which the D6R gene, encoding the 70-kDa subunit of VETF, is under stringent Escherichia coli lac operator control. When cells were infected with the recombinant virus in the absence of an inducer, synthesis of the 70-kDa protein was undetectable and the yield of infectious virus was severely reduced. Under these nonpermissive conditions, DNA replication and synthesis of viral proteins other than the one encoded by D6R occurred, suggesting that de novo synthesis of VETF is not required for expression of early or late genes during the virus growth cycle. Electron microscopy, however, revealed that immature virus particles and masses of electron-dense material accumulated in the absence of an inducer. We concluded that VETF has a direct role in virion morphogenesis or is required for expression of a novel subset of genes that have such a role.

Animals↗
PubMed1996

The scabrous gene encodes a secreted glycoprotein dimer and regulates proneural development in Drosophila eyes.

R8 photoreceptor cells play a primary role in the differentiation of Drosophila eyes. In scabrous (sca) mutants, the pattern of R8 photoreceptor differentiation is altered. The sca gene is predicted to encode a secreted protein related in part to fibrinogen and tenascins. Using expression in Drosophila Schneider cells, we showed that sca encoded a dimeric glycoprotein which was secreted and found in soluble form in the tissue culture medium. The sca protein contained both N- and O-linked carbohydrates and interacted with heparin. This Schneider cell protein was similar to protein detected in embryos. We showed that sca mutations, along with conditional alleles of Notch (N) and Delta (Dl), each affected the pattern of cells expressing atonal (ato), the proneural gene required for R8 differentiation. In normal development, about 1 cell in 20 differentiates into an R8 cell; in the others, ato is repressed. N and Dl were required to repress ato in the vicinity of R8 cells, whereas sca had effects over several cell diameters. Certain antibodies detected uptake of sca protein several cells away from its source. The overall growth factor-like structure of sca protein, its solubility, and its range of effects in vivo are consistent with a diffusible role that complements mechanisms involving direct cell contact. We propose that as the morphogenic furrow advances, cell secreting sca protein control the pattern of the next ommatidial column.

Amino Acid Sequence↗
PubMed1996

IGF-I stimulation of muscle protein synthesis in the awake rat: permissive role of insulin and amino acids.

Infusion of insulin-like growth factor I (IGF-I) lowers plasma amino acid and insulin concentrations, which may limit the capacity of IGF-I to promote muscle protein synthesis in vivo. We measured heart and skeletal muscle incorporation of continuously infused L-[ring-2,6-3H]phenylalanine in awake postabsorptive rats receiving 4-h intravenous infusions of saline (n = 11), IGF-I (1 microgram.kg-1.min-1) with (n = 10) or without (n = 11) amino acid replacement, or IGF-I with insulin replacement (n = 8). There were no significant increases in muscle protein synthesis during the infusion of IGF-I alone, which was associated with decreases in both plasma insulin (52 +/- 5%, P < 0.001) and amino acids (25 +/- 5%, P < 0.05). When IGF-I was given together with amino acids, protein synthesis was significantly increased in gastrocnemius (4.7 +/- 0.4 vs. 2.5 +/- 0.3%/day, P < 0.001), oblique (4.5 +/- 0.4 vs. 2.8 +/- 0.4%/day, P < 0.05), and soleus (8.8 +/- 0.7 vs. 6.4 +/- 0.3%/day, P < 0.01) and tended to be higher than saline control values in heart (10.9 +/- 0.9 vs. 8.8 +/- 0.7%/day, P = 0.08). Amino acid replacement prevented plasma concentrations from falling and also blunted the decline in plasma insulin (22 +/- 5%, P < 0.01 vs. IGF-I alone). When IGF-I and insulin replacement were given, protein synthesis was increased in heart (13.0 +/- 0.6%/day), gastrocnemius (4.7 +/- 0.4%/day), and oblique (4.5 +/- 0.4%/day) (P < 0.001 for each, compared with saline). We conclude that the action of IGF-I to acutely stimulate muscle protein synthesis in the awake rat is limited by the fall in circulating insulin and/or amino acid concentrations that accompanies IGF-I infusion in vivo and is prevented by co-infusion of insulin or amino acids.

Amino Acids↗
PubMed1996

Pilot trial of infusional cyclophosphamide, doxorubicin, and etoposide plus didanosine and filgrastim in patients with human immunodeficiency virus-associated non-Hodgkin's lymphoma.

PURPOSE: To determine the following: (1) the feasibility of combining the antiretroviral didanosine (ddl) with a 96-hour continuous intravenous (IV) infusion of cyclophosphamide (800 mg/m2), doxorubicin (50 mg/m2), and etoposide (240 mg/m2) (CDE) plus filgrastim in patients with non-Hodgkin's lymphoma (NHL) associated with human immunodeficiency virus (HIV) infection; (2) the effect of ddl on CDE-induced myelosuppression and CD4 lymphopenia; and (3) the effect of CDE on serum p24 antigen and quantitative HIV blood cultures. METHODS: Twenty-five patients with HIV-related NHL received CDE every 28 or more days. Consecutive patients were assigned in an alternating fashion to group A (ddl given at a standard dose during cycles one, two, five, and six) or group B (ddl given during cycles three, four, five, and six). RESULTS: ddl use was associated with less leukopenia (mean nadir, 3.33 v 1.49 x 10(3)/microL; p = .03), neutropenia (2.38 v 1.07 x 10(3)/microL; p = .03), and thrombocytopenia (76 v 48 x 10(3)/microL; p = .059), and fewer RBC (1.6 v 3.1 per cycle; p < .01) and platelet transfusions (0.7 v 1.5 per cycle; p < .01), but had no significant effect on CD4 lymphopenia. Furthermore, lymphomatous bone marrow involvement and low CD4 count were associated with significantly greater myelosuppression. Although there was no substantial change in serum p24 antigen, the HIV blood culture became quantitatively more positive or converted from negative to positive in seven patients (64%). Complete response (CR) occurred in 58% of patients (95% confidence interval, 38% to 78%), median CR duration exceeded 18 months, tumor-related mortality was 20%, and median survival was 18.4 months. CONCLUSION: Our results suggest that the CDE and filgrastim regimen is tolerable and effective for patients with HIV-associated NHL, and that combination with ddl is feasible and may result in less myelosuppression.

Acquired Immunodeficiency Syndrome↗
PubMed1996

The expression and characterization of human recombinant proinsulin-like growth factor II and a mutant that is defective in the O-glycosylation of its E domain.

In humans, newly synthesized proinsulin-like growth factor II (pro-IGF-II), i.e. IGF-II with an E domain extension of 89 amino acids, is 0-glycosylated on Thr75. As an approach to define the role that glycosylation of the E domain serves in the processing, secretion, and biological activities of IGF-II and to identify the sites of endoproteolytic processing, we constructed a mutant that encodes carbohydrate-free prepro-IGF-II. The mutant and wild-type prepro-IGF-II were expressed in NIH-3T3 cells, and the protein products were analyzed by SDS-PAGE followed by immunoblots with antipeptide antibodies to human and homologous rat E domain sequences. Transfectants that express glycosylated pro-IGF-II, i.e. xz97 and G11 cells, have intracellular forms of the growth factor with apparent Mr (appMr) of 21, 23, and 27K. NIH-3T3 xz95 cells, i.e. transfected with DNA that is missing the 0-glycosylation sequence, could also synthesize pro-IGF-II with an appMr of 21K. However, they did not accumulate the 23K and 27K forms of presumably glycosylated growth factor. None of the transfected NIH 3T3 cells processed much pro-IGF-II intracellularly, as the appMr 21K, 23K, and 27K forms had terminal E domain amino acid sequences that were recognized by antibodies to the homologous rat peptide sequence Met117 to Gln156. Subsequent to their secretion, the IGF-II in xz97 and G11 cells accumulated in the conditioned medium mostly as two partially processed species with appMr, of 17K and 14K, respectively. The IGF-II that accumulated in the conditioned medium of the xz95 cells had an appMr of 11K. As evidenced by a decrease in mass after treatment with neuraminidase and 0-glycosidase, the 17-kDa form of pro-IGF-II secreted by the NIH-3T3 xz97 cells was 0-glycosylated, whereas that secreted by the xz95 cells was oligosaccharide free. All of the pro-IGF-II forms have E domain amino acid sequences that reacted with antipeptide Ab to the Asp69 to Lys88 sequence. However, appMr 17K IGF-II, but not 14K IGF-II, also contained a larger E domain that was recognized by Ab to the sequence Phe89 to Arg101. The final step in the processing of 11- to 17-kDa IGF-II at Arg68 and the generation of mature IGF-II did not occur in the NIH-3T3 transfectants and is similar to what has been observed in human embryonic cells and mesenchymal tumors. The failure to remove the glycosylated E domain peptide from appMr, 14K and 17K IGF-II did not affect their binding to IGF-II/cation-independent mannose-6 phosphate receptors or presumably to IGF-I receptors, because in in vitro mitogenic assays they were equipotent with mature IGF-II. Unglycosylated pro-IGF-II from the NIH-3T3 xz95 cells also bound to these receptors. However, it was about 10 times more potent than IGF-II in stimulating thymidine incorporation into NIH-3T3 i24 IGF-IR cells, possibly because of the absence of negatively charged sialic acid and/or steric occlusion.

3T3 Cells↗