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Biomedical subjects

S Peng

Publications and source records attributed to S Peng.

107 records · Page 6Linked to original sources
PubMed1993

Rapid purification of recombinant baculovirus using fluorescence-activated cell sorting.

Expression of foreign proteins in the baculovirus-insect cell expression system has been limited by difficulties in rapid identification and purification of recombinant virus. Although the process of identifying recombinant virus has been greatly facilitated by the introduction of vectors that lead to insect cell co-expression of beta-galactosidase with foreign genes of interest, isolation of pure recombinant virus using plaque purification may still take several weeks to months to accomplish. Using a fluorescent beta-galactosidase substrate, we have established that insect cells harboring recombinant virus can be rapidly isolated using fluorescence-activated cell sorting. Pure recombinant virus can then be readily obtained using this cellular fraction, with a pure viral culture generally obtained within 2-3 weeks of insect cell transfection.

Animals↗
PubMed1992

Recombinant synthesis, purification, and nucleotide binding characteristics of the first nucleotide binding domain of the cystic fibrosis gene product.

The majority of mutations which lead to clinical cystic fibrosis are located within the two predicted nucleotide binding domains of the cystic fibrosis gene product. We have used a prokaryotic expression system to synthesize and purify the first nucleotide binding domain (NBD-1, amino acids 426-588) with and without the most common mutation associated with the disease (the deletion of phenylalanine at position 508, delta F508). Both wild type and delta F508 NBD-1 bind ATP-agarose in a quantitatively comparable manner; this binding was inhibited by excess Na2ATP, trinitrophenol-ATP, or 8-azido-ATP. Irreversible NBD-1 labeling by an ATP analog was demonstrated using [32P]8-azido-ATP. This covalent labeling was inhibited by preincubation with Na2ATP, with half-maximal inhibition for Na2ATP occurring at approximately 5 mM for both the wild type and delta F508 nucleotide binding domain. These experiments are among the first to confirm the expectation that the cystic fibrosis transmembrane conductance regulator NBD-1 binds nucleotide. Since, under the conditions used in our study, NBD-1 without phenylalanine 508 displays very similar nucleotide binding characteristics to the wild type protein, our results support previous structural models which predict that the delta F508 mutation should not cause an alteration in ATP binding.

Adenosine Triphosphate↗
PubMed1992

Determination of the number of polypeptide subunits in a functional VDAC channel from Saccharomyces cerevisiae.

Genes encoding VDAC proteins containing specific site-directed amino acid alterations were introduced into wild-type Saccharomyces cerevisiae. The mutant VDAC proteins form channels with ion selectivities very different from that of the wild-type channel. Therefore, the resulting yeast strains express two different genes capable of coding for functional, yet distinct, VDAC channels. If VDAC were an oligomeric channel, analysis of VDAC from these strains should have revealed not only the presence of channels with wild-type or mutant selectivity but also channels with intermediate selectivities. While channels with wild-type and mutant selectivities were observed with approximately equal frequency, no channels with intermediate selectivity were observed. Sufficient observations were performed with two different mutant genes K61E.K65E and K19E.K61E) that the likelihood of having missed hybrid channels was less than 1 in 10(7). These findings favor the hypothesis that each functional VDAC channel is composed of a single 30-kDa polypeptide chain.

Fungal Proteins↗
PubMed1992

Large scale rearrangement of protein domains is associated with voltage gating of the VDAC channel.

The VDAC channel of the mitochondrial outer membrane is voltage-gated like the larger, more complex voltage-gated channels of the plasma membrane. However, VDAC is a low molecular weight (30 kDa), abundant protein, which is readily purified and reconstituted, making it an ideal system for analyzing the molecular basis for ion selectivity and voltage-gating. We have probed the VDAC channel by subjecting the cloned yeast (S. cerevisiae) VDAC gene to site-directed mutagenesis and introducing the resulting mutant channels into planar bilayers to detect the effects of specific sequence changes on channel properties. This approach has allowed us to formulate and test a model of the open state structure of the VDAC channel. Now we have applied the same approach to analyzing the structure of the channel's low-conducting "closed state" (essentially closed to important metabolites). We have identified protein domains forming the wall of the closed conformation and domains that seem to be removed from the wall of the pore during channel closure. The latter can explain the reduction in pore diameter and volume and the dramatically altered channel selectivity resulting from the channel closure. This process would make a natural coupling between motion of the sensor and channel gating.

Amino Acid Sequence↗
PubMed1992

A case-control study of dietary factors in patients with lung cancer.

A case-control study was designed to investigate association of dietary factors with the risk of lung cancer in Sichuan, China. The cases consisted of 135 patients with preinvasive lung cancer which had been confirmed with histopathology, fiber bronchoscope, CT and X-ray film in three provincial hospitals in the recent one year. Controls were healthy subjects who went to one of these hospitals for health check-up; patients with pulmonary diseases was excluded. Controls were matched to cases for sex and age with a ratio of 1:1. Nutrient intakes, the eating habit and other relevant factors were investigated. The data analyzed with the conditional logistic regression model indicated that dietary beta-carotene intakes had a significantly inverse association with the risk of lung cancer. Vitamin C had a less significantly inverse association with the risk. Association of protein, fat, energy, retinol intakes or diet-balance index with the risk was not significant. Association of tea, alcohol, garlic or mushroom, respectively, with the risk was also not observed. Consumption of more processed foods and deep-fried foods were found to be risk factors. Smoking and air pollution from coal burning stoves were also observed as independent risk factors of lung cancer in the present study. The mental stress incidence in the case was significantly higher than that in the control.

Adenoma↗
PubMed1992

[Improving the method of processing Polygonum cuspidatum Sieb. et Zucc. and Spatholobus suberectus Dunn].

This paper is concerned with a comparison of Polygonum cuspidatum with Spatholobus suberectus in cutting, drying time, products, chief compositions and aqueous extracts. The results showed that fresh cut was better than the customary cut, i. e. re-cut pieces, for the former could not only raise the quality specifications and contents of ingredients of the drinking tablets, but also avoid repeated labour, save energy for drying and eliminate waste of drug materials.

Anthraquinones↗
PubMed1991

Determination of dietary cadmium-induced metallothioneins in rabbit kidneys and cadmium in metallothioneins by anion-exchange high-performance liquid chromatography coupled with graphite furnace atomic absorption spectrometry.

A rapid method is described for the determination of dietary cadmium-induced metallothioneins (MTs) in rabbit kidneys by anion-exchange high-performance liquid chromatography. Rabbit kidney MT-I and MT-II were eluted at ca. 15.0 and 18.8 min, respectively, from a DEAE-5PW anion-exchange column with a Tris-HCl buffer (0.01-0.25 M, pH 8.6) and detected by ultraviolet absorbance at 254 nm. A standard calibration curve was constructed using purified standard MT isoforms, which demonstrated an excellent linear correlation between UV absorbance peak heights and the amounts of MT isoforms. Feeding a dose of cadmium for some days resulted in an increase in MT concentrations in rabbit kidneys, but not in the livers. The cadmium concentrations in MT-I and MT-II elutions were determined by graphite furnace atomic absorption spectrometry. MT-I and MT-II showed some differences associated with the oral intake of cadmium. Dietary cadmium also caused zinc to accumulate in kidneys to some extent. The effects of dietary oleic acid on the synthesis of MTs were also studied. Based on the method of standard additions, the recovery of MTs exceeded 93% and replicated injection of samples yielded a relative standard deviation of 2.4% at an MT level of 280 micrograms/g.

Animals↗
PubMed1990

Selectivity changes in site-directed mutants of the VDAC ion channel: structural implications.

The gene encoding the yeast mitochondrial outer membrane channel VDAC was subjected to site-directed mutagenesis to change amino acids at 29 positions to residues differing in charge from the wild-type sequence. The mutant genes were then expressed in yeast, and the physiological consequences of single and multiple amino acid changes were assessed after isolation and insertion of mutant channels into phospholipid bilayers. Selectivity changes were observed at 14 sites distributed throughout the length of the molecule. These sites are likely to define the position of the protein walls lining the aqueous pore and hence, the transmembrane segments. These results have been used to develop a model of the open state of the channel in which each polypeptide contributes 12 beta strands and one alpha helix to form the aqueous transmembrane pathway.

Amino Acid Sequence↗
PubMed1990

Mitogen-activation of spleen cells in aged animals is potentiated by dietary restriction: a preliminary report.

Dietary restriction has been reported to be associated with increased life span and increased DNA repair capacity in both male and female rats and mice. We examined dietary restriction effects on immune system function and prolongation of life span in specific pathogen free rats and mice. In this preliminary report the authors show that dietary restriction is correlated with a dramatic increase in the lifespan of both male and female rats and mice in a pathogen free environment, and in the capacity of cultured splenocytes from those animals to initiate blastogenesis in response to antigenic stimulation.

Aging↗
PubMed1989

Age-related changes in DNA polymerase alpha expression.

DNA polymerase alpha isozymes differing in specific activity and affinity of binding to DNA were purified from human fibroblasts derived from donors of different ages. Fetal-derived fibroblasts expressed a single, high-activity enzyme (A2), with high affinity of binding to DNA. Adult-derived fibroblasts exhibited two forms of DNA polymerase alpha, one identical to the fetal enzyme, and a second with about tenfold less activity showing low affinity of binding to DNA (A1). The ratio of DNA polymerase A2/A1 decreased dramatically with age, from 100% A2 in fetal-derived fibroblasts to about 94% A1 in fibroblasts derived from a 66-year-old donor. The DNA binding affinity of polymerase alpha A1 from adult-derived fibroblasts increased concomitant with a significant increase in activity when the enzyme was treated with phosphatidylinositol-4-monophosphate (PIP), or with inositol-1, 4-bisphosphate (I(1,4)P2). The enzyme reverted back to a less active form, with loss of the noncovalently bound I(1,4)P2, as a function of time. When permeabilized human fibroblasts with low DNA excision repair capacity were treated with 7,8-dihydrodiol-9,10-epoxybenzo(a)-pyrene (BPDE) in the presence of 32P-ATP, phosphatidylinositol, and cycloheximide, excision repair was initiated and 32P-labeled DNA polymerase alpha was recovered in the absence of de novo protein synthesis. DNA synthesis associated with either scheduled DNA synthesis or BPDE-initiated excision repair declined as a function of increased age in human cells. The data suggest that the decline in both DNA excision repair-associated and mitogen-activated DNA synthesis may be correlated with decreased total intracellular levels of DNA polymerase and with the decline in polymerase alpha activity as a function of age, that DNA repair-associated initiation of DNA synthesis in adult-derived cells may increase with activation of a pool of low activity DNA polymerase alpha, and that DNA polymerase alpha activity increases as a function of enzyme interaction with a component of the PI phosphorylation cascade.

Cell Survival↗
PubMed1988

Effects of gonadotropin-releasing hormone analogs on cis-platinum-induced spermatogenic damage.

In order to test the hypothesis that pretreatment with gonadotropin-releasing hormone (GnRH) analogs might ameliorate cytotoxic drug-induced testicular damage, mature male Wistar rats were pretreated for 2 weeks with a GnRH superactive agonist or a pure GnRH antagonist prior to, and for 1 week after, a 5 mg/kg intraperitoneal dose of cis-platinum. Despite inhibition of testicular function by both GnRH analogs prior to cis-platinum administration, there was no evidence of protection or enhanced recovery of spermatogenesis at 6 and 12 weeks after cis-platinum treatment, and spermatogenesis was significantly further depressed at both time-points by both GnRH agonist and antagonist pretreatment. This suggests that pretreatment with GnRH analogs in the rat does not protect spermatogenesis from cis-platinum-induced testicular damage within up to two spermatogenic cycles and that hypogonadism at the time of cytotoxic drug treatments may aggravate testicular damage.

Animals↗
PubMed1984

Effects of chronic D-Leu6, des-Gly10-gonadotropin releasing hormone ethylamide on male sex tissues.

The chronic administration of superactive agonists of gonadotropin releasing hormone (GnRH-A) have been reported to have a direct inhibitory effect on the sex tissues of the male rat. In an attempt to confirm or refute this statement, adult male rats were either left intact or were castrated and then treated daily for 14 days with either testosterone (T), dihydrotestosterone (DHT) or sesame oil (vehicle). Half of the intact and castrate animals also received daily injections of 200 ng of the GnRH agonist, D-Leu6, des-Gly10-GnRH ethylamide for 14 days. Twenty-four hours after completing treatment, blood levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH) and T were measured by radioimmunoassay and the ventral prostate gland (VP), seminal vesicle (SV) and penis were weighed. After 2 weeks of GnRH-A treatment, the plasma T level was reduced from 2506 +/- 170 (pg/ml +/- SEM) in the intact, nontreated animals to 907 +/- 69 in the intact, GnRH-A-treated group, indicating that the dosage of GnRH-A used in this study had an inhibiting effect on T secretion. No differences were observed in the VP, SV and penile weights between the castrate, GnRH-A and the castrate, nontreated groups. When exogenous T or DHT was given for 14 days to these castrated animals, the concomitant administration of GnRH-A did not appear to have any effect on the plasma T levels or the sex accessory tissue weights. These data suggest that GnRH-A itself does not appear to have a direct inhibitory or stimulatory effect on the sex tissues of the adult male rat.

Animals↗
PubMed1983

Synergy of abdominal pressure and androgens in testicular descent.

The potential synergistic activity between intra-abdominal pressure and androgens in facilitating testicular descent was investigated in the rat. In the rat, the testis normally descends on or about the 21st day of age. If one testis is excised at 14 days of age and replaced by a silicone prosthesis, the silicone prosthesis will descend into the scrotum in approximately 11/20 (55%) of the animals by 28 days of age. If both testes are excised at 14 days of age and a solitary silicone prosthesis is then placed into the abdominal cavity, the prosthesis will descend into the scrotum in approximately 4/18 (22%) of the animals. However, if both testes are excised at 14 days of age and a silicone prosthesis is placed into the abdominal cavity but the animals are treated daily from Day 14 to Day 27 of age with 2 mg of dihydrotestosterone, the prosthesis will at 28 days of age descend into the scrotum in 10/16 (63%) of the animals treated. These data suggest that: 1) androgens do not appear to be solely responsible for testicular descent in the rat; 2) another mechanism such as intra-abdominal pressure may be operative in facilitating testicular descent in this animal species; and 3) there may be some synergism between androgens and intra-abdominal pressure in promoting testicular descent.

Abdomen↗
PubMed1976

Ultraviolet photoelectron studies of biological purines: the valence electronic structure of adenine.

The UV photoelectron spectra of adenine, 9-methyladenine, and 6-methylaminopurine contain highly resolved bands arising from the six highest occupied molecular orbitals. The spectra have been analyzed using UV absorption data, photoelectron data from previous studies of heterocyclic compounds, and results from both semi-empirical and ab initio molecular orbital calculations. The analysis indicates that the first, third, and fifth photoelectron bands in adenine and the two methyl substituted derivatives arise from pi orbitals. The second, fourth, and sixth bands arise from nitrogen atom lone-pair orbitals. Compared to adenine, the six uppermost orbitals of 9-methyladenine and 6-methylaminopurine have lower ionization potentials. This destabilization of the valence electrons is expected to play an important role in causing the increase in base stacking forces observed in methyl substituted adenines.

2-Aminopurine↗
PubMedDate not supplied

Temporal neocorticectomy in management of intractable epilepsy: long-term outcome and predictive factors.

We report the results of a long-term follow-up study of 50 patients who underwent removal of temporal neocortex with preservation of deeper limbic structures as surgical therapy for intractable temporal lobe epilepsy. The follow-up period ranged from 3 to 15 years. Preoperative EEG investigations were based on interictal discharges alone. Three factors were predictive of a good outcome: (a) A clear unilateral anterior-midtemporal focus (p less than 0.01), (b) stereotypical onset of temporal lobe seizure (p less than 0.005), and (c) greater volume of tissue removed at operation (p less than 0.05). Overall results showed that 62% of patients experienced an outcome of "cure" or "almost cure," as classified according to a modified version of Crandall's criteria (Crandall's I and II). Those who experienced a significant reduction in seizures but who continued to have intractable epilepsy (Crandall's III) were not considered to have had a good result. Overall outcome compares favorably with other that of centers using different surgical approaches and indicates that neocorticectomy is a suitable procedure in a highly selected population even when limited resources are available.

Adaptation, Psychological↗