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J Ding

Publications and source records attributed to J Ding.

At least 235 records · Page 13Linked to original sources
PubMed1994

Buried surface analysis of HIV-1 reverse transcriptase p66/p51 heterodimer and its interaction with dsDNA template/primer.

The p66/p51 human immunodeficiency virus type 1 reverse transcriptase is a heterodimer with identical N-terminal amino acid sequences. The enzyme contains two polymerization domains and one RNase H domain, which is located at the C-terminus of the p66 subunit. Both polymerization domains fold into four individual subdomains that are not arranged in a similar fashion, forming an unusually asymmetric dimer. The complexity of the RT p66/p51 heterodimer structure is simplified using solvent-accessibility surface areas to describe the buried surface area of contact among the different subdomains. In addition, the RT/DNA contacts in the recently published RT/DNA/Fab structure [Jacobo-Molina et al., Proc. Natl Acad. Sci. USA, 90, 6320-6324 (1993)] are described using the same approach. Finally, the RT/DNA complex is compared with other dimeric DNA-binding proteins. It was found that the size of the protein and the extent of the dimer interface were not directly related to the extent of contact between the protein and the DNA. Furthermore, RT, the only protein that is not a sequence-specific DNA binding protein in this analysis, had the largest surface of interaction with the nucleic acid.

Binding Sites↗
PubMed1994

Epidermal growth factor enhances oocyte maturation in pigs.

Epidermal Growth Factor (EGF) has been reported to stimulate nuclear maturation in porcine oocytes (Sommer et al., 1992). The objective of this experiment was to test the effect of EGF alone or in combination with gonadotropins and follicular shell coculture, on cytoplasmic maturation of oocytes in vitro. A preliminary experiment tested the effective dose of EGF for stimulation of oocyte nuclear maturation in our culture system. Nuclear maturation rates (MII%) of 67.2 +/- 4.6, 81.1 +/- 8.6, and 80.7 +/- 5.1 for oocyte complexes cultured in the presence of 0.1, 1, and 10 ng/ml EGF, respectively, were significantly higher (P < 0.05) than for oocytes cultured in the absence of EGF (18.1 +/- 9.4%). In the main experiment a 2 x 2 x 2 factorial random complete block design was used to examine the effect of EGF (1.0 ng/ml) alone or in combination with gonadotropins and follicular shell coculture on cytoplasmic maturation. Cytoplasmic maturation was evaluated by the ability of oocytes to decondense sperm nuclei after sperm penetration. EGF alone did not stimulate cytoplasmic maturation in oocytes in vitro (P > 0.05). However, EGF showed a positive interaction (P < 0.05) with gonadotropin treatment on male pronuclear formation. We conclude that EGF alone can stimulate nuclear maturation in pig oocytes, and EGF can interact with gonadotropins to enhance cytoplasmic maturation. A high level of cytoplasmic maturation of in vitro matured pig oocytes could be achieved using a combination of EGF, gonadotropin, and follicular shell supplementation to the culture environment.

Animals↗
PubMed1994

Identification of phosphorylated peptides from complex mixtures using negative-ion orifice-potential stepping and capillary liquid chromatography/electrospray ionization mass spectrometry.

A rapid method for identifying and characterizing sites of phosphorylation of peptides and proteins is described. High-performance capillary liquid chromatography (HPLC) coupled with electrospray ionization mass spectrometry (ESI-MS) is used to distinguish non-phosphorylated and phosphorylated peptides originating from mixtures as complex as enzyme digests. The method relies on the ability to produce a fragment ion characteristic and unique to phosphopeptides (m/z 79, PO3) by stepping the orifice potential of the mass spectrometer as a function of mass. At low m/z values, a high orifice potential is applied to induce extensive fragmentation of the peptide, leading to the formation of the m/z 79 phosphate-derived ion. This method is analogous to that described by Carr et al. for the identification of glycopeptides from enzymatic digestion of glycoproteins (S.A. Carr, M.J. Huddleston, M.F. Bean, Protein Science 2, 183 (1993)). The method was first evaluated and validated for a mixture of non-, mono- and di-phosphorylated synthetic peptides. Both mono- and di-phosphorylated peptides were found to generate fragment ions characteristic of PO3 whereas the non-phosphorylated peptide did not. Application of the method was extended to identifying phosphopeptides generated from an endoprotease Lys-C digestion of beta-casein. Both the expected mono- and tetra-phosphorylated Lys-C peptides were observed and identified rapidly in the LC/SEI-MS analysis. The procedure was used additionally to identify the site(s) of phosphorylation of the cytosolic non-receptor tyrosine kinase, pp60(c-src).

Amino Acid Sequence↗
PubMed1994

FSH-stimulated follicular secretions enhance oocyte maturation in pigs.

Follicular secretions can support cytoplasmic maturation in vitro in the pig. The effects of follicular secretions stimulated in vitro by different combinations of gonadotropins and over different culture periods on cytoplasmic maturation of the pig oocyte were studied. In Experiment 1, follicular shells (including theca and mural granulosa cells) from 5 to 7-mm follicles were cultured in vitro under the stimulation of different combinations of gonadotropins for 48 h, and then the obtained conditioned media were used for oocyte maturation. Oocytes cultured in conditioned medium harvested after treatment of follicular shells with 2.5 mug/ml FSH (FSH-stimulated conditioned medium) yielded a higher percentage of male pronuclear formation than those matured in conditioned medium harvested after culture of follicular shells with a combination of hormones (2.5mug/ml FSH, 2.5 mug/ml LH and 20 ng/ml PRL, FSH-LH-PRL-stimulated conditioned medium; 54.1 vs 28.5%; P=0.001). Addition of the combination of FSH, LH and PRL during the period of oocyte maturation marginally improved male pronuclear formation rates (41.3 vs 55.6%; P=0.06). In Experment 2, follicular shells were cultured under the stimulation of FSH only. Conditioned media were harvested after the first 24 h and the second 24 h of culture. The rates of male pronuclear formation in oocytes matured in these 2 conditioned media did not differ (P=0.65), but were higher than those of oocytes matured in fresh control medium (P<0.03). It is concluded that factors secreted by follicular cells stimulated by FSH alone provide better support for full oocyte maturation in the pig than by combined FSH, LH and PRL treatment.

Journal Article↗
PubMed1994

Cloning and characterization of a cDNA encoding phosphofructokinase from Schistosoma mansoni.

Schistosoma mansoni, a human parasitic worm, depends on anaerobic glycolysis as the main source of energy. Phosphofructokinase (ATP: D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11; PFK) limits the rate of glycolysis in these organisms and it has been found to be a target for some antischistosomal agents. A cDNA clone from this parasite has been isolated and characterized. The cDNA is 3046 base pairs long, contains an open reading frame of 2346 bp and codes for a deduced protein of 781 amino acids. The putative protein encoded by the clone has an exact match with the human muscle PFK of 58% and a 73% match when conserved amino acid substitutions are considered. ATP and Fructose-6-P sites have been identified by crystallographic data in the Escherichia coli and Bacillus stearothermophilus PFKs. There is excellent homology between those PFKs and the schistosome PFK at those sites. The PFK-coding cDNA was expressed in insect cells and was shown to be enzymatically active. Western blot analysis of the recombinant protein in cell extracts gave a positive band with the expected molecular weight of 86 kDa.

Amino Acid Sequence↗
PubMed1994

A monoclonal antibody marker for Alport syndrome identifies the Alport antigen as the alpha 5 chain of type IV collagen.

The nephropathy of Alport syndrome is associated with unique abnormalities of glomerular basement membranes and is caused in many families by mutations in the X-chromosomal gene COL4A5, which encodes the alpha 5 chain of type IV collagen. We have previously reported that Alport epidermal and glomerular basement membranes fail to bind a monoclonal antibody, Mab A7, that reacts with normal epidermal and glomerular basement membranes, and that this abnormality is unique to Alport syndrome. The molecule in normal tissues that reacts with Mab A7 was termed the "Alport antigen". In the present study we used recombinant carboxyterminal noncollagenous (NC1) domains of the alpha 1, alpha 2, alpha 3, alpha 4 and alpha 5 chains of type IV collagen to determine the molecular identity of the Alport antigen. Mab A7 was found to bind specifically to the NC1 domain of the alpha 5 chain of type IV collagen, by ELISA and immunoblotting studies. This finding provides a molecular explanation for the utility of Mab A7 as a marker for the Alport basement membrane defect. Mab A7 can identify the Alport basement membrane defect in those patients in whom COL4A5 mutations prevent incorporation of alpha 5(IV) into basement membranes.

Antibodies, Monoclonal↗
PubMed1994

Conditioned media produced by follicular shells of different maturity affect maturation of pig oocytes.

The objective of this study was to further test the hypothesis that follicular secretions produced by follicular cells with different maturity affect oocyte cytoplasmic maturation differently. A group of nine gilts were slaughtered on Day 17 of the estrous cycle and another group of ten were slaughtered on Day 20. The five largest and five smallest follicles were selected from the fifteen largest healthy follicles dissected from each pair of ovaries and categorized as large and small follicles, respectively. The five follicular shells in each category were pooled and cultured in 10 ml of modified TCM 199 supplemented with 2.5 micrograms FSH/ml for 48 h with use of a mobile culture system. Afterwards, conditioned media were collected and used for maturing immature porcine oocytes. Each of six replicated experiments consisted of two culture dishes for each conditioned medium: Day 17 small (S17) and Day 17 large (L17), and Day 20 small (S20) and Day 20 large (L20) follicular shells. After 47 h culture, oocytes were inseminated with boar spermatozoa and were fixed 11 h post-insemination to examine fertilization. The average diameters of follicles used were 4.4, 5.7, 6.2, and 7.5 mm for S17, L17, S20, and L20, respectively. Nuclear maturation was slightly higher in oocytes matured in conditioned media produced by large follicular shells than in those produced by small follicular shells (97.0 vs. 93.5%, p = 0.045). Penetrability of oocytes was not affected by treatments.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Subunit specificity of mutations that confer resistance to nonnucleoside inhibitors in human immunodeficiency virus type 1 reverse transcriptase.

We constructed plasmid vectors that simultaneously express both the p66 and p51 subunits of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) in Escherichia coli. These vectors allow us to generate HIV-1 RT heterodimers in which either the p66 or the p51 subunit has the wild-type sequence and the other subunit has a specific amino acid substitution. We used these vectors to express HIV-1 RT heterodimers containing several different amino acid substitutions reported to confer resistance to nonnucleoside inhibitors. Most of the amino acid substitutions conferred resistance to nonnucleoside inhibitors R86183 (TIBO) and TSAO-m3T only when present in the p66 subunit of the p66-p51 heterodimer; heterodimers that contained a wild-type p66 subunit and a mutant p51 subunit remained sensitive to the inhibitors. However, there was one mutation, E138K, that conferred drug resistance when the mutation was present in the p51 subunit. The corresponding heterodimer with the E138K mutation in the p66 subunit and a wild-type p51 subunit remained sensitive to the inhibitors. Analysis of the three-dimensional structure of HIV-1 RT indicated that residue 138 of the p51 subunit is in the nonnucleoside inhibitor-binding pocket while residue 138 of the p66 subunit is not. The mutagenesis results, combined with structural data, support the idea that the nonnucleoside inhibitors exert their effects by binding to a hydrophobic pocket in the RT heterodimer and that mutations which give rise to drug resistance directly interfere with the interactions between the nonnucleoside inhibitors and HIV-1 RT.

Antiviral Agents↗
PubMed1994

Linkage of stress-induced hypocalcemia, gastric lesions, and emotional behavior in Wistar-Kyoto rats.

The effects of water-restraint stress on blood calcium levels and gastric pathology and their behavioral relevance were examined in Wistar-Kyoto (WKY) and Wistar rats. The stress induced more severe hypocalcemia (0.32 mM decrease) and gastric lesions (34.6 mm in mean length) in WKY rats than in Wistar rats (0.19 mM and 17.7 mm, respectively). The magnitude of hypocalcemia correlated positively with that of gastric lesions in both strains (WKY, r = 0.59; Wistar, r = 0.69). In the forced-swimming test, WKY rats exhibited a longer immobility time (6.53 min) and a shorter struggling time (0.54 min) than Wistar rats (3.33 and 1.90 min, respectively). The severity of hypocalcemia and gastric lesions correlated positively (r = 0.59 and 0.69, respectively) with the length of immobility time in the WKY rats, while it correlated negatively (r = -0.70 and -0.61, respectively) with the length of struggling time in the Wistar rats. These results suggest that stress-induced hypocalcemia and gastric lesions are closely related and are also influenced by behavioral responsiveness in a strain-dependent manner.

Animals↗
PubMed1994

COL4A5 deletions in three patients with Alport syndrome and posttransplant antiglomerular basement membrane nephritis.

Mutations in the COL4A5 gene encoding the alpha 5 chain of Type IV collagen were identified in three men with Alport syndrome and posttransplant antiglomerular basement membrane (GBM) nephritis by the use of Southern analysis, polymerase chain reaction amplification of genomic DNA, and reverse transcription and amplification of lymphocyte RNA. Two related patients (M.C. and J.M.) exhibited deletion of COL4A5 beginning in the 5'-most portion of the gene and extending through the 3' untranslated region, whereas the third patient (J.E.) had an intragenic deletion encompassing Exons 4 through 47. Combined with previously reported data, these findings suggest that the incidence of deletions of COL4A5, as opposed to other COL4A5 mutations, is much higher in Alport patients who develop posttransplant anti-GBM nephritis than in the general Alport population. Immunofluorescence studies of kidney from Patient J.E. showed no reactivity of GBM with monoclonal antibodies directed against the alpha 3, alpha 4, and alpha 5 chains of Type IV collagen. This finding confirms that the mutation affecting the alpha 5(IV) chain can interfere with the incorporation of alpha 3(IV) and alpha 4(IV) into the GBM.

Adult↗
PubMed1994

[Fang Yizhi, beginner of thinking of confluence and western medicine].

As a famous scholar of late Ming and early Qing Dynasties, Fang Yizhi's academic achievements have been highly praised. However, little was known about his advocation of confluence of Chinese and Western medicine. Through the author's study, it is evident that Fang Yizhi introduced new ideas on human anatomy and physiology spread by western missionaries. His records that "brain is responsible for memory", "brain governs movement and sensation" embodies the description of confluence of Chinese and Western medicine and is the earliest of its kind of his contemporaries. His summary of "zhiche", "tonji" on the art of Chinese and western knowledge is a conclusion in principle and universally accepted in the academic field. The so-called "superior-inferior-spring-autumn", "configuation-nature and Qihua" conception from main exponents of confluence school, Wang Xuequan, Tang Roagehuan, Zhuk Peiwen is derived from Fan's idea.

China↗
PubMed1994

[A new serotype of Salmonella III b].

A new serotype of Salmonella No. S. 3337 was isolated from the intestinal content of reptile a snake in August 1989. Providing with Salmonella biological characteristics, it could be classified into subspecies III b because it utilized sodium malonate, attacked lactose promptly, ONPG positive, and did not ferment dulcitol. H antigens appeared diphasic. Antigenic analysis shows that it represents a new serotype with an antigenic formula of 65 : z : z55.

Animals↗
PubMed1993

Stimulation of neutrophils with a chemoattractant activates several novel protein kinases that can catalyze the phosphorylation of peptides derived from the 47-kDa protein component of the phagocyte oxidase and myristoylated alanine-rich C kinase substrate.

Novel protein kinases that may participate in the signal transduction pathways of neutrophils were sought by a procedure based on the ability of these enzymes to undergo renaturation and catalyze the phosphorylation of a peptide substrate fixed in a gel. We report that neutrophils contain four uncharacterized protein kinases with molecular masses of about 69, 63, 49, and 40 kDa, which are rapidly activated upon stimulation of these cells with the chemoattractant fMet-Leu-Phe. These kinases can catalyze the phosphorylation of a peptide that corresponds to residues 297-331 of the 47-kDa subunit of the NADPH oxidase system (p47-phox). A peptide that corresponds to residues 153-178 of the human myristolyated alanine-rich C kinase substrate (MARCKS) protein was also a substrate for the 69- and 63-kDa kinases. The time course for the activation of these enzymes was similar to the phosphorylation of p47-phox and MARCKS in intact neutrophils. In contrast, stimulation of these cells with 4 beta-phorbol 12-myristate 13-acetate, the calcium ionophore A23187, or the combination of these agonists did not activate these enzymes. Activation of the 63- and 40-kDa protein kinases was blocked by pertussis toxin, calyculin A, and staurosporine. Several other unidentified protein kinases were also active with these peptides but did not exhibit enhanced activity after cell stimulation with this method.

Amino Acid Sequence↗
PubMed1993

Protein kinases potentially capable of catalyzing the phosphorylation of p47-phox in normal neutrophils and neutrophils of patients with chronic granulomatous disease.

A procedure for uncovering novel protein kinases was used to search for enzymes in neutrophils that may catalyze the phosphorylation of the 47-Kd subunit of the NADPH oxidase system (p47-phox). This component of the oxidase can undergo phosphorylation on multiple sites. The method is based on the ability of renatured kinases to recognize exogenous substrates fixed in gels. We report that neutrophils contain several uncharacterized protein kinases that catalyze the phosphorylation of a peptide substrate that corresponds to amino acid residues 297 through 331 of p47-phox. Some of these enzymes are strongly activated on stimulation of the cells with phorbol 12-myristate 13-acetate (PMA). The results indicate that the phosphorylation of p47-phox in neutrophils may be more complicated than previously appreciated and may involve multiple protein kinases. In addition, we have examined both the renaturable protein kinases and the properties of protein kinase C (PKC) in neutrophils from patients with chronic granulomatous disease (CGD) who are deficient in cytochrome b558. Previous studies have shown that these cells exhibit incomplete phosphorylation of p47-phox on stimulation. In this study, we were unable to detect any alterations in the renaturable protein kinases or PKC in CGD neutrophils that could explain these defects in the phosphorylation of p47-phox.

Amino Acid Sequence↗
PubMed1993

Crystal structure of human immunodeficiency virus type 1 reverse transcriptase complexed with double-stranded DNA at 3.0 A resolution shows bent DNA.

The crystal structure of a ternary complex of human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) heterodimer (p66/p51), a 19-base/18-base double-stranded DNA template-primer, and a monoclonal antibody Fab fragment has been determined at 3.0 A resolution. The four individual subdomains of RT that make up the polymerase domains of p66 and p51 are named fingers, palm, thumb, and connection [Kohlstaedt, L. A., Wang, J., Friedman, J. M., Rice, P. A. & Steitz, T. A. (1992) Science 256, 1783-1790]. The overall folding of the subdomains is similar in p66 and p51 but the spatial arrangements of the subdomains are dramatically different. The template-primer has A-form and B-form regions separated by a significant bend (40-45 degrees). The most numerous nucleic acid interactions with protein occur primarily along the sugar-phosphate backbone of the DNA and involve amino acid residues of the palm, thumb, and fingers of p66. Highly conserved regions are located in the p66 palm near the polymerase active site. These structural elements, together with two alpha-helices of the thumb of p66, act as a clamp to position the template-primer relative to the polymerase active site. The 3'-hydroxyl of the primer terminus is close to the catalytically essential Asp-110, Asp-185, and Asp-186 residues at the active site and is in a position for nucleophilic attack on the alpha-phosphate of an incoming nucleoside triphosphate. The structure of the HIV-1 RT/DNA/Fab complex should aid our understanding of general mechanisms of nucleic acid polymerization. AIDS therapies may be enhanced by a fuller understanding of drug inhibition and resistance emerging from these studies.

Base Sequence↗
PubMed1993

Effect of follicle cells and steroidogenesis on maturation and fertilization in vitro of pig oocytes.

Follicle cell numbers, steroidogenesis, and culture conditions were manipulated in a series of experiments designed to identify their effect during in vitro maturation of pig oocytes upon meiotic cycle progression, sperm penetration and male pronuclear formation. In Experiment 1 a static culture system was used to determine the effect of follicle cell numbers on maturation. When the follicle cell number was 3.5 x 10(3) cells per complex or less, cell cycle progression to metaphase II (M-II) and male pronuclear formation were both low (< 30%). Increasing follicle cell numbers to 15 x 10(3) per complex significantly enhanced both meiotic maturation (93%) and male pronuclear formation (83%). A further 10-fold increase in follicle cell numbers (150 x 10(3)), however, reduced subsequent male pronuclear formation (33%). The purpose of Experiment 2 was to determine whether the observed follicle cell effects were influenced by the type of culture system used for oocyte maturation. Comparisons were made between both static and non-static forms of culture. Meiotic progression was significantly depressed when non-static culture systems were used for the maturation of oocyte-cumulus complexes containing low numbers of somatic cells (< 15 x 10(3) cells). However, the addition of somatic cells (follicle shells) to non-static, but not static, culture systems significantly enhanced male pronuclear formation in all groups irrespective of the number of cumulus cells in the oocyte complex. Experiment 3 investigated the effects of the steroid environment on maturation and male pronuclear formation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗