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Biomedical subjects

J Dong

Publications and source records attributed to J Dong.

At least 181 records · Page 10Linked to original sources
PubMed1996

Growth differentiation factor-9 is required during early ovarian folliculogenesis.

Growth factors synthesized by ovarian somatic cells directly affect oocyte growth and function, but it is unclear whether oocyte-secreted factors play a reciprocal role in modulating somatic cell functions in vivo. During the functional analysis of members of the transforming growth factor-beta superfamily in mouse development, we have uncovered a new family member, growth differentiation factor-9 (GDF-9), which is required for ovarian folliculogenesis. GDF-9 messenger RNA is synthesized only in the oocyte from the primary one-layer follicle stage until after ovulation. Here we analyse ovaries from GDF-9-deficient female mice and demonstrate that primordial and primary one-layer follicles can be formed, but there is a block in follicular development beyond the primary one-layer follicle stage which leads to complete infertility. Oocyte growth and zona pellucida formation proceed normally, but other aspects of oocyte differentiation are compromised. Thus, GDF-9 is the first oocyte-derived growth factor required for somatic cell function in vivo.

Animals↗
PubMed1996

Characterization of the mononickel metallocenter in H134A mutant urease.

A mutant form of Klebsiella aerogenes urease possessing Ala instead of His at position 134 (H134A) is inactive and binds approximately half the normal complement of nickel (Park, I.-S., and Hausinger, R. P.(1993) Protein Sci. 2, 1034-1041). The crystal structure of the H134A protein was obtained at 2.0-A resolution, and it confirms that only Ni-1 of the two nickel ions found in the native enzyme is present. In contrast to the pseudotetrahedral geometry observed for Ni-1 in native urease (where it is liganded by His-246, His-272, one oxygen atom of carbamylated Lys-217, and a water molecule at partial occupancy), the mononickel metallocenter in the H134A protein was found to possess octahedral geometry and was coordinated by the above protein ligands plus three water molecules. The nickel site of H134A urease was probed by UV-visible, variable temperature magnetic circular dichroism, and x-ray absorption spectroscopies. The spectroscopic data are consistent with the presence of Ni(II) in octahedral geometry coordinated by two histidylimidazoles and additional oxygen and/or nitrogen donors. These data underscore the requirement of Ni-2 for formation of active urease and demonstrate the important role of Ni-2 in establishing the proper Ni-1 coordination geometry.

Binding Sites↗
PubMed1996

The role of arsenic-thiol interactions in metalloregulation of the ars operon.

The ars operon of the Escherichia coli plasmid R773 that confers arsenical and antimonial resistance is negatively regulated by the ArsR repressor. ArsR residues Cys-32 and Cys-34 were previously identified as involved in induction by arsenite and antimonite, suggesting coordination between As(III) and the two cysteine thiolates. However, in small molecule thiolate-As(III) complexes, arsenic is frequently three-coordinate. A site-directed mutagenic approach was employed in a search for a third arsenic ligand. ArsR proteins with C32G, C34G, and C32G/C34G substitutions were active repressors, but were not inducible in vivo. In vitro, the altered repressor-ars DNA complexes could not be dissociated by inducers. Alteration of Cys-37 and Ser-43, residues located in or near the putative helix-turn-helix DNA-binding region of the protein, had no effect on the inducibility of the operon. While these results indicated that neither the thiolate of Cys-37 nor the hydroxyl oxygen of Ser-43 is required for induction, they did not eliminate either atom as a potential arsenic ligand. Another approach involved reaction with an alternative inducer, phenylarsine oxide, which can form only two coordinations. Phenylarsine oxide was shown to be as effective as or more effective than arsenite or antimonite in induction in vivo. In vitro, the organic arsenical was more effective than either arsenite or antimonite in dissociating the repressor-promoter complex. Thus, two ArsR-arsenic bonds are sufficient for induction. The interaction of ArsR proteins with As(III) was examined using a phenylarsine oxide affinity resin. ArsR proteins containing any two of the three cysteine residues Cys-32, Cys-34, and Cys-37 bound to the resin. Alteration of any two of the three resulted in loss of binding. Arsenic X-ray absorption spectroscopy of ArsR treated stoichiometrically with arsenite confirmed the average arsenic coordination as AsS3 These results suggest that all three cysteine thiolates are arsenic ligands, but binding to only two, the Cys-32 and Cys-34 thiolates, is required to produce the conformational change that results in release of the repressor from the DNA and induction.

Amino Acid Sequence↗
PubMed1996

Coexpression of mammalian cytochrome P450 and reductase in Escherichia coli.

cDNAs for human cytochrome P450 2E1 and rat NADPH-cytochrome-P450 reductase were cloned separately and in tandem into bacterial expression vectors, and expression of the two proteins in Escherichia coli was monitored by immunoblotting, spectroscopy, and catalytic assays. The cDNAs were separated on the coexpression plasmid by 22 nucleotides, with the P450 cDNA preceding the reductase cDNA. P450 content in solubilized cell membranes, whether expressed alone or coexpressed with P450 reductase, was approximately 0.11 nmol/mg of protein, and approximately 0.8 nmol could be obtained per liter of culture. Reductase content was five- to sixfold greater than P450 content when coexpressed, but severalfold less than that obtained when expressed without the upstream P450 cDNA, indicating differences in both stability and translatability between the two proteins. Solubilized membranes from cells expressing both proteins catalyzed aniline hydroxylation, p-nitrophenol hydroxylation, and N-nitrosodimethylamine demethylation at rates equivalent to those obtained by combining P450 and reductase preparations; addition of purified reductase to these membranes did not augment the activity. However, in contrast to results obtained with P450 2E1 expressed in other heterologous systems, addition of rabbit liver cytochrome b5 to preparations catalyzing p-nitrophenol or N-nitrosodimethylamine oxidation did not increase turnover, and, although activity could be shown with unsolubilized membranes, oxidation of these substrates in vivo could not be demonstrated. Nonetheless, the ability to coexpress P450 and reductase in E. coli so as to generate a functional monooxygenase system in vitro enhances the utility of this organism for the expression and characterization of cloned P450 isoforms.

Animals↗
PubMed1996

Microsurgical replantation of the avulsed scalp: report of 20 cases.

Our 4-year experience with 20 patients who had suffered avulsion of 75 percent or more of the scalp is reviewed. All patients underwent replantation using microsurgical technique with 100 percent survival in 16, partial survival in 3, and failure in only 1 case. The emergency management and indications for replantation are demonstrated. The roles of sufficient preoperative preparation, generous debridement of damaged vessels, interpositional vein grafts, and the shortening of operative time in contributing to this success are emphasized. We developed a new surgical procedure called simultaneous vein grafts on donor and recipient sites in an effort to use less time in the anastomosis of interpositional vein grafts. Furthermore, we anastomosed the extra artery of the scalp to the vein on the recipient head when no suitable vein could be found. Intraoperative repair of the scalp sensory nerve and no postoperative use of any vasodilator or anticoagulant are discussed.

Adolescent↗
PubMed1996

Ultra low temperature cryopreservation of somatic embryogenic cell line of foxtail millet [Setaria italica (L.) Beauv].

Ultra low temperature cryopreservation is one of the methods for preservation of biological material. Until now, a major problem of protoplast culture of Gramineae is the instability of state of the somatic embryogenic cell line. In our experiments, elements affecting the ultra low temperature cryopreservation of somatic embryogenic cell line were studied: components of cryopreserve solution, somatic embryogenic cell line of different subculture time, growth recovery of cryopreserved cell line, and their protoplast cultures. Results demonstrated that the ultra low temperature cryopreservation did not change the properties of protoplast culture, and by using the cryopreserve method, plating efficiency of protoplast culture of cryopreserved cell line was maintained or enhanced.

Cell Line↗
PubMed1996

[Studies on relationship between toxicity of trichothecene toxin T-2 and its structure].

Toxic action of mycotoxin T-2 and its metabolite T-2 tetraol and deepoxy T-2; tetraol was studied by cytotoxicity and animal toxicity tests to explore the relationship between toxin T-2 and its structure. Restults showed that proliferation of LLC-PK1 cells and synthesis of DNA could be inhibited by both toxin T-2 and T-2 tetraol. Toxicity of toxin T-2 was 100 times greater than that of T-2 tetraol. There was no obvious toxicity to the proliferation of LLC-PK1 cells and synthesis of DNA in a dose of 10 mg/L deepoxy T-2 tetraol. But, toxin T-2 caused obvious damage in heart muscle and articular cartilage of chicken embryos, and T-2 tetraol and deepoxy T-2 tetraol in heart muscle, but not in articular cartilage. T-2 tetraol produced by hydrolysis of toxin T-2 had toxicity to certain extent, but its strength was significantly less than that of the latter. It suggests that the epoxide group in toxin T-2 played a determinant role for its toxicity. If epoxy cycle of the basic nucleus of the molecule is open, its toxicity will change significantly.

Animals↗
PubMed1996

[Inferior mesocaval shunt].

The results of inferior mesenteric vein-inferior vena cava shunt coupled with splenectomy and gastroesophageal devascularization in 16 patients with cirrhotic portal hypertention were reviewed. The mean diameter of the shunt stoma was 6.46 +/- 0.78 mm, and the portal pressure decreased from 4.25 +/- 0.54 kPa to 3.39 +/- 0.41 kPa. Variceal bleeding stopped in all 16 patients with no severe postoperative complications. This procedure should preserve satisfactory portal perfusion to the liver, and technically is easier than other shunt procedures.

Adult↗
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↗
PubMed1996

[A study on the expression of three cytokine-receptors and airway hyperreactivity in asthmatics after specific provocation].

OBJECTIVE: In order to study the role of T and B lymphocytes activation in the pathogenesis of asthma. METHOD: Pulmonary function tests were performed and percentages of three cytokine receptors and the levels of sIL-2R, sIgE and TIgE were measured in 31 allergic asthmatics and 12 healthy subjects before and after specific provocation. RESULTS: It was found that the serum levels of sIL-2R (t = 5,719, 5,647 P < 0.01), TIgE were measured in 31 allergic asthmatics and 12 healthy (t = 6,306, 7,218 P < 0.01) and percentages of CD+23 (chi(2) -23.43, 18.56 P < 0.01) or CD+(25) (chi(2) = 27.59, 15. 28) cells were significantly higher in asthmatics after challenge than those in controls or in asthmatics before challenge as well; There were significantly negative correlations between sIL-2R and FEV1 or sGaw (r = -0.573, -0.426 P < 0.01), whereas positive correlations between sIL-2R and Raw or percent of IL-2R/CD+(25) cells (r = 0.417, 0.532 P < 0.01); TIgE and percent of Fc epsilon R II/CD+(23) cells (r = 0.603 P < 0.01), respectively. CONCLUSION: These findings suggest that T cell activation, increased serum levels of sIL-2R and increased expression of CD+(25) or CD+(23) on cells, may lead to enhanced AHR in allergic asthmatics.

Adult↗
PubMed1995

Bursting and oscillating neurons of the cat basolateral amygdaloid complex in vivo: electrophysiological properties and morphological features.

1. To characterize the physiological properties of lateral and basolateral (BL) amygdaloid neurons, intracellular recordings were performed in barbiturate-anesthetized cats. Morphological identification of recorded cells was achieved by intracellular injection of neurobiotin. Two types of physiologically identified projection neurons were distinguished in the BL and lateral nuclei. 2. The first type of neurons prevailed in the BL nucleus (80% of BL cells). Their resting membrane potential (Vm) averaged -66 +/- 4.9 (SE) mV. They generated stereotyped spike doublets or bursts in response to threshold depolarizing pulses. In most cells, depolarizing pulses of higher amplitude elicited spike bursts or doublets at a shorter latency followed by a nonadapting train of single spikes whose frequency rose with the amplitude of the current pulses. However, 15% of BL bursting neurons generated repetitive spike bursts or doublets in response to prolonged depolarizing current pulses. The response of BL bursting neurons to hyperpolarizing current pulses revealed the presence of slow inward rectification in the form of a depolarizing sag, thus suggesting the presence of a hyperpolarization-activated current. 3. The second type of neurons prevailed in the lateral nucleus. Their resting Vm was quite polarized (-74 +/- 2.85 mV) and they generated slow Vm oscillations (2-10 Hz) upon steady depolarization beyond congruent to -62 mV. The frequency of the oscillation increased with the amount of depolarizing current. In the majority of cells, analysis of voltage responses to subthreshold current pulses revealed the presence of voltage- and time-dependent rectification in the depolarizing direction. Current pulses that brought the Vm to -65 mV and beyond elicited a voltage response that reached an early peak and then decayed. Increasing the amplitude of the pulse decreased the latency of the early peak until it triggered an action potential. Current-voltage plots demonstrated inward rectification in the depolarizing direction. At the break of hyperpolarizing current pulses applied at depolarized levels, the Vm overshot prepulse values and generated one or more oscillatory cycles. 4. An important proportion of bursting and oscillating neurons (45.8% and 29%, respectively) were physiologically identified as projection neurons by antidromic invasion from the basal forebrain, entorhinal cortex, or perirhinal cortex. The conduction velocity of bursting and oscillating neurons estimated from the latency of antidromic spikes was low (< or = 2.5 m/s). 5. Most bursting and oscillating neurons of the BL nucleus were spiny cells with a pyramidal morphology. Four to eight dendritic trunks emerged from the apex, base, and sides of their triangular soma.(ABSTRACT TRUNCATED AT 400 WORDS)

Amygdala↗
PubMed1995

Cationic lipids for reporter gene and CFTR transfer to rat pulmonary epithelium.

Increasing evidence indicates that cationic liposomes are capable of safely transferring foreign genes to pulmonary epithelium in vitro and in vivo. To transfer reporter genes and the cystic fibrosis transmembrane conductance regulator (CFTR) to mammalian respiratory epithelium we used two cationic lipid formulations: N-[1-(2,3-dioleoyloxy)propyl] N,N,N-triethylammonium chloride (DOTMA), and 1,2-dimyristyloxy-propyl-3-dimethylhydroxyethylammonium bromide (DMRIE) at a 1:1 molar ratio with dioleoyl phosphatidylethanolamine (DOPE). Lipid-DNA conjugates containing either CFTR or LacZ were instilled directly into the airways of Sprague-Dawley rats. Rats treated with LacZ cDNA in vivo demonstrated expression in 30-50% of the large and medium-sized airways, with some airways showing high efficiency gene transfer and expression (in the most proximal airways, 70-80% of surface epithelial cells were positive for expression of a nuclear targeted LacZ). While control and LacZ treated tracheas mounted in Ussing chambers showed minimal stimulation of transepithelial chloride (Cl)-currents by cAMP (suggesting low levels of endogenous rat CFTR activity), tracheas taken from animals receiving CFTR exhibited significant forskolin-stimulated currents at 72 h after gene transfer. Human CFTR gene expression was also detected by polymerase chain reaction (PCR) analysis of reverse transcribed lung RNA. These results, together with previous studies using lipid-mediated gene transfer in mice, help confirm the potential for cationic lipid-mediated gene transfer in the gene therapy of cystic fibrosis in humans.

Animals↗