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Biomedical subjects

Y Gong

Publications and source records attributed to Y Gong.

At least 163 records · Page 9Linked to original sources
PubMed1996

Invasive phenotype of Trypanosoma cruzi restricted to a population expressing trans-sialidase.

Trypanosoma cruzi expresses a developmentally regulated trans-sialidase implicated in the pathogenesis of Chagas' disease. On inhabitation of the extracellular milieu of cultured cells by infective trypomastigotes, the enzyme is restricted to a small (20 to 30%) population of parasites. The biological significance of trans-sialidase expression on this subset, termed TS+, and not on the majority (70 to 80%) of morphologically similar trypanosomes, named TS-, is unknown. To determine the roles of the TS+ and TS- subsets in T. cruzi invasion, we prepared pure populations of TS- and TS+ trypanosomes using magnetic beads coated with a monoclonal antibody specific for the tandem repeat unit of the trans-sialidase C terminus. After removal of nonadherent TS- trypomastigotes, the TS+ trypomastigotes were isolated from the beads by specific elusion with a synthetic peptide epitope of the trans-sialidase monoclonal antibody. Confirmation of TS+ and TS- phenotypes was obtained by immunofluorescence, immunoblotting, and sialidase or sialyl transferase activity measurements. The TS+ trypanosomes were highly invasive, as they attached to, penetrated, and thrived in cultured mammalian cells much more efficiently than did unfractionated parasites. The critical role of the trans-sialidase in invasion was underscored by the observation that infection was neutralized by human antibodies to transsialidase. What's more, the TS- parasites, in sharp contrast to their TS+ counterparts, were extremely inefficient in invading epithelial cells and fibroblasts. Further, introduction of small amounts of exogenous trans-sialidase into suspensions of nonpenetrating TS- parasites converted them to a highly invasive phenotype indistinguishable from that of the TS+ population. Rescue of the invasive phenotype was specific for the T. cruzi enzyme, for it didn't happen with bacterial and viral sialidases. The in vitro results were confirmed in the murine model of Chagas' disease, as TS- trypomastigotes were relatively avirulent while TS+ trypomastigotes were more virulent than unfractionated parasites.

Animals↗
PubMed1996

Osteoporosis-pseudoglioma syndrome, a disorder affecting skeletal strength and vision, is assigned to chromosome region 11q12-13.

Osteoporosis-pseudoglioma syndrome (OPS) is an autosomal recessive disorder characterized by severe juvenile-onset osteoporosis and congenital or juvenile-onset blindness. The pathogenic mechanism is not known. Clinical, biochemical, and microscopic analyses suggest that OPS may be a disorder of matrix homeostasis rather than a disorder of matrix structure. Consequently, identification of the OPS gene and its protein product could provide insights regarding common osteoporotic conditions, such as postmenopausal and senile osteoporosis. As a first step toward determining the cause of OPS, we utilized a combination of traditional linkage analysis and homozygosity mapping to assign the OPS locus to chromosome region 11q12-13. Mapping was accomplished by analyzing 16 DNA samples (seven affected individuals) from three different consanguineous kindreds. Studies in 10 additional families narrowed the candidate region, supported locus homogeneity, and did not detect founder effects. The OPS locus maps to a 13-cM interval between D11S1298 and D11S971 and most likely lies in a 3-cM region between GSTP1 and D11S1296. At present, no strong candidate genes colocalize with OPS.

Alleles↗
PubMed1996

Oncogenic transformation of Syrian hamster embryo cells by 5.3-MeV alpha particles and a tumor promoter phorbol ester.

The primary Syrian hamster embryo (SHE) cells were used to study the oncogenic transformation by 238pu alpha particles or X-rays alone or in combination with a chemical promoter phorbol ester. Survival curves of SHE cells following exposure to alpha-particles or X-rays were fitted to single-or multi-target models, respectively. Model parameters were: Do = 0.55 Gy, n = 1 for alpha particles; Do = 1.44 Gy, Dq = 3.0 Gy, n = 7.7 for X-rays. Incidence of alpha particles or X-rays induced cell transformation was dose-dependent. alpha particles were more efficient in inducing cell transformation than that of X-rays. The enhancement of SHE cell transformation by phorbol 12-myristate 13-acetate (PMA) following exposure to alpha particles of 0.25-1.00 Gy was observed.

Animals↗
PubMed1996

[The effect of IL2 gene transfection on the growth and metastatic characteristics of muous B16 melanoma cells].

In order to investigate the antitumor effect of IL2 gene transfected tumor vaccine, gene transfected tumor cells which secrete IL2 in a comparatively sustained manner must be obtained. Their growth and metastatic characteristics must be identified. This study used the retrovirus infection method to introduce human IL2 cDNA into mouse B16 melanoma cells. Southern blot analysis confirmed the establishment of IL2-integrated B16 cells (B16-IL2). Northern blot also showed the expression of IL2 gene in mRNA level. IL2 secretion of B16-IL2 was comparatively stable for a period of 6 months with the highest production rate of 53u/ml. No obvious influence was observed on the morphology and growth of the tumor cells in vitro after the transfection of IL2-cDNA. But, their oncogenicity in vivo was reduced and the tumor growth induced by B16-IL2 cells was inhibited. Lung metastasis rate and extent was also reduced. This study laid the foundation for the preparation of an IL2-secreting tumor vaccine.

3T3 Cells↗
PubMed1995

Molecular cloning of the alpha 3 chain of human type IX collagen: linkage of the gene COL9A3 to chromosome 20q13.3.

Type IX collagen is composed of three polypeptides derived from the human genes COL9A1, COL9A2, and COL9A3 that assemble to form a mature collagen molecule with the structure alpha 1(IX)alpha 2(IX)alpha 3(IX). We have identified overlapping cDNA and genomic clones that encode for the entire alpha 3 chain of human type IX collagen. Tryptic peptides from the human alpha 3(IX) collagen chain were subjected to N-terminal amino acid sequencing, and a stretch of 124 contiguous amino acids that included the NC1, COL1, and NC2 domains was obtained. Degenerate oligonucleotide primers were designed based on the amino acid sequences of the human tryptic peptides as well as bovine peptides and sequences from chicken cDNA clones. These primers were used to amplify three overlapping PCR products that covered the majority of the human alpha 3(IX) collagen. PCR products were then used to identify overlapping cDNA clones from a human chondrocyte library. A lambda genomic clone was identified that contained the 5'-most exon that encodes the signal peptide to complete the entire structure of the human alpha 3(IX) collagen chain. Genomic amplification identified a single-strand conformational polymorphism in COL1 that was used to map COL9A3 to chromosome 20q13.3 by linkage analysis. The present study completes the structure of human type IX collagen, and linkage for COL9A3 completes the genomic mapping of cartilage collagen genes. These data will greatly assist the genetic screening of families with degenerative cartilage and eye diseases by allowing investigators to screen for a complete set of candidate collagen gene markers.

Amino Acid Sequence↗
PubMed1995

Reactive-site hydrolyzed Cucurbita maxima trypsin inhibitor-V: function, thermodynamic stability, and NMR solution structure.

Reactive-site (Lys44-Asp45 peptide bond) hydrolyzed Cucurbita maxima trypsin inhibitor-V (CMTI-V*) was prepared and characterized: In comparison to the intact form, CMTI-V* exhibited markedly reduced inhibitory properties and binding affinities toward trypsin and human blood coagulation factor XIIa. The equilibrium constant of trypsin-catalyzed hydrolysis, Khyd, defined as [CMTI-V*]/[CMTI-V], was measured to be approximately 9.4 at 25 degrees C (delta G degrees = -1.3 kcal.mol-1). From the temperature dependence of delta G degrees, the following thermodynamic parameters were estimated: delta H degrees = 1.6 kcal.mol-1 and delta S degrees = 9.8 eu. In order to understand the functional and thermodynamic differences between the two forms, the three-dimensional solution structure of CMTI-V* was determined by a combined approach of NMR, distance geometry, and simulated annealing methods. Thus, following sequence-specific and stereospecific resonance assignments, including those of beta-, gamma-, delta-, and epsilon-hydrogens and valine methyl hydrogens, 809 interhydrogen distances and 123 dihedral angle constraints were determined, resulting in the computation and energy-minimization of 20 structures for CMTI-V*. The average root mean squared deviation in position for equivalent atoms between the 20 individual structures and the mean structure obtained by averaging their coordinates is 0.67 +/- 0.15 A for the main chain atoms and 1.19 +/- 0.23 A for all the non-hydrogen atoms of residues 5-40 and residues 48-67.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗
PubMed1995

Three-dimensional solution structure of Cucurbita maxima trypsin inhibitor-V determined by NMR spectroscopy.

The solution structure of Cucurbita maxima trypsin inhibitor-V (CMTI-V), which is also a specific inhibitor of the blood coagulation protein, factor XIIa, was determined by 1H NMR spectroscopy in combination with a distance-geometry and simulated annealing algorithm. Sequence-specific resonance assignments were made for all the main-chain and most of the side-chain hydrogens. Stereospecific assignments were also made for some of the beta-, gamma-, delta-, and epsilon-hydrogens and valine methyl hydrogens. The ring conformations of all six prolines in the inhibitor were determined on the basis of 1H-1H vicinal coupling constant patterns; most of the proline ring hydrogens were stereospecifically assigned on the basis of vicinal coupling constant and intraresidue nuclear Overhauser effect (NOE) patterns. Distance constraints were determined on the basis of NOEs between pairs of hydrogens. Dihedral angle constraints were determined from estimates of scalar coupling constants and intraresidue NOEs. On the basis of 727 interproton distance and 111 torsion angle constraints, which included backbone phi angles and side-chain chi 1, chi 2, chi 3, and chi 4 angles, 22 structures were calculated by a distance geometry algorithm and refined by energy minimization and simulated annealing methods. Both main-chain and side-chain atoms are well-defined, except for a loop region, two terminal residues, and some side-chain atoms located on the molecular surface. The average root mean squared deviation in the position for equivalent atoms between the 22 individual structures and the mean structure obtained by averaging their coordinates is 0.58 +/- 0.06 A for the main-chain atoms and 1.01 +/- 0.07 A for all the non-hydrogen atoms of residues 3-40 and 49-67. These structures were compared to the X-ray crystallographic structure of another protein of the same inhibitor family-chymotrypsin inhibitor-2 from barley seeds [CI-2; McPhalen, C. A., & James, M. N. G. (1987) Biochemistry 26, 261-269]. The main-chain folding patterns are highly similar for the two proteins, which possess 62% sequence differences. However, major differences are noted in the N- and C-terminal segments, which may be due to the presence of a disulfide bridge in CMTI-V, but not in CI-2.

Amino Acid Sequence↗
PubMed1995

Changing the inhibitory specificity and function of Cucurbita maxima trypsin inhibitor-V by site-directed mutagenesis.

Cucurbita maxima trypsin inhibitor-V (CMTI-V) is also a specific inhibitor of human blood coagulation factor beta-factor XIIa. A recombinant version of CMTI-V has allowed probing of roles of individual amino acid residues including the reactive site residue, lysine (P1), by site-directed mutagenesis. The K44R showed at least a 5-fold increase in inhibitory activity toward human beta-factor XIIa, while there was no change toward bovine trypsin. This result demonstrates that beta-factor-XIIa prefers an arginine residue over lysine residue, while trypsin is non-specific to lysine or arginine in its binding pocket. On the other hand, the specificity of CMTI-V could be changed from trypsin to chymotrypsin inhibition by mutation of the P1 residue to either leucine or methionine (K44L or K44M).

Animals↗
PubMed1995

Structural analysis of the N- and C-termini in a peptide with consensus sequence.

We present a structural analysis of a peptide, the sequence of which includes amino acids that show preferences for specific positions near the N- and C-termini in protein helices. This peptide has the sequence ac-YMSEDELKAAEAAFKRHGVP-amide, which includes a strong version of an N-terminal Harper-Rose capping box structure as well as a Gly located close to the C-terminus designed to elucidate its role in C-terminal capping. The sequence of five residues at the middle is inserted to separate effects at the two ends via a helix-stabilizing linker. Application of a simulated annealing procedure using interproton distance constraints derived from 1H NOESY experiments in water reveals the presence of a C-terminal structure in this model. The C-terminus forms a folded back structure in a significant fraction of structures generated by the annealing, in most of which Gly assumes an alpha L conformation. This structure occurs within a highly flexible region of the molecule and hence is occupied only a fraction of the time.

Amino Acid Sequence↗
PubMed1995

A practical method for stereospecific assignments of gamma- and delta-methylene hydrogens via estimation of vicinal 1H-1H coupling constants.

Stereospecific assignments are made for gamma- and delta-methylene hydrogens in a protein by means of estimation of vicinal 1H-1H spin-spin coupling constants from a short-mixing-time TOCSY experiment. 3J alpha beta coupling constants, as measured from a P.E. COSY map, are shown to be well correlated with alpha-beta cross-peak intensities of a short-mixing-time (10 ms) TOCSY map. The procedure is illustrated by application to a trypsin-inhibitor protein (M(r) approximately 7 Kd). Thus, gamma-methylene hydrogens of isoleucine residues have been stereospecifically assigned on the basis of 3J beta gamma 1H-1H coupling patterns and intraresidue cross-peak intensities in a NOESY map; gamma-hydrogens of other residues, such as lysine and arginine, have been stereospecifically assigned solely on the basis of cross-peak intensity patterns resulting from coupling of two beta-hydrogens to two gamma-hydrogens, and in conjunction with stereospecific assignments of beta-methylene hydrogens. However, intraresidue NOE intensities are needed if one or two pairs of coupling constants cannot be estimated because of cross peaks either overlapping or occurring proximal to the diagonal. The delta-methylene hydrogens have been stereospecifically assigned on the basis of coupling between two gamma-hydrogens and two delta-hydrogens, in combination with stereospecific assignments of gamma-hydrogens. Stereospecific assignments of side chains should contribute to the overall precision and accuracy of NMR-determined three-dimensional solution structures of proteins.

Arginine↗
PubMed1995

Use of endotoxin as a positive (toxic) control in the mouse embryo assay.

PURPOSE: The mouse embryo assay (MEA) is used to test media used for in vitro fertilization (IVF). Negative controls usually consist of previously tested media known to support growth of embryos to the blastocyst stage by 72 h. Often, no concurrent positive (toxic) controls are reported. Thus, any unusually hardy cohort of embryos may go undetected. Endotoxin was tested for its suitability as a positive control in the MEA. RESULTS: Female mice were stimulated with gonadotropins mated with males, and embryos flushed from their oviducts 36 h after HCG injection. Two-cell embryos were pooled and randomly distributed to culture dishes containing media without protein supplement. Endotoxin inhibited blastocyst growth beginning at 50 micrograms/ml, with complete suppression of development at 5000 micrograms/ml. With 500 micrograms/ml endotoxin, an average of 34.8% of the embryos developed to the blastocyst stage for eight separate assays. The interassay coefficient of variation (CV) was 76%, while the intraassay CV was 9.4%. At 48 h the zona pellucida was absent from all of the embryos exposed to the endotoxin. A large difference was found between two lots of endotoxin with the same claimed potency. CONCLUSIONS: These studies demonstrate the importance for inclusion of a well-defined positive control when performing the mouse embryo assay.

Animals↗
PubMed1995

Quantitative analysis of HIV-1 RNA in plasma preparations.

HIV-1 RNA extraction methodology, stability and cellular location in plasma were studied by quantitative analysis using reverse transcriptase (RT) and polymerase chain reaction (PCR). HIV-1 RNA as intact virus was stable in plasma at room temperature for at least for 24 h, or stable in RNAzol (Tel-Test, Inc. Texas) at -70 degrees C for at least 6 months. The HIV-1 RNA PCR signal did not decline significantly after freezing and thawing of the virus in plasma or in RNAzol. To assess the effect of plasma constituents from different individuals upon quantitative PCR, identical copy members of HIV LAI were spiked into plasma from 9 different, normal individuals. PCR detection of HIV-1 RNA did not show any significant variation in quantitative signals. Additionally, platelet-rich plasma from three seropositive subjects was fractionated into a platelet-free plasma fraction and a platelet pellet fraction. The quantitative analysis of HIV-1 RNA in these fractions, and in the corresponding peripheral blood lymphocytes (PBLs) from each patient, demonstrated that the majority of the HIV-1 RNA was distributed in the plasma, and the HIV-1 RNA in the plasma of these patients seemed not to be strongly platelet associated.

Blood Platelets↗
PubMed1995

Calcium regulation of androgen receptor expression in the human prostate cancer cell line LNCaP.

Elevation of intracellular calcium levels in the presence of normal androgen levels has been implicated in apoptotic prostate cell death. Since the androgen receptor (AR) plays a critical role in the regulation of growth and differentiation of the prostate, it was of interest to determine whether Ca2+ would affect the expression of androgen receptor messenger RNA (mRNA) and protein, thus affecting the ability of androgens to control prostate function. AR-positive human prostate cancer cells, LNCaP, were incubated with either the calcium ionophore A23187 or the intracellular endoplasmic reticulum Ca(2+)-ATPase inhibitor thapsigargin. Subsequently, AR mRNA and protein levels were assessed by Northern and Western blot analysis. Both A23187 and thapsigargin were found to down-regulate steady state AR mRNA levels in a time- and dose-dependent manner. AR mRNA began to decrease after 6-8 h of incubation with 10(-6) M A23187 or 10(-7) M thapsigargin, reaching a nadir at 16 and 10 h of incubation, respectively. In contrast, control mRNA (glyceraldehyde 3-phosphate dehydrogenase) did not change significantly during the treatments with either A23187 or thapsigargin. AR protein levels were found to be decreased after 12 h of incubation with either 10(-6) M A23187 or 10(-7) M thapsigargin. The decrease in AR mRNA and protein seemed to precede apoptosis, since neither A23187 (24 h) nor thapsigargin (30 h) was found to alter cell morphology within the treatment time. Cycloheximide and actinomycin D were unable to change the calcium-mediated decrease in AR mRNA, ruling out the necessity for de novo protein synthesis or a change in mRNA stability. Moreover, the decrease in AR mRNA induced by calcium does not seem to involve protein kinase C- or calmodulin-dependent pathways, since inhibitors of these cellular components had no effect. Nuclear run-on assays demonstrated little or no effects of either A23187 or thapsigargin treatment on AR gene transcription (8 h and 10 h). In conclusion, these studies show that intracellular calcium seems to be a potent regulator of AR gene expression in LNCaP cells.

Alkaloids↗
PubMed1995

Radiotherapy of midline malignant reticulosis.

The results of radiotherapy made on 23 patients with midline malignant reticulosis showed that the 1-, 3-, 5- and 8-year survival rates were 91.3%, 63.2%, 50.0% and 40.0%, respectively. The optimum radiation dose was 50-60 Gy / 5-6 weeks. Chemotherapy with CCNU following the radiotherapy facilitated the healing of lesion, reduced local recurrence and distant metastasis, improved the therapeutic effect and raised the survival rate.

Adolescent↗
PubMed1995

[Radiolabelling and assay of Chinese agkistrodon acutus venom with carrier-free Na 125I].

Chinese agkistroden acutus venom (CAAV) was radiolabelled with carrier-free Na 125I by the method of Iodogen. The specific activity and radiochemical purity for radiolabelled products were 4236.5 x 10(10) Bq/mmol and 98%, respectively. Each CAAV molecule carried 0.52 125I atom. Physical and chemical characterization of radiolabelled CAAV was similar to unradiolabelled CAAV. Binding analysis showed that 125I-CAAV was bound to platelet in a saturable manner. Binding sites per platelet were 13,255 +/- 6292/platelet. The dissociation constant (Kd) was 3.2 +/- 0.69 x 10(-10) mol/L. These results are similar to binding sites of other snake venom on platelet. The investigation showed that radiolabelled CAAV made by our laboratory was useful for radioligand binding assay.

Agkistrodon↗