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Biomedical subjects

P Huang

Publications and source records attributed to P Huang.

At least 127 records · Page 7Linked to original sources
PubMed1998

Excision of mismatched nucleotides from DNA: a potential mechanism for enhancing DNA replication fidelity by the wild-type p53 protein.

The tumor suppressor p53 plays a critical role in the regulation of the cell cycle and the maintenance of genetic stability. The 3'-->5' exonuclease activity of p53 has recently been recognized as a novel biochemical function of this molecule, but the biological significance of this activity remains elusive. Using an in vitro DNA replication assay with purified human DNA polymerases, p53 protein, and defined DNA primer/templates, we demonstrated that the wild-type (wt) p53 protein, but not the mutant p53 protein, specifically enhanced the DNA replication fidelity of polymerase (pol) alpha, an enzyme that lacks 3'-->5' exonuclease activity. The misincorporation of non-complementary deoxynucleotides into DNA by pol alpha was substantially decreased by p53. In contrast, wt p53 showed no significant effect on replication fidelity or the rates of DNA synthesis by human pol epsilon or the bacterial enzyme pol I. Inhibition of 3'-->5' exonuclease activity by guanosine monophosphate (GMP) abolished the ability of p53 to enhance the replication fidelity of pol alpha. Quantitative analyses revealed that the 3'-->5' exonuclease activity of p53 effectively removed mismatched nucleotides from DNA in preference over matched nucleotides. Furthermore, study in intact cells revealed that the wt p53 protein was co-localized with DNA synthesis activity in S phase cells. These results suggest a possibility that the 3'-->5' exonuclease of the wt p53 protein might provide the proof-reading function for DNA pol alpha. The preferential excision of mismatched nucleotides from the replicating DNA strand appears to be a potential biochemical mechanism by which p53 enhances DNA replication fidelity and thereby helps to maintain genomic integrity.

Cell Nucleus↗
PubMed1998

Stroke protection by 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitors mediated by endothelial nitric oxide synthase.

The treatment of ischemic strokes is limited to prophylactic agents that block the coagulation cascade. Here, we show that cholesterol-lowering agents, 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitors, protect against cerebral injury by a previously unidentified mechanism involving the selective up-regulation of endothelial NO synthase (eNOS). Prophylactic treatment with HMG-CoA reductase inhibitors augments cerebral blood flow, reduces cerebral infarct size, and improves neurological function in normocholesterolemic mice. The up-regulation of eNOS by HMG-CoA reductase inhibitors is not associated with changes in serum cholesterol levels, but is reversed by cotreatment with L-mevalonate and by the downstream isoprenoid, geranylgeranyl pyrophosphate and not by farnesyl pyrophosphate. The blood flow and neuroprotective effects of HMG-CoA reductase inhibitors are completely absent in eNOS-deficient mice, indicating that enhanced eNOS activity by HMG-CoA reductase inhibitors is the predominant if not the only mechanism by which these agents protect against cerebral injury. Our results suggest that HMG-CoA reductase inhibitors provide a prophylactic treatment strategy for increasing blood flow and reducing brain injury during cerebral ischemia.

Animals↗
PubMed1998

Separation of tryptic digests using a modified buffer in pressurized capillary. Electrochromatography with an ion trap storage/reflectron time-of-flight mass spectrometer.

A rapid and efficient separation method using pressurized capillary electrochromatography (pCEC) has been developed to separate protein digests. The effects of mobile-phase ion conductivity, pH, and column size on separation speed and column efficiency were studied and optimized. The pCEC method was demonstrated to provide enhanced speed with more efficient and selective separation than HPLC by performing separations of a bovine beta-lactoglobulin A digest. A human hemoglobin digest was separated by pCEC under optimized conditions, and more than 20 peaks were separated in less than 20 min. Using an ion trap storage/reflectron time-of-flight mass spectrometer, coeluting components were clearly identified, and the resolving power of the pCEC method was further enhanced.

Animals↗
PubMed1998

Muscarinic cholinergic regulation of cardiac myocyte ICa-L is absent in mice with targeted disruption of endothelial nitric oxide synthase.

Cardiac myocytes have been shown to express constitutively endothelial nitric oxide synthase (eNOS) (nitric oxide synthase 3), the activation of which has been implicated in the regulation of myocyte L-type voltage-sensitive calcium channel current (ICa-L) and myocyte contractile responsiveness to parasympathetic nervous system signaling, although this implication remains controversial. Therefore, we examined the effect of the muscarinic cholinergic agonist carbachol (CCh) on ICa-L and contractile amplitude in isoproterenol (ISO)-prestimulated ventricular myocytes isolated from adult mice, designated eNOSnull mice, with targeted disruption of the eNOS gene. Although both eNOSnull and wild-type (WT) ventricular myocytes exhibited similar increases in ICa-L in response to ISO, there was no measurable suppression of ICa-L by CCh in cells from eNOSnull mice, in contrast to cells from WT mice. These results were reflected in the absence of an effect of CCh on the positive inotropic effect of ISO in eNOSnull myocytes. Also, unlike myocytes from WT animals, eNOSnull myocytes failed to exhibit an increase in cGMP content in response to CCh. Nevertheless, the pharmacologic nitric oxide donors 3-morpholino-sydnonimine and S-nitroso-acetyl-cystein increased cGMP generation and suppressed ISO-augmented ICa-L in eNOSnull cells, suggesting that the signal transduction pathway(s) downstream of eNOS remained intact. Of importance, activation of the acetylcholine-activated K+ channel by CCh was unaffected in atrial and ventricular eNOSnull myocytes. These results confirm the obligatory role of eNOS in coupling muscarinic receptor activation to cGMP-dependent control of ICa-L in cardiac myocytes.

Animals↗
PubMed1998

Lysophosphatidylglycerol: a novel effective detergent for solubilizing and purifying the cystic fibrosis transmembrane conductance regulator.

Similar to the recombinant cystic fibrosis transmembrane conductance regulator (CFTR) expressed in Sf9 insect cells, underglycosylated CFTR expressed in yeast is not effectively solubilized by a variety of commonly used detergents, requiring instead harsh alkali and SDS treatments, which would denature most proteins. Moreover, solubilized CFTR has a strong tendency to aggregate and form high-molecular-weight aggregates during subsequent purification. We report here that the mild detergent, lysophosphatidylglycerol (LPG), is a very effective detergent for solubilizing the CFTR expressed in both yeast and Sf9 insect cells. LPG solubilizes nearly 100% of the CFTR in yeast in the absence of NaCl and none in the presence of 1 M NaCl. It is also very potent in preventing aggregation of the CFTR during subsequent purification. Exploiting these characteristics, a rapid simple procedure for the purification of functional recombinant CFTR expressed in yeast has been developed. It includes selective CFTR solubilization in the presence and the absence of NaCl followed by nickel-chelate chromatography of His-tagged CFTR. The CFTR produced by this procedure is about 70% pure. Purified CFTR molecules were reconstituted into liposomes and then fused to planar lipid bilayers for single-channel recording. The reconstituted CFTR exhibits regulatory chloride channel activities with a slope conductance of 7.1 pS and a reversal potential of -32 mV. The effectiveness and simplicity of this new purification procedure for the CFTR should greatly facilitate a variety of biochemical and biophysical studies of this important protein. Furthermore, the potency of LPG in solubilizing the notoriously intractable underglycosylated CFTR suggest that this detergent may be useful for solubilizing the CFTR from other sources and for other difficult membrane proteins as well.

Animals↗
PubMed1998

On-line analysis by capillary separations interfaced to an ion trap storage/reflectron time-of-flight mass spectrometer.

The interface of high-resolution capillary separation methods to time-of-flight mass spectrometry (TOF-MS) has generated considerable interest since TOF can provide the rapid and sensitive detection required by high resolution separations. In recent years, our laboratory has developed a variety of high-resolution capillary separation methods interfaced to TOF-MS via an ion trap storage/reflectron time-of-flight (IT/reTOF) instrument. Using this hybrid configuration, detection of fast separations at very low detection levels has been successfully performed for on-line separations of peptides and protein digests using electrospray ionization. In this report, we review the current status in our laboratory of interfacing high-performance liquid chromatography, capillary electrophoresis, and capillary electrochromatography to an IT/reTOF-MS instrument and various applications that have been developed involving this technology.

Chromatography, High Pressure Liquid↗
PubMed1998

Use of the comet assay for assessment of drug resistance and its modulation in vivo.

Drug resistance is generally considered to be a major impediment to successful cancer chemotherapy, yet it is generally not possible to predict the degree or timing of the emergence of tumour resistance in most chemotherapy protocols. Recent developments with the single-cell gel electrophoresis or 'comet' assay for DNA damage at the single-cell level suggest that this technique might provide a method for identifying and potentially monitoring tumour cell responsiveness to many anti-cancer agents in situ. In principle, this assay could be applied to any accessible tumour being treated with chemotherapeutic agents that cause overt DNA damage. We have investigated that supposition using several rodent and human tumour cell lines exhibiting a spectrum of resistance to the DNA strand-breaking drug, etoposide. By assessing cells grown as monolayers, spheroids and xenografted tumours in immunodeficient mice, we found that the comet assay can provide not only an index of sensitivity to etoposide, but, additionally, can demonstrate the efficacy (or lack thereof) of multidrug resistance (MDR) reversing agents for cells in vitro, and tumours in vivo.

Animals↗
PubMed1998

Production of longer acting tissue plasminogen activator of humans in milk of transgenic mice.

An exogenous gene was expressed in the mammary epithelium of transgenic mice in the hope that the encoded protein could be secreted into milk. The transgenic mice carrying the promoter and upstream regulatory sequences from the sheep BLG gene which were fused to cDNA encoding human tissue plasminogen activator (tPA) with its endogenous secretion signal sequence were generated. The hybrid genes were microinjected into mouse embryos. Two mice were identified as being transgenic by Southern blot. Milk obtained from lactating females contained biologically active tPA, and its concentration was calculated to be about 1.5 microg/ml. This result establishes the feasibility of secreting protein into the milk of transgenic animals for production of biologically active tPA proteins, and may provide a powerful method to produce such proteins on a large scale.

Animals↗
PubMed1998

[A preliminary report of antiscar formation effect by subconjunctival injection of mitomycin C before trabeculectomy in glaucoma].

To improve the function of filtering blebs and increase the successful rate of the trabeculectomy, mitomycin C injection 0.02 mg.ml-1 was given subconjunctivally at the operative site before trabeculectomy in 32 patients (38 eyes) with moderate or late glaucoma. All patients were followed up for 6 to 12 months. The results showed that the rate of functional blebs were 93.7%. The successful rate of the operation was 94.7%. The common postoperative complication was shallow anterior chamber (7.9%). These findings indicate that the subconjunctival injection of mitomycin C before operation is a safe, effective method for antiscar formation.

Adult↗
PubMed1998

[Relationship between IL-1 beta and TNF-alpha in subretinal fluids of rhegmatogenous retinal detachment with PVR].

The concentrations of IL-1 beta and TNF-alpha in subretinal fluids from 49 patients with rhegmatogenous retinal detachment with proliferative vitreoretinopathy(PVR) were measured by Enzyme-linked immunosorbent assay (ELISA). The results showed that all subretinal fluids contained IL-1 beta, their concentrations were positive correlation with severity of PVR (r = 0.677) and no correlation with age, sex, holes, ranges and times of retinal detachment, TNF-alpha was detected in 18/49 subretinal fluids including 2 patients with PVR-1 beta and 16 patients with PVR-C and the above cases. It suggested that IL-1 beta and TNF-a are involved in the pathogenesis of PVR.

Adolescent↗
PubMed1998

[Cytotoxic effect of mitomycin C on the nonpigmented epithelium of ciliary body in rabbit eyes].

OBJECTIVE: To observe pathomorphological changes of nonpigmented epithelial cells (NECs) of ciliary body after local single application of mitomycin C (MMC) during sclerectomy in rabbit eyes, and to explore cytotoxic effect of MMC on ciliary body. METHODS: A single five minutes intraoperative exposure to the agent MMC (0.2 mg/ml, 0.1 ml) was used during sclerectomy in rabbit eyes. The treated area was rinsed with 20 ml normal saline. The NECs of ciliary body at the operated area were observed under light microscope and transmission electron microscope on the 7th and 28th day postoperatively. RESULTS: On the 7th day postoperatively, the NECs were swollen, and the intracellular abundant mitochondria were swollen and vacuolized. String pearls-like membrane-encapsulated vacuoles were present in the intervals of beta-cell membrane of the NECs. On the 28th day postoperatively, these damages were gradually recovered. CONCLUSIONS: These findings suggest a toxic effect of MMC on the NECs of ciliary body, possibly that can decrease the aqueous production and intraocular pressure, even causing persistent hypotony.

Animals↗
PubMed1998

[Effects of biomaterials on immunoglobulins and biochemistry in serum of extracoporal circulation].

The polyvinyl chloride(PVC) tube coated with cellulose acetate(CA), polyether suffone(PES), polyurethane(PU), silicon rubber(SR) and polyester fibre(PETF) were put in contact with circular serum at 37 degrees C. According to different time-periods of exposure, the immunoglobulins (G.A.M), albumin(ALB), total protein (TP) and total cholesteral(Tch) in serum were measured by an IL-Monarch biomedical analyser. The results showed that the levels of immunoglobutins significantly decreased after the serum came in contact with CA, PETF, SR and that the levels of ALB, TP and Tch were significantly lower in the serum exposed to PETF while those in the serum exposed to other materials were normal. These changes may relate to the complement activation induced by different materials.

Biocompatible Materials↗
PubMed1998

[A comparison of amino acid composition of proteins from thermophiles and mesophiles].

The thermophilic feature of thermophiles are due to the high thermostability of their proteins. In order to investigate the mechanism of the high thermostability of thermophilic proteins and compare the difference of amino acid composition between thermophilic and mesophilic proteins, 110 pairs of homologous protein sequences from thermophiles and mesophiles respectively are collected and the amino acid composition, percentage of hydrophobic amino acids, hydropathy index, percentage of charged amino acids of the two protein groups are compared. It reveals that the two groups have evidently different composition in several kinds of amino acids, and thermophilic proteins have higher average hydropathy and charged amino acids composition. A comparison of the aliphatic indices between the two protein groups indicates that the reason that thermophilic proteins have higher aliphatic index is that thermophilic proteins have higher leucine composition, and thus sheds doubt on the validity of aliphatic index. It shows that it is possible to disclose some general rules with regard to the mechanism of protein thermostability by comparing a large amount of protein sequence data.

Amino Acids↗
PubMed1998

[A preliminary report of the effect of levodopa and carbidopa for childhood amblyopia].

PURPOSE: To explore the short-term effect of levodopa and carbidopa in 36 children (4.5-14 years) with refractory amblyopia. METHODS: 36 cases of incurable childhood amblyopia treated with occlussion with visual acuity stably for 6 months were retreated by levodopa and carbidopa 1.5 mg/kg, 0.38 mg/kg respectively for 3 months. Visual acuity, fusional and stereoscopic function, retinal light sensitivity and scotomas were observed at 1 and 3 months after tablet taken. Medicine side effects were observed. RESULTS: At 3 months, the visual acuity of 88.89% eyes were improved(average 2.27 +/- 1.26 line), fusional and stereoscopic function were restored in 10 and 6 cases respectively. Retinal light sensitivity significantly increased, scotomas disappeared in 19 eyes and contracted in 6 eyes. The basic cure rate was 42.86% Less side effect were found. CONCLUSION: Average dose levels(1.5/0.38 mg/kg) of levodopa and carbidopa were an effective and secuity method to improve visual function of refractory childhood amblyopia.

Adolescent↗
PubMed1998

[Effect of AHMED valve implantation surgery for the treatment of neovascular glaucoma].

PURPOSE: To evaluate the efficacy of the valve implantation for the treatment of neovascular glaucoma. METHODS: 15 cases (15 eyes) neovascular glaucoma were treated with AHMED valve implantation surgery, and followed up for 6 to 42 months. RESULTS: The IOP of the cases is lowered from 6.01 +/- 1.28 kPa (1 kPa = 7.5 mmHg) preoperatively to 1.96 +/- 0.68 kPa postoperatively. The success rate was 80.0% (The criteria of success is 0.80 kPa < or = IOP < or = 2.80 kPa and without severe complication that influence the visual acuity). The common postoperative complications included hyphema, shallow anterior chamber. CONCLUSION: The AHMED valve implantation surgery can lower the IOP, increase the operative success rate and preserve the visual function effectively. It is regarded to be an effective, safe method to treat neovascular glaucoma.

Adult↗
PubMed1998

Homology modeling of tissue-type plasminogen activator K1 domain and studies on the interactions between kringles and lysine.

The 3-D structure of t-PA K1 domain was predicted by the method of homology modeling. The putative lysine-binding pockets of t-PA K1, UK K, PLG K1 and K4 were determined by superimposing their 3-D structures to that of t-PA K2 domain, of which the lysine-binding pockets had been revealed previously. After that the key residues of lysine-binding pockets of kringles were identified. The structural analyses showed that both the electrostatic potential and hydrophobic complementarity were well matched between lysine and the binding pockets of t-PA K2, PLG K1 and K4, but for t-PA K1 and UK K domains the complementarity did not match well in one or both respects. It is proposed that this is the reason that t-PA K1 and UK K domains do not bear the ability to bind lysine. With the respect of improving the affinity for fibrin, new types of mutants of t-PA K1 and UK K domains were designed, and structural changes caused by mutation were predicted by simulating the residue replacements. The mutant structural models demonstrated that the molecular design was reasonable.

Amino Acid Sequence↗
PubMed1997

Biochemical characterization of the protein activity responsible for high molecular weight DNA fragmentation during drug-induced apoptosis.

Cleavage of cellular DNA into high molecular weight (predominantly 50 kb) fragments is an early event during apoptosis. We previously reported that this fragmentation was a Ca2+-independent process during apoptosis, which was induced by anticancer agents in human leukemia cells. The present study demonstrated that a high molecular weight DNA fragmentation activity (HDFA) was induced in the drug-treated cells and, upon fusion of the drug-treated cells with untreated target cells prelabeled with [14C]thymidine, caused fragmentation of the labeled DNA in the target cells. Furthermore, extracts of the drug-treated cells caused high molecular weight DNA fragmentation in nuclei isolated from untreated cells. Biochemical characterization of HDFA revealed the following properties: HDFA was proteinaceous in nature, as evidenced by its inactivation by heating or by digestion with proteinase K; HDFA required Mg2+ for optimal activity but was inhibited by Zn2+ and K+; HDFA was active in vitro at pH 6.0-8.0 and was inactive under more acidic conditions (pH < 6.0); addition of ATP (0.5-2 mM) substantially potentiated HDFA activity in isolated nuclei; and HDFA was not inhibited by actin (an inhibitor of DNase I) but was inhibited by the extracts from K562 cells, which were resistant to drug-induced apoptosis. The specific inhibitor of cysteine proteases (interleukin 1beta-converting enzyme protease family) blocked the generation of drug-induced high molecular weight DNA fragmentation in whole cells, whereas in isolated nuclei, the cysteine protease inhibitors did not prevent the cleavage of chromatin by exogenous HDFA. These results suggest that, once HDFA is activated during apoptosis, it does not require the presence of cysteine proteases for its endonucleolytic activity and that the cysteine proteases may be involved in the apoptotic process upstream of the activation of HDFA in whole cells.

Antineoplastic Agents↗