Biomedical subjects

Y Huang

Publications and source records attributed to Y Huang.

At least 1,063 records · Page 59Linked to original sources

PubMed1993

Purification and characterization of fatty acid-binding proteins from brown adipose tissue of the rat.

Fatty acid-binding proteins (FABPs) have been identified and purified from interscapular brown adipose tissue of the rat. The proteins were characterized and their properties compared with the FABP present in white adipose tissue. FABP was purified to electrophoretic homogeneity from brown adipose tissue by a procedure involving precipitation with 70% ammonium sulphate, followed sequentially by ion-exchange chromatography and gel filtration chromatography. The purified fraction migrated as a single band on SDS-PAGE with an apparent molecular mass of 14,200. Scatchard analysis of [14C]oleate-binding to purified FABP gave a Kd value of approx. 0.80 +/- 0.02 microM and a maximal binding of 0.65 +/- 0.03 mol per mol of protein; these values were similar to that found with the FABP purified from white fat. The FABP concentration in brown adipose tissue was almost twice that of FABP in white adipose tissue. Fatty acid analysis of FABP from brown adipose tissue revealed that the intrinsic arachidonic acid content was proportionately higher than that present in FABP of white adipose tissue. Isoelectric focusing of delipidated FABP indicated that it existed with two charge isoforms (pI 6.85 and 7.35). The purified FABP additionally emerged in two peaks (FABP-I and FABP-II) from a reverse phase HPLC column. Amino acid analysis showed that Gly, Thr, and Ser residues in FABP-I were almost twice as high as in FABP-II. The N-terminals of both FABP-I and -II were not blocked. These components have been partially sequenced and showed a sequence homology only between 25-31 residues from the N-terminal. Further studies are required to elucidate the precise function of the two different isoforms of FABP in brown adipose tissue.

Adipose Tissue, Brown↗

PubMed1993

Clonal ordering of 17p and 5q allelic losses in Barrett dysplasia and adenocarcinoma.

Both 17p and 5q allelic losses appear to be involved in the pathogenesis or progression of many human solid tumors. In colon carcinogenesis, there is strong evidence that the targets of the 17p and 5q allelic losses are TP53, the gene encoding p53, and APC, respectively. It is widely accepted that 5q allelic losses precede 17p allelic losses in the progression to colonic carcinoma. The data, however, supporting this proposed order are largely based on the prevalence of 17p and 5q allelic losses in adenomas and unrelated adenocarcinomas from different patients. We investigated the order in which 17p and 5q allelic losses developed during neoplastic progression in Barrett esophagus by evaluating multiple aneuploid cell populations from the same patient. Using DNA content flow cytometric cell sorting and polymerase chain reaction, 38 aneuploid cell populations from 14 patients with Barrett esophagus who had high grade dysplasia, cancer or both were evaluated for 17p and 5q allelic losses. 17p allelic losses preceded 5q allelic losses in 7 patients, both 17p and 5q allelic losses were present in all aneuploid populations of 4 patients, and only 17p (without 5q) allelic losses were present in the aneuploid populations of 3 patients. In no patient did we find that a 5q allelic loss preceded a 17p allelic loss. Our data suggest that 17p allelic losses typically occur before 5q allelic losses during neoplastic progression in Barrett esophagus.

Adenocarcinoma↗

PubMed1993

Altered messenger RNA and unique mutational profiles of p53 and Rb in human esophageal carcinomas.

Seventy-nine esophageal carcinoma patients were studied for genetic abnormalities in the p53 and Rb tumor suppressor genes. Single-strand conformation polymorphism analysis and DNA sequencing were used to detect p53 point mutations, Northern blotting was used to examine abnormal expression of p53 and Rb, and polymerase chain reaction and Southern blotting were used to analyze allelic loss. Twenty-five cases were analyzed by DNA sequencing to detect mutations in p53. Fourteen samples contained mutations within exons 5 through 9 of p53; seven had missense mutations giving rise to single amino acid substitutions. The remaining seven (50%) contained nonsense mutations leading to premature termination, five due to single base pair substitutions, and two that were the result of frameshift mutations. In other human tumors, p53 mutations are predominantly missense mutations, but our data as well as those from other groups show that nonsense mutations are common in human esophageal cancer. All but one of the constitutionally heterozygous samples containing mutations also manifested loss of the normal p53 allele; the one exception without allelic loss contained a silent mutation, which should not have had any affect on the p53 protein product. In addition, Northern blotting analysis revealed abnormalities (altered transcript size or mRNA levels) in 5 of 7 cases involving p53 and in 2 of 7 cases analyzed for Rb. Thirty-four cases were informative for allelic loss studies of both p53 and Rb; of these, 25 (74%) lost heterozygosity of p53, Rb, or both. When point mutations and mRNA expression abnormalities were also considered, 33 of 45 (73%) tumors informative for allelic loss assays of both genes as well as for mRNA or point mutation studies showed one or more abnormalities in p53 or Rb. Our results strongly suggest that a unique profile of molecular alterations involving p53 and Rb characterizes human esophageal cancer and that these specific genetic lesions are important in the development and/or progression of most human esophageal carcinomas.

Adenocarcinoma↗

PubMed1993

Antibodies to a human alpha 2-C10 adrenergic receptor fusion protein confirm the cytoplasmic orientation of the V-VI loop.

DNA encoding the hydrophilic region between transmembrane domains V and VI of the human platelet alpha 2-adrenergic receptor (alpha 2-C10) was amplified using the polymerase chain reaction and was cloned in-frame with a portion of the gene encoding glutathione-S-transferase (GST). Expression of the recombinant plasmid in E. coli resulted in the production of a GST/alpha 2-C10 fusion protein which was purified by preparative SDS-PAGE. Chickens inoculated with the fusion protein produced antibodies that were present in their eggs. In cells expressing the alpha 2-C10, these antibodies recognized the receptor in both Western Blots and indirect immunofluorescence. For the immunofluorescence studies, antibody recognition required permeabilization of the cells with detergent. This evidence establishes the cytoplasmic orientation of the V-VI loop and supports the general model for G-protein coupled receptors.

Animals↗

PubMed1993

Higher-plant cofactor-independent phosphoglyceromutase: purification, molecular characterization and expression.

Cofactor-independent phosphoglyceromutase (PGM) was purified to homogeneity from developing castor seed endosperm. Immunological characterization using monospecific antisera raised against this protein indicates that the enzyme is located in the cytosol and that there is no immunologically related polypeptide in the leucoplast from this tissue. Isolation and sequence determination of full-length cDNA clones for castor and tobacco PGM demonstrate that the protein is highly conserved in these plants and is closely related to the maize enzyme. A comparison of the amino acid sequence of peptides derived from Neurospora crassa PGM with the cofactor-independent enzyme from higher plants demonstrated that they are related and may have diverged from a common ancestral gene. The previously proposed relationship between higher-plant PGM and alkaline phosphatases is not supported by sequence analysis of the castor and tobacco enzymes. Expression of the single castor cytosolic PGM gene correlates well with other cytosolic glycolytic genes in developing and germinating castor seeds, and with the appearance of enzyme activity and PGM polypeptides in these tissues.

Amino Acid Sequence↗

PubMed1993

p53 point mutations in dysplastic and cancerous ulcerative colitis lesions.

BACKGROUND: The molecular basis of colorectal dysplasia and carcinoma arising in ulcerative colitis is poorly understood. Loss of heterozygosity involving the tumor suppressor gene p53 occurs frequently in neoplastic ulcerative colitis lesions. Point mutation affecting p53 is associated with loss of heterozygosity in other cancers. Therefore, it was determined whether p53 point mutation occurs in ulcerative colitis-associated neoplasia. METHODS: Single-strand conformation polymorphism analysis, DNA sequencing, and loss of heterozygosity studies were performed on 45 patients with ulcerative colitis-associated dysplasia and carcinoma. RESULTS: Point mutations were detected in 26 lesions from 20 patients, including 18 carcinomas, 6 dysplasia-associated masses, 1 flat dysplasia, and 1 lymph node metastasis. In two cases, identical p53 mutations were observed in both carcinoma and adjacent dysplasia. Missense mutations causing amino acid substitutions as well as nonsense mutations resulting in premature stop codons were seen. Tandem mutations, in which more than 1 sequence alteration occurred on the same allele of p53, were also detected. Point mutation was accompanied by loss of the other p53 allele in 8 of 10 patients informative for both loss of heterozygosity and mutation assays. CONCLUSIONS: These findings suggest that inactivation of p53 by mutation and loss of heterozygosity is a common mechanism of malignant transformation in ulcerative colitis. They also imply that in contrast to sporadic colorectal carcinoma, ulcerative colitis-associated neoplastic progression may involve p53 inactivation at relatively early, noninvasive stages.

Chromosome Deletion↗

PubMed1993

Single channel study of a Ca(2+)-activated K+ current associated with ras-induced cell transformation.

1. Ras-transformed fibroblasts have a whole-cell Ca(2+)-activated K+ current which is either absent or unavailable for activation in their non-transformed counterparts. To better understand the physiological significance of this K+ current the single channel basis for the current was characterized in ras-transformed cells. 2. More than 90% of inside-out patches from ras-transformed balb 3T3 cells had a channel type which was Ca(2+)-activated (threshold < 0.2 microM internal Ca2+), K(+)-selective (permeability ratio PNa:PK < 0.02), and inwardly rectifying in symmetric 150 mM KCl solutions (conductances at -60 and 60 mV of 33 +/- 1 and 17 +/- 1 pS respectively). Channel opening probability increased 25-50% between -60 and 60 mV due to an increase in the frequency of opening. Single K+ channels in outside-out patches were blocked by externally applied 10 mM TEA or 100 nM charybdotoxin, as were whole-cell Ca(2+)-activated K+ currents. The properties of this class of K+ channel are sufficient to account for the whole-cell Ca(2+)-activated current in ras-transformed cells. 3. Inside-out patches from C3H10T1/2 and NIH 3T3 fibroblasts transformed by the H-ras oncogene had Ca(2+)-activated K+ channels identical to those observed in K-ras-transformed balb 3T3 cells. 4. As predicted from whole-cell experiments Ca(2+)-activated K+ channels were not observed in inside-out patches from non-transformed balb 3T3 cells. The purpose of the excised patch recordings was, instead, to rule out potential technical complications with the whole-cell experiments. For instance A23187, which evoked whole-cell K+ currents in transformed cells, may not have elevated Ca2+ sufficiently to allow K+ channel activation in non-transformed cells. Another possibility was that trypsin pretreatment used to round-up cells for whole-cell recording may have preferentially disabled channels in non-transformed cells. The first problem was addressed by exposing patches from non-transformed cells to 100-1000 microM Ca2+. Excised patches were also taken from non-transformed cells which had not been exposed to trypsin. K+ channel activity was not observed under either condition. 5. Patches from both ras-transformed and non-transformed cells had a type of non-specific cation channel which was activated at internal Ca2+ concentrations > or = 100 microM. This channel was sensitive to membrane voltage, mean open time increasing from 12 to 72 ms between -90 and 90 mV.

Animals↗

PubMed1993

A new de novo mutation (A113T) in HMG box of the SRY gene leads to XY gonadal dysgenesis.

We describe a new point mutation in the SRY gene of a Chinese XY female with gonadal dysgenesis (Swyer syndrome). Using the double stranded DNA cycle sequencing method, a single nucleotide substitution of G-->A was identified at codon 113 of the patient's SRY gene, resulting in a conservative amino acid change from alanine (A) to threonine (T) at a residue that lies within the putative DNA binding motif. With this mutation, one MnlI recognition site is abolished and a new BsmAI site is present in the DNA sequence of the SRY gene; therefore, it is easily detected by analysis of the digestion of the amplified SRY DNA fragment on an electrophoretic agarose gel. In situ hybridisation to the XY female's chromosomes showed that her mutant SRY gene was indeed located on the short arm of her Y chromosome. The SRY mutation in the XY female reported here occurred de novo, as sequence analysis showed that it was not present in her father or other family members.

Adult↗

PubMed1993

Monitoring in vivo oxidation of 14C-labelled substrates to 14CO2 by brain microdialysis.

Cultured brain cells oxidize glucose and numerous alternate substrates to CO2 for energy production, however, the importance of these observations to the intact brain have not been established. We have adapted in vivo brain microdialysis procedures to measure the rate of 14CO2 formation from 14C-glutamate, 14C-glutamine, and 14C-glucose in the hippocampus of awake unanesthetized free-moving rats. Two, 9 and 16 days after surgery (to implant guide cannulae) microdialysis probes were inserted into the guide cannulae and perfused with artificial CSF containing either 14C-glutamate, 14C-glutamine or 14C-glucose. Dialysate fractions were collected during 20 min intervals for determination of 14CO2. The amount of labelled 14CO2 increased for 40 to 60 min and then plateaued and remained relatively constant for at least 6 hours. When the probe was removed from the hippocampus and inserted into a vial containing non-radioactive CSF, 14CO2 production dropped rapidly. The quantity of 14CO2 recovered from glutamate was greater than from glucose or glutamine reflecting pool sizes, uptake characteristics and point of entry into oxidative pathways. The microdialysis system was verified by using model systems with cultured astrocytes suspended in media to simulate the brain. The present results indicate brain microdialysis may be used to study the role of alternate substrates in specific brain regions under varying physiological states.

Amino Acids↗

PubMed1993

Cell-derived unesterified cholesterol cycles between different HDLs and LDL for its effective esterification in plasma.

Pulse-chase incubations of human plasma with [3H]cholesterol-laden skin fibroblasts or low density lipoproteins (LDL) and nondenaturing two-dimensional electrophoresis were used to study the transfer and esterification of cell-derived unesterified cholesterol (UC) in human plasma lipoproteins. Specific radioactivities ([3H]UC per microgram of UC) were calculated, and net cholesterol mass transfer was quantified using a fluoro-enzymatic assay to validate productive transfers of UC between high density lipoprotein (HDL) and LDL. Cellular UC was initially taken up by pre-beta 1-HDL and subsequently transferred in the sequence pre-beta 2-HDL-->pre-beta 3-HDL-->alpha-HDL-->LDL. During the first 5 minutes of this process, only 5% of cellular cholesterol was esterified in pre-beta 3-HDL and alpha-HDL; the remainder reached LDL as UC. Cellular UC accumulating in LDL was then redistributed to various HDL particles via two pathways: 1) the partially LDL receptor-mediated uptake and re-secretion of UC by cells and 2) the direct transfer of UC to HDL, mostly to alpha-HDL and a small amount to pre-beta-HDL. UC was not transferred from LDL to HDL after inhibition of lecithin:cholesterol acyltransferase (LCAT). The esterification of cellular [3H]cholesterol in plasma was competitively inhibited by the addition of excess unlabeled LDL but not of excess HDL. However, both excess LDL and excess HDL prevented the esterification of cell-derived cholesterol in apolipoprotein B-free plasma. This demonstrated that LDL is the major source of UC to the LCAT reaction and that the transfer of UC from LDL to HDL is LCAT dependent. In conclusion, the effective esterification of cell-derived cholesterol in plasma involves a rapid transfer of UC via HDL particles to LDL, from which it is distributed to pre-beta-HDL and alpha-HDL. Furthermore, we hypothesize that the transfer per se of cellular UC to LDL forms a cholesterol concentration gradient between cell membranes and HDL and thus a second, reverse cholesterol transport mechanism in addition to the esterification of cholesterol by LCAT.

Cells↗

PubMed1993

Effect of hemopoietic growth factors G-CSF and pIXY 321 on the activity of high dose Ara-C in human myeloid leukemia cells.

Recently, high dose Ara-C (HIDAC) has been shown to induce leukemic cell death in vitro by the alternative process of programmed cell death (PCD) or apoptosis which correlates with the inhibition of their clonogenic survival. Since co-treatment with hemopoietic growth facts (HGFs) GM-CSF and IL-3 have been demonstrated to enhance the metabolism and cytotoxic effects of HIDAC against leukemic progenitor cells, we examined the effect of HGFs pIXY 321 (a GM-CSF/IL3 fusion protein) and G-CSF on HIDAC induced PCD and related gene expressions as well as HIDAC mediated colony growth inhibition of human myeloid leukemia cells. Treatment with G-CSF or pIXY 321 alone for up to 24 hours neither suppressed nor induced PCD in HL-60 or KG-1 cells. However, exposure to either of the HGFs for 20 hours followed by a combined treatment for 4 hours with HIDAC plus either of the HGFs versus HIDAC alone significantly enhanced the intracellular Ara-CTP accumulation and the oligonucleosomal DNA fragmentation characteristic of PCD. This was temporally associated with a marked induction of C-jun expression but a significant repression in BCL-2 and c-myc expressions. In addition, the treatment with either of the HGFs plus HIDAC versus HIDAC alone produced a significantly greater inhibition of the clonogenic survival of the myeloid leukemia cells. These findings underscore an additional mechanism of leukemic cell death induced by HIDAC which can be modulated by the HGFs to improve the antileukemic activity of HIDAC.

Arabinofuranosylcytosine Triphosphate↗

PubMed1993

Management of major arterial injuries of the limbs in 166 cases.

The charts of 166 patients with major arterial injuries (175 arterial repairs) inflicted during periods of peace and war time were reviewed. One hundred and sixty-seven (95%) of the arteries repaired remained patent. Nine patients had limbs which developed ischemic contracture. Nine patients required amputations. Follow-up data was obtained from seventy-five patients with an average follow-up of five years and one month (six months to thirty years). Early diagnosis, prompt treatment including fasciotomies when indicated, complete debridement, appropriate coverage, meticulous surgical technique, and simultaneous treatment of concomitant injuries are all crucial factors in successful limb salvage. Major arterial injuries of the limb occur frequently in our patient population, especially in time of war. Successful treatment of major arterial injuries may be life-saving, as well as allowing limb salvage and restoration of limb function. The purpose of this study is to review our experience with repair of major arterial injuries.

Adolescent↗

PubMed1993

Taxol induces internucleosomal DNA fragmentation associated with programmed cell death in human myeloid leukemia cells.

The present results demonstrate that the exposure of human myeloid leukemia HL-60 and KG-1 cells to clinically achievable concentrations of taxol produced internucleosomal DNA fragmentation of approximately 200 base-pair multiples, and the morphologic changes characteristic of cells undergoing programmed cell death (PCD) or apoptosis. Taxol-induced PCD was associated with a marked inhibition of suspension culture growth and clonogenic survival of HL-60 cells. In addition, taxol treatment decreased BCL-2 oncogene expression, which is known to block PCD. The exposure to taxol moderately decreased c-myc expression, but did not induce c-jun expression--which has been previously noted for a variety of DNA interactive, antileukemic drugs. These findings indicate that taxol may induce leukemic cell death partly by the alternative but gene-directed and active mechanism of PCD.

Apoptosis↗

PubMed1993

[Determination of ceftizoxime in serum by high performance liquid chromatography].

This paper reports the determination of ceftizoxime, a new cephalosporin antibiotic, in serum by RP-HPLC. The technique for serum protein precipitation by adding perchloric acid is adapted for sample preparations. After centrifugation, a 100 microliters portion of aqueous phase is injected into chromatographic column of ultrasphere CN, the mobile phase being a mixture of methanol and 1% acetic acid (15:85). The standard curve is linear within the range of 2.5-120 mg/L in serum. The detection limit of ceftizoxime in serum stands at 0.1 mg/L. The recoveries from serum samples after protein precipitation reach 95.4-96.3%. The within day CVs and interday CVs are 2.7-3.3% and 4.8-7.6%, respectively. This method is rapid, reliable and reproducible. It has been utilized to measure the concentration of ceftizoxime in patient serum for clinical research and pharmacokinetic studies.

Ceftizoxime↗

PubMed1993

Further study on the magnesium-mediated change in physical state of phospholipid modulates mitochondrial F0-F1-ATPase activity.

We have postulated that magnesium may play a role in altering the lipid fluidity of the bilayers, which would induce a change of conformation of the F0-ATPase portion (buried in the lipid core) of mitochondrial F0-F1-ATPase. Such change could be transmitted to the soluble F1 portion, resulting in higher enzymatic activity. The assumption was further supported by the results presented in the following: (1) A conformational difference for the F0-ATPase-containing proteoliposomes induced by the magnesium effect could be detected using a fluorescent probe acrylodan; (2) H(+)-translocation activity of F0-ATPase-incorporated proteoliposomes with magnesium, monitoring by fluorescence quenching of 9-aminoacridine or the bulk phase pH change, was higher than that without magnesium; (3) The magnesium effect on the reconstituted F0-F1-ATPase activity was greatly enhanced when the reconstitution was carried out in the presence of oligomycin sensitivity conferring protein (OSCP, a main component of the connecting link between the F1 and F0 sector of F0-F1-ATPase).

2-Naphthylamine↗

PubMed1993

[Studies on the pleomorphism of trachoma inclusion].

A series of trachoma inclusion color photograph is taken from the conjunctival smears of 48 trachoma cases. These smears were Giemsa-stained and stored in the slide-boxes for more than 30 years with no distinct changes in color. A description is given to the Pleomorphism of trachoma inclusion and its relation to the host cells. According to the gradual morphological changes of Chlamydia trachomatis multiplied in the infected cells, a 5-staged classification for trachoma inclusions is provided which may be benefit to understanding the pleomorphism of the trachoma inclusion and clinical laboratory diagnosis.

Chlamydia trachomatis↗

PubMed1993

Effect of combined treatment with interleukin-3 and interleukin-6 on 4-hydroperoxycyclo-phosphamide-induced programmed cell death or apoptosis in human myeloid leukemia cells.

In autologous bone marrow transplantation in patients with acute myeloid leukemia (AML), 4-hydroperoxycyclophosphamide (4-HC) is a commonly used ex vivo purging agent for leukemic blasts. In the present report, we demonstrate that exposure to high concentrations of 4-HC for 1 hour, as used in ex vivo bone marrow purging, produces internucleosomal DNA fragmentation characteristic of apoptosis, or programmed cell death (PCD), in human myeloid leukemia HL60 cells. Lower concentrations of 4-HC (10, 20, or 50 microM/L) failed to cause this effect, while higher concentrations (> or = 200 microM/L) produced random DNA fragmentation. 4-HC-mediated internucleosomal DNA fragmentation was associated with a marked induction in c-jun and significant reductions in bcl-2 and c-myc oncogene expressions. A combined treatment with interleukin-3 (IL-3) plus IL-6 for 18 hours before an additional, 1-hour concurrent treatment with 4-HC (100 microM/L) significantly increased 4-HC-induced DNA fragmentation as well as colony growth inhibition of HL60 cells. The effects of cotreatment with IL-3 plus IL-6 were also associated with a further, modest decrease in bcl-2 and c-myc and augmentation of c-jun expression. These findings highlight an alternative mechanism of 4-HC-induced leukemic cell death that can be potentially enhanced by cotreatment with IL-3 plus IL-6.

Apoptosis↗

PubMed1993

Effect of intra-vas deferens injection of gossypol-polylactic acid on fertility and spermatogenesis in rats.

In order to reduce gossypol's side effects, we applied a controlled drug release system. In this system gossypol is carried on a polymer of polylactic acid which controls drug release and can also be injected into the vas deferens so that the drug can act continuously and directly on spermatogenesis. We used a single dose of gossypol-polylactic acid (0.35 mg/0.1 ml for each vas deferens) which is only 0.2% of the usual antifertility dose. Fertility tests showed that male rats became infertile 4 weeks after intravasal injection of gossypol-polylactic acid. Gross and microscopic findings in the heart, liver, kidneys and lungs revealed no evidence of pathologic change. Treated rats gained body weight as fast as did the controls. The majority of spermatozoa showed breakage of the axial fibers, indicating that gossypol had diffused into the testes. The fertility of the treated rats recovered four to eight months after treatment. Gossypol-polylactic acid injected into the vas deferens exhibited a low degree of toxic side effects and a high antifertility activity, and is therefore an important approach to decreasing the side effects while enhancing the antifertility specificity of gossypol.

Animals↗