Search PubMed⌕ Search

Biomedical subjects

X Fang

Publications and source records attributed to X Fang.

At least 163 records · Page 9Linked to original sources
PubMed1995

An improved method for determination of acid dissociation constants of peptides.

PURPOSE: The method described here enables the proton dissociation constants of several amino acid residues of a peptide to be determined simultaneously in aqueous solution without prior knowledge of the exact concentration of the peptide. METHODS: The method used here employs a non-linear fitting program, the BEST program, or a linear least-squares method in combination with the BEST program. These methods are discussed in detail with an emphasis on the quality of the potentiometric titration data that are obtained. Two representative peptides, one with two proton dissociation constants (Ka1, Ka2) and the other with four proton dissociation constants (Ka1-Ka4) were used to illustrate the advantages and the limitations of these two complementary methods. RESULTS: The pKa values of TVL, a schizophrenia-related tripeptide, were found to be 3.62 (+/- 0.02) and 7.17 (+/- 0.05); the pKa values of ELTLQE, a hexapeptide, were found to be 2.32, 3.77, 4.58 and 7.74. CONCLUSIONS: The methods reported here are generally applicable to a variety of peptides. The possibility of integrating these procedures into a preparative chromatographic system for the "on-line" assessment of the pKa values of peptides during the purification stage is an attractive and novel feature of this method.

Amino Acid Sequence↗
PubMed1995

Generation and reactions of the disulphide radical anion derived from metallothionein: a pulse radiolytic study.

OH-radicals were generated by pulse radiolysis of aqueous solutions of rabbit (Zn,Cd)-metallothionein (MT). They react with MT mainly by forming a thiyl radical with a rate constant of 1.7 x 10(12) dm3 mol-1 s-1. The thiyl radical reacts rapidly but reversibly with a thiolate function to form RSSR.-: RS + RS- reversible RSSR.-. The kinetics of the formation and decay of this radical anion have been studied pulse radiolytically by monitoring the evolution of the optical absorption of RSSR.- at 450 nm. This process is mostly intermolecular, i.e. bimolecular in MT. In the absence of O2, RSSR.- decays bimolecularly: RSSR.(-)+RS.-->RSSR + RS-. In the presence of O2, RS. may be scavenged by O2 and thus the yield of RSSR.- decreases: RS.+O2 reversible RSOO.. Under these conditions RSSR.- decays by first-order kinetics: RSSR.(-)+O2-->RSSR + O2.-. The rate constants of these reactions have been determined at room temperature: k4 = 1.8 x 10(9) dm3 mol-1 s-1, k5 = 7 x 10(4) s-1, k10 = 9.2 x 10(8) dm3 mol-1 s-1, and k18 about 3 x 10(7) dm3 mol-1 s-1. From the dependence of the maximal absorbance at 450 nm on the thiolate concentration in the absence of oxygen, epsilon (RSSR.-) = 9 x 10(3) dm3 mol-1 cm-1 and the stability constant (K4/5) of 2.3 x 10(4) dm3 ml-1 was determined. K4/5 is in good agreement with that determined kinetically, k4/k5 = 2.6 x 10(4) dm3 mol-1. The stability constant K15/16 of the thiylperoxyl radical, RSOO., was determined to be 5.5 x 10(3) dm3 mol-1.

Animals↗
PubMed1995

[Clinical characteristics of hepatic tuberculosis].

The clinical characteristics of hepatic tuberculosis in 52 cases diagnosed pathologically were analyzed. Fever, abdominal pain and hepatomegaly were the major clinical manifestations; they were present in 96.2%, 42.3% and 88.5% of the patients respectively. The fever had no consistent pattern and the abdominal pain was usually localized to the right hypochondrium and not related to overwork. Hypergrammaglobulinaemia, elevated alkaline phosphatase level and increased ESR were noted in most of the patients (76.9%, 75.0%, 76.5% respectively). 62.5% of the 52 patients was diagnosed by percutaneous liver biopsy. Since there is no consistent clinical pattern in patients with hepatic tuberculosis, the diagnosis should be considered in patients with unexplained fever associated especially with hepatomegaly or hepatosplenomegaly, elevated alkaline phosphatase level, hypergrammaglobulinaemia and increased ESR, Liver biopsy is the most valuable method to confirm the diagnosis of hepatic tuberculosis.

Adolescent↗
PubMed1995

Epoxyeicosatrienoic acid metabolism in arterial smooth muscle cells.

Epoxyeicosatrienoic acids (EETs) are eicosanoids synthesized from arachidonic acid by the cytochrome P450 eposygenase pathway. The present studies demonstrate that 8,9-, 11,12-, and 14,15-EET are rapidly taken up by porcine aortic smooth muscle cells. About half of the uptake is incorporated into phospholipids, and saponification indicates that most of this remains in the form of EET. The EETs also are converted to the corresponding dihydroxyeicosatrienoic acids (DHETs) and during prolonged incubations, additional metabolites that do not retain the EET carboxyl group are formed. Most of these products are released into the medium. However, some DHET and metabolites less polar than EET are incorporated into the phospholipids, and a small amount of unesterified EET is also present in the cells. The incorporation of 14,15-EET and its conversion to DHET did not approach saturation until the concentration exceeded 10-20 microM, indicating that vascular smooth muscle has a large capacity to utilize this EET. These findings suggest that certain vasoactive effects of EETs may be due to their incorporation by smooth muscle cells. Furthermore, through conversion to DHET and other oxidized metabolites, smooth muscle apparently has the capacity to inactivate EETs that are either formed in or penetrate into the vascular wall.

8,11,14-Eicosatrienoic Acid↗
PubMed1994

The region encompassing the procyclic acidic repetitive protein (PARP) gene promoter plays a role in plasmid DNA replication in Trypanosoma brucei.

We have previously reported the construction and characterization of an autonomously replicating plasmid in Trypanosoma brucei. In this plasmid the procyclic acidic repetitive protein (PARP) gene promoter drives the transcription of a selectable marker. Deletion of this promoter incapacitates the plasmid, suggesting its utilization as a promoter-trap. Three independent libraries were created by inserting variously digested T.brucei genomic DNA into this promoterless construct. Transfection of these libraries into procyclic T.brucei and the subsequent isolation of episomes led only to the reisolation of the PARP promoter. Additionally, a ribosomal RNA promoter failed to keep the construct as an episome, although it can sustain mRNA transcription in T.brucei and was shown to be an efficient promoter in this construct. Finally, by using a transient replication assay involving the methylation-sensitive restriction endonuclease DpnI to distinguish between input and replicated DNA, we showed that the PARP promoter-bearing construct could replicate autonomously in procyclic T.brucei, but the corresponding construct with the rRNA promoter could not. The close association between elements that sustain transcription and DNA replication in T.brucei mirrors results observed in several higher eukaryotes and their viruses and suggests an ancient origin of this feature.

Animals↗
PubMed1994

Purity control of different bee venom melittin preparations by capillary zone electrophoresis.

A rapid capillary zone electrophoresis method was developed for the determination of melittin during its purification from bee venom by column chromatography. Low-pH buffer was selected to decrease the adsorption of the analytes on capillary wall. The average relative standard deviations of the migration time of melittin and of the peak area were 1.2 and 2.3%, respectively.

Bee Venoms↗
PubMed1994

Preformulation studies with melanotan-II: a potential skin cancer chemopreventive peptide.

Melanotan-II (1) is a cyclic heptapeptide analogue of alpha-melanocyte-stimulating hormone (alpha-MSH) which tans the skin and is currently being evaluated for the prevention of sunlight-induced skin cancers. The dissociation constants of 1 were determined using potentiometric titration and ultraviolet spectrophotometry. The pKa1 (histidine) and pKa2 (arginine) were estimated to be 6.54 and 11.72, respectively. The apparent partition coefficient (PC) was measured at three pH values using both n-octanol and isooctane as the nonpolar phase. The PC(octanol) and delta log PC at pH 7.35 were 2.82 and 1.05, respectively. These data, together with the observance of a bioavailability of 4.6% in the rat, indicate that 1 may be a suitable candidate for oral delivery. The data presented here are useful in developing an appropriate dosage form for 1.

Amino Acid Sequence↗
PubMed1994

High-efficiency gene transfer and high-level expression of wild-type p53 in human lung cancer cells mediated by recombinant adenovirus.

A replication-defective and helper-independent recombinant p53 adenovirus was generated. The virus, Ad5CMV-p53, carries an expression cassette that contains human cytomegalovirus E1 promoter, human wild-type p53 cDNA, and SV40 early polyadenylation signal. Four human non-small-cell lung cancer cell lines representing differences in p53 configuration were used to evaluate the Ad5CMV-p53 virus. In the H358 cell line, which has a homozygous deletion of p53, the p53 gene was transferred with 97% to 100% efficiency, as detected by immunohistochemical analysis, when the cells were infected with Ad5CMV-p53 at a multiplicity of infection of 30 to 50 plaque-forming units/cell. Western blots showed that the p53 protein was expressed at a high level. The protein expression peaked at day 3 after infection and lasted for at least 15 days. Growth of the Ad5CMV-p53 virus-infected H358 cells was inhibited 79%, whereas that of noninfected cells or the cells infected with the control virus was not inhibited. Growth of cell line H322, which has a point mutation in p53, was inhibited 72% by Ad5CMV-p53, while that of cell line H460 containing wild-type p53 was less affected (28% inhibition). Tests in nude mice demonstrated that tumorigenicity of the Ad5CMV-p53-treated H358 cells was greatly inhibited. In a mouse model of orthotopic human lung cancer, the tumorigenic H226Br cells, with a point mutation in p53, were inoculated intratracheally 3 days before the virus treatment. Intratracheal instillation of Ad5CMV-p53 prevented tumor formation.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenoviruses, Human↗
PubMed1994

Radiolysis of metallothionein in deaerated and oxygen-saturated solutions.

Gamma radiolysis of metallothionein (MT) in phosphate-buffered solutions at pH 8.0 leads to the formation of intra- and intermolecular disulfide bridges as well as the release of chelated metal ions. When MT solutions deaerated with N2 or saturated with N2O were irradiated, G values for RS- loss as determined by Ellman's reagent were 4.2 and 7.0, respectively, whereas those for dimerization were only 0.32 and 0.67. A short chain reaction existed in the decrease in RS- when radiolysis was performed in O2-saturated solutions. The rate constants for reactions of MTs with OH radical and H2O2 were measured to be about 10(12) and 100 M-1 s-1, respectively. A mechanism for the radiolysis of MT-1 is proposed.

Animals↗
PubMed1993

Molecular approaches to the diagnosis and treatment of cancer.

We have used autologous bone marrow transplantation (ABMT) as a setting to develop the genetic therapy of cancer in hematopoietic neoplasms based on the use of the bone marrow as a conduit through which to introduce regulatory molecules into the patient. This has involved three developmental phases: 1) learning how to develop genetic modification techniques; 2) learning how to develop in vivo selection techniques for the genetically modified cells; and 3) developing molecular vectors for modification of hematopoietic cells for therapy of cancer. These programs will be summarized in terms of their progress at the conference.

Bone Marrow Transplantation↗
PubMed1993

Generation and identification of recombinant adenovirus by liposome-mediated transfection and PCR analysis.

A simplified method for generating recombinant adenovirus was developed by using liposome-mediated co-transfection and by directly observing for the cytopathic effect caused by recombinant adenovirus in transfected 293 cells. This approach avoided difficulties associated with calcium-phosphate precipitation and agarose overlays for plaque assays. The ease of generating recombinant adenovirus was considerably improved. Analysis by PCR of DNA samples from the supernatant of the cell cultures with the cytopathic effect was also developed, which made identification of any newly generated recombinant virus rapid and specific.

Adenoviruses, Human↗
PubMed1992

A family of erythrocyte binding proteins of malaria parasites.

Malaria erythrocyte binding proteins use the Duffy blood group antigen (Plasmodium vivax and Plasmodium knowlesi) and sialic acid (Plasmodium falciparum) on the erythrocyte surface as receptors. We had previously cloned the one P. vivax gene, the one P. falciparum gene, and part of one of the three P. knowlesi genes encoding these erythrocyte binding proteins and described the homology between the P. knowlesi and P. vivax genes. We have completed the cloning and sequencing of the three P. knowlesi genes and identified introns in the P. vivax and P. falciparum genes that correct the previously published deduced amino acid sequences. All have similar structures, with one or two exons encoding the signal sequence and the erythrocyte binding domain, an exon encoding the transmembrane domain, and two exons encoding the cytoplasmic domain with the exception of the P. knowlesi beta gene. The regions of amino acid sequence homology among all the genes are the 5' and 3' cysteine-rich regions of the erythrocyte binding domain. On the basis of gene structure and amino acid homology, we propose that the Duffy binding proteins and the sialic acid binding protein are members of a gene family. The level of conservation (approximately 70%) of the deduced amino acid sequences in the 5' cysteine-rich region between the P. vivax protein and the three P. knowlesi proteins is as great as between the three P. knowlesi proteins themselves; the P. knowlesi beta protein just 3' to this cysteine-rich region is homologous to the P. vivax protein but not to the other P. knowlesi proteins. Conservation of amino acid sequences among these organisms, separated in evolution, may indicate the regions where the adhesin function resides.

Amino Acid Sequence↗
PubMed1991

Fluorometric labeling of tetrahydroprogesterones.

7-Diethylaminocourmarin-3-carbohydrazide was used to label the ketone group of the tetrahydroprogesterones to form fluorescent derivatives with high sensitivity. The four isomeric 3-hydroxypregnanes separated readily on high-performance, thin-layer chromatography after derivatization. This separation was not possible with the underivatized isomers. Standards and steroids from biologic mixtures were separated and showed similar characteristics. The methods used are described.

Chromatography, High Pressure Liquid↗
PubMed1990

Antagonists of luteinizing hormone releasing hormone with novel unnatural amino acids at position six.

Five new antagonists of luteinizing hormone releasing hormone (LHRH) containing novel unnatural amino acids at position six are reported. They are very effective in the rat antiovulatory assay. Using saline as vehicle, antagonist-[N-Ac-D-2-Nal1, D-4-Cl-Phe2, D-3-Pal3, Arg5, D-A26, D-Ala10]-LHRH inhibited ovulation completely at 1 micrograms/rat and three of the other antagonists showed some antiovulatory activity at 0.5 micrograms/rat.

Amino Acids↗