Biomedical subjects

T Huang

Publications and source records attributed to T Huang.

At least 19 recordsLinked to original sources

PubMed1999

Messenger RNAs encoding mouse histone macroH2A1 isoforms are expressed at similar levels in male and female cells and result from alternative splicing.

Two protein isoforms of histone macroH2A1 (mH2A1) are found in mammalian cells. One isoform, mH2A1.2 is highly concentrated on the heterochromatinized inactive X chromosome (Xi) of female cells. mH2A1.2 protein is also present in male cells, but fails to form dense concentrations. Another protein isoform, mH2A1.1, differs from mH2A1.2 by a single short segment of amino acids. In this study, we cloned and characterized the genomic locus of the mouse mH2A1 gene and mapped it to chromosome 13. Two alternatively spliced transcripts derived from the mH2A1 locus are responsible for the generation of the two mH2A1 protein isoforms with mH2A1.2 mRNA being the most abundant spliced form in all tissues examined. The absolute amount of mH2A1 mRNA is similar in male and female cells for most tissues with the exception of testes where it is par-ticularly abundant. Both spliced forms are present in all adult tissues analyzed as well as in female embryonic stem cells. In contrast, male embryonic stem cells expressed mH2A1.1 at low levels if at all. The relatively abundant expression of mH2A1 in both sexes suggests that mH2A1 has functions in addition to a possible involvement in X chromosome inactivation.

Aging↗

PubMed1999

The novel Mrf-2 DNA-binding domain recognizes a five-base core sequence through major and minor-groove contacts.

Recent NMR studies of the purified Mrf-2 DNA-binding domain peptide have shown that its structure differs significantly from previously characterized classes of DNA-binding domains. Here we report biochemical studies of the DNA-binding properties of this peptide. Binding interference and binding site selection assays indicated that Mrf-2 requires the core sequence AATA(C/T) for high affinity binding. Kinetic analyses of several selected sequences indicated that the core sequence alone is not sufficient for high affinity binding, however. Kinetic analyses were also performed using a series of synthetic oligonucleotides with single base analogues at each position in the core sequence. Base analogues that altered the major groove structure reduced or eliminated Mrf-2 binding when present in the second, third, and fourth base-pairs of the core sequence, but had little or no effect in the first and fifth positions. These results suggest that Mrf-2 contacts both the major and minor grooves of its target sequences.

Base Sequence↗

PubMed1999

The alpha-helical domain of Galphat determines specific interaction with regulator of G protein signaling 9.

RGS proteins (regulators of G protein signaling) are potent accelerators of the intrinsic GTPase activity of G protein alpha subunits (GAPs), thus controlling the response kinetics of a variety of cell signaling processes. Most RGS domains that have been studied have relatively little GTPase activating specificity especially for G proteins within the Gi subfamily. Retinal RGS9 is unique in its ability to act synergistically with a downstream effector cGMP phosphodiesterase to stimulate the GTPase activity of the alpha subunit of transducin, Galphat. Here we report another unique property of RGS9: high specificity for Galphat. The core (RGS) domain of RGS9 (RGS9) stimulates Galphat GTPase activity by 10-fold and Galphai1 GTPase activity by only 2-fold at a concentration of 10 microM. Using chimeric Galphat/Galphai1 subunits we demonstrated that the alpha-helical domain of Galphat imparts this specificity. The functional effects of RGS9 were well correlated with its affinity for activated Galpha subunits as measured by a change in fluorescence of a mutant Galphat (Chi6b) selectively labeled at Cys-210. Kd values for RGS9 complexes with Galphat and Galphai1 calculated from the direct binding and competition experiments were 185 nM and 2 microM, respectively. The gamma subunit of phosphodiesterase increases the GAP activity of RGS9. We demonstrate that this is because of the ability of Pgamma to increase the affinity of RGS9 for Galphat. A distinct, nonoverlapping pattern of RGS and Pgamma interaction with Galphat suggests a unique mechanism of effector-mediated GAP function of the RGS9.

3',5'-Cyclic-GMP Phosphodiesterases↗

PubMed1999

Different TBX5 interactions in heart and limb defined by Holt-Oram syndrome mutations.

To better understand the role of TBX5, a T-box containing transcription factor in forelimb and heart development, we have studied the clinical features of Holt-Oram syndrome caused by 10 different TBX5 mutations. Defects predicted to create null alleles caused substantial abnormalities both in limb and heart. In contrast, missense mutations produced distinct phenotypes: Gly80Arg caused significant cardiac malformations but only minor skeletal abnormalities; and Arg237Gln and Arg237Trp caused extensive upper limb malformations but less significant cardiac abnormalities. Amino acids altered by missense mutations were located on the three-dimensional structure of a related T-box transcription factor, Xbra, bound to DNA. Residue 80 is highly conserved within T-box sequences that interact with the major groove of target DNA; residue 237 is located in the T-box domain that selectively binds to the minor groove of DNA. These structural data, taken together with the predominant cardiac or skeletal phenotype produced by each missense mutation, suggest that organ-specific gene activation by TBX5 is predicated on biophysical interactions with different target DNA sequences.

Adult↗

PubMed1999

Angiopoietins 3 and 4: diverging gene counterparts in mice and humans.

The angiopoietins have recently joined the members of the vascular endothelial growth factor family as the only known growth factors largely specific for vascular endothelium. The angiopoietins include a naturally occurring agonist, angiopoietin-1, as well as a naturally occurring antagonist, angiopoietin-2, both of which act by means of the Tie2 receptor. We now report our attempts to use homology-based cloning approaches to identify new members of the angiopoietin family. These efforts have led to the identification of two new angiopoietins, angiopoietin-3 in mouse and angiopoietin-4 in human; we have also identified several more distantly related sequences that do not seem to be true angiopoietins, in that they do not bind to the Tie receptors. Although angiopoietin-3 and angiopoietin-4 are strikingly more structurally diverged from each other than are the mouse and human versions of angiopoietin-1 and angiopoietin-2, they appear to represent the mouse and human counterparts of the same gene locus, as revealed in our chromosomal localization studies of all of the angiopoietins in mouse and human. The structural divergence of angiopoietin-3 and angiopoietin-4 appears to underlie diverging functions of these counterparts. Angiopoietin-3 and angiopoietin-4 have very different distributions in their respective species, and angiopoietin-3 appears to act as an antagonist, whereas angiopoietin-4 appears to function as an agonist.

Amino Acid Sequence↗

PubMed1999

Isolation of oncogenes from rat mammary tumors by a highly efficient retrovirus expression cloning system.

A majority of mammary tumors induced with N-methyl-N-nitrosourea in rats contain G to A transitional mutation of c-Ha-ras at the 12th codon. Additional oncogene activation is known to be necessary for further tumor progression. To isolate novel oncogenes, we used an expression cloning system utilizing the pMX retroviral vector in combination with BOSC23 packaging cells. First, we elucidated the sensitivity of this system in the NIH 3T3 focus assay; foci were detectable even after 10(-6) dilution using v-Ha-ras, neuT, and beta-galactosidase constructs in pMX vector. This system is sensitive enough to detect low copy number cDNAs. We used the pMX/BOSC23 expression cloning system to clone novel oncogenes from rat mammary tumors harboring an activated c-Ha-ras and isolated several candidate oncogenes that caused transformation of NIH 3T3 cells and/or generated tumors when transplanted to nude mice.

3T3 Cells↗

PubMed1999

Bioavailability of carotenoids in human subjects.

There is growing need for accurate information regarding the bioavailability of carotenoids, both with respect to carotenoids per se and to the vitamin A value of provitamin A carotenoids in foods or supplement preparations. Little quantitative information is currently available, owing primarily to the lack of adequate methods to assess carotenoid bioavailability. Methods applied to xenobiotic drugs are in most cases not useful for carotenoids, many of which circulate in appreciable quantities in human plasma. Reported ranges of carotenoid bioavailability (% dose absorbed) range from 1-99, and variability is generally high both within and between treatments. With the current methods, relative bioavailability is more readily assessed than absolute bioavailability. The most commonly applied methods include measuring the increase in plasma carotenoid concentration following chronic intervention, and use of postprandial chylomicron (PPC) carotenoid or retinyl ester response following a single dose of carotenoid. The advantages and limitations of these approaches, together with examples of each, are discussed. A new PPC approach utilizing extrinsic-stable-isotope-labelled vitamin A (2H4-labelled retinyl acetate) is under development in our laboratory, and examples of its application are presented. The currently available data suggest that oil solutions of carotenoids are more bioavailable than those from food matrices, and heating can improve the bioavailability of carotenoids from some food products. Increased availability of labelled carotenoids and retinoids should aid the development of reliable methods of carotenoid bioavailability assessment. Such data are needed for dietary recommendations, supplement formulation, and design of intervention strategies involving carotenoids.

Biological Availability↗

PubMed1999

Concurrent improvements in ambulatory cardiac catheterization practices following inpatient interventions.

Questions have been increasingly raised about the value of performing right heart catheterization. A preliminary analysis done in 1992 revealed significant interhospital variation in the frequency of the procedure among Medicare Part A and Medicaid patients in New York State, and it also suggested that the procedure was being performed routinely in some hospitals. In 1993, IPRO initiated a cooperative health care quality improvement program involving the state's 53 catheterization laboratories. As a result of this educational intervention, the rate of bilateral catheterization among Medicare Part A patients fell from 89/100,000 beneficiaries in 1992 to 65/100,000 in 1996, and the overall percentage of catheterized Medicare patients undergoing bilateral catheterization fell from 30.5% in 1992 to 17.4%. A major question was whether a corresponding decrease had occurred among ambulatory patients (Medicare Part B). To determine the answer, the Medicare Part B database was analyzed for the identical period of time. It was found that the percentage of ambulatory Medicare patients who underwent bilateral catheterization at the 53 laboratories fell from 37.6% in 1992 to 17.0% in 1996, paralleling the decline observed among inpatients. The results of this quality improvement study show that an educational intervention directed at inpatient practice patterns can have a similar impact on outpatient patterns.

Ambulatory Surgical Procedures↗

PubMed1998

Thermodynamic analysis of the binding of the polyglutamate chain of 5-formyltetrahydropteroylpolyglutamates to serine hydroxymethyltransferase.

The thermodynamic parameters for the binding of 5-formyltetrahydrofolate (5-CHO-H4PteGlun) and its polyglutamate forms to rabbit liver cytosolic serine hydroxymethyltransferase (SHMT) were determined by a combination of isothermal titration calorimetry and spectrophotometry. Binding of 5-CHO-H4PteGlun to SHMT exhibits both positive enthalpy and entropy, showing that binding is entropically driven. 5-CHO-H4PteGlu5 has a 300-fold increased affinity for SHMT compared to 5-CHO-H4PteGlu. This increase in affinity is due primarily to a decrease in the positive enthalpy with little change in entropy. A variety of anions inhibit the binding of 5-CHO-H4PteGlu5 with Ki values in the 10-20 mM range. Anions are ineffective inhibitors of 5-CHO-H4PteGlu binding to SHMT, showing that anions compete for the polyglutamate binding site. There was little difference in the Ki values for a series of dicarboxylic acids as inhibitors of 5-CHO-H4PteGlu5, suggesting that spacing of the negative charges may not be important in determining their effectiveness as inhibitors. Both the mono- and pentaglutamate derivatives of 5-CHO-H4PteGlun were cross-linked to SHMT by a carbodiimide reaction to Lys-450 which resides in a stretch of Lys, His, and Arg residues.

Amino Acid Sequence↗

PubMed1998

Optimization of post-column photolysis and electrochemical detection for the liquid chromatographic determination of 3-nitro-L-tyrosine.

A new post-column photolysis technology has been developed based on the use of a low pressure, low temperature UV lamp and TiO2 coated knitted reaction coil. As a test case the developed technique was used for the determination of 3-nitro-L-tyrosine by liquid chromatography with electrochemical detection. Different photolysis lamps and reactor tubing lengths were evaluated in terms of their effect on the separation efficiency and/or photolysis efficiency. A detection limit of 0.5 nM (10 fmol) for 3-nitro-L-tyrosine was achieved under optimized conditions, with a linear correlation coefficient of R2 = 0.9898 over a concentration range of 2-100 microM. Pre-injection photolysis of 3-nitro-L-tyrosine indicated that dihydroxyphenylalanine is the main photolysis product. In general, use of the photoreactor prior to liquid chromatography is an excellent method for exploring photodegradation products of an analyte in conjunction with the full range of available liquid chromatography detectors.

Animals↗

PubMed1998

Comparison of detection methods for liquid chromatographic determination of 3-nitro-L-tyrosine.

A liquid chromatographic method has been developed for the determination of 3-nitro-L-tyrosine. Different detection methods, including UV, oxidative and redox electrochemistry, and postcolumn photolysis followed by electrochemical detection, have been optimized and compared in terms of analysis time, detection limit and dynamic range. It was demonstrated that liquid chromatography with postcolumn photolysis followed by electrochemical detection is the most effective method, with an analysis time of 5 min, detection limit of 0.01 pmol, and a linear dynamic range from 2 nM to 100 microM.

Animals↗

PubMed1998

Defective retrovirus insertion activates c-Ha-ras protooncogene in an MNU-induced rat mammary carcinoma.

Endogenous retrovirus sequences are present in the genome of a wide variety of animal species. The activation of the proto-oncogenes of the ras family, particularly c-Ha-ras, by either point mutation or overexpression, has been shown to be associated with a vast number, of different cancers. here we report that the insertion of a defective retrovirus in the -1 intron of rat c-Ha-ras is responsible for the activation of the gene by over 10-fold overexpression in an MNU-induced rat mammary cancer. A portion of the 3' end of the retroviral sequence is expressed as a part of the c-Ha-ras transcript in the carcinoma tissue, indicating the direct involvement of this element in the transcription of the c-Ha-ras gene. The c-Ha-ras structural gene transcribed by the promoter of the defective retroviral element can neoplastically transform the NIH 3T3 cell line upon transfection.

3T3 Cells↗

PubMed1998

[Interference of human processed pseudogene and corresponding counter-measures in reverse transcriptase-polymerase chain reaction].

OBJECTIVE: This study aimed to set up an effective method to find the interference of processed pseudogene and overcome it in RT-PCR. METHODS: Using RT-PCR methods, the authors amplified the cytokeratin 19 cDNA or DNA sequence in human lung squamous cell carcinoma samples. RESULTS: They could find the influence of pseudogene through performing genomic DNA and nonreversed transcribed RNA controls. By digesting the extracted RNA sample with RNase-free DNase or regulating the annealing temperature based on mismatched primers, they could effectively eliminate the interference. CONCLUSION: The presence of processed pseudogene usually interferes in the experimental protocol in RT-PCR. This problem can be resolved with the method aforementioned.

Carcinoma, Squamous Cell↗

PubMed1998

Determination of L-phenylalanine based on an NADH-detecting biosensor.

An enzyme carbon paste electrode containing three different enzymes was developed for the determination of L-phenylalanine. This sensor is based on the enzymatic/electrochemical recycling of tyrosinase in combination with salicylate hydroxylase and L-phenylalanine dehydrogenase (PADH). The enzymes salicylate hydroxylase and tyrosinase were coimmobilized first in a carbon paste electrode for the sensitive detection of NADH. The principle of the bienzyme scheme is as follows: the first enzyme, salicylate hydroxylase, converts salicylate to catechol in the presence of oxygen and NADH. The second enzyme, tyrosinase, then oxidizes the catechol to o-quinone, which is electrochemically detected and reduced back to catechol at the electrode at an Eappl = -50 mV vs Ag/AgCl. This results in an amplified signal due to the recycling of the catechol and o-quinone between tyrosinase and the surface of the electrode. Prior to adding PADH, the salicylate hydroxylase-tyrosinase carbon paste electrode was characterized in terms of its sensitivity to NADH, pH dependence, buffer composition, interferences, and stability. Interference from ascorbic acid and uric acid was found to be minimal. Human serum was used to investigate whether this bienzyme system was suitable for the detection of NADH in serum and blood samples. The sensitivity for NADH was increased by a factor of 33 times using the bienzyme amplification scheme (electroreduction of o-quinone at Eappl = -50 mV) as opposed to the salicylate hydroxylase single-enzyme system (at which catechol would have been oxidized at Eappl = +150 mV vs Ag/AgCl). The detection limit for NADH achieved by the bienzyme carbon paste electrode was 1 vs 100 microM for the single-enzyme carbon paste electrode. The salicylate hydroxylase-tyrosinase system was then coupled with phenylalanine dehydrogenase for L-phenylalanine determination. This multienzyme sensor was able to achieve a linear range of 20-150 microM and a detection limit of 5 microM for L-phenylalanine. The sensitivity is sufficient since the reference clinical range for L-phenylalanine is 78-206 microM.

Amino Acid Oxidoreductases↗

PubMed1998

Long-term results of percutaneous balloon mitral valvuloplasty for mitral stenosis: a follow-up study to 11 years in 202 patients.

We studied the first 202 patients with rheumatic mitral stenosis (MS) who underwent percutaneous balloon mitral valvuloplasty (PBMV) with the Inoue balloon catheter for a follow-up (FU) period of 5-11 years. Pre- and post-PBMV and at FU, the mean left atrial pressure was 21.3+/-7.4, 10.2+/-5.6, and 11.2+/-4.1 mm Hg; mean diastolic mitral gradient was 18.4+/-7.3, 2.9+/-3.2, and 5.1+/-4.3 mm Hg; and mitral valve area was 1.0+/-0.3, 2.1+/-0.6, and 1.7+/-0.5 cm2. Functional status improved from New York Heart Association (NYHA) class IV in 3, class III in 119, and class II in 80 pre-PBMV to class I in 163, class II in 37, and class III in 2 post-PBMV, and was class I in 146, class II in 39, and class III in 17 patients at FU. In the 17 patients with NYHA class III at FU, mitral restenosis was the culprit; 4 underwent repeat PBMV, 12 had mitral valve replacement for severe mitral calcification and subvalvular fusion, and 1 refused further intervention. Thus PBMV using the Inoue balloon catheter is an effective method of relieving MS with excellent long-term results in patients without severe mitral calcification and subvalvular fusion.

Adolescent↗

PubMed1998

Improving the fixation method for preimplantation genetic diagnosis by fluorescent in situ hybridization.

PURPOSE: Our purpose was to modify a fixation method using Tween-20 and HCl (TH) and to compare it with a protocol using methanol and acetic acid (MA) for the improvement of preimplantation genetic diagnosis by fluorescence in situ hybridization (FISH). METHODS: Single blastomeres were allocated to either the TH or the MA procedure. The two methods were compared to evaluate efficiency of fixation and the intensities of FISH signals. RESULTS: With the TH method, 123 (93.9%) of 131 blastomeres were fixed, while only 95 (78.5%) of 121 were fixed with MA. Average scores for the intensity of FISH signals were significantly stronger for TH than for MA (P < 0.05). There was also a significant difference in signal intensity scores between the two methods for type-3 nuclei. CONCLUSIONS: Our results indicate that not only are fewer nuclei lost during fixation but also stronger FISH signals can be obtained with the TH method. Thus, modified TH can improve the overall efficiency of preimplantation genetic diagnosis.

Acetic Acid↗

PubMed1998

Effects of pre-exposure of mouse testis with low-dose (16)O8+ ions or 60Co gamma-rays on sperm shape abnormalities, lipid peroxidation and superoxide dismutase (SOD) activity induced by subsequent high-dose irradiation.

PURPOSE: To investigate the effects of pre-exposure of mouse testis with low-doses of (16)O8+ ions or 60Co gamma-rays on sperm shape abnormalities, lipid peroxidation and superoxide dismutase (SOD) activity induced by subsequent high-dose irradiation. MATERIALS AND METHODS: Testes of the B6C3F1 hybrid strain mice were pre-irradiated with 0.05 Gy of (16)O8+ ions or 60Co gamma-rays and then after 4 h given a test irradiation with 2 Gy of the same radiation type. SOD activity and thiobarbituric acid reactive substances (TBARS) in the testes were determined by spectrophotometric and TBA methods respectively at 4 h after irradiation. Testis weight, sperm count and sperm morphology were analysed at day 35 after irradiation. RESULTS: Compared with controls, there was a significant increase in SOD activity and a significant decrease in TBARS level of pretreated testes. Testis weight loss, sperm count reduction and sperm abnormalities were significantly lower in the pretreated testes. The bioeffects of a 2 Gy dose of (16)O8+ ions relative to 60Co gamma-rays were 1.84 +/- 0.28 for testis weight, 1.22 +/- 0.25 for sperm count and 1.29 +/- 0.10 for sperm abnormalities. CONCLUSIONS: These data suggest that pre-exposure of testes with a low dose of heavy ions or gamma-rays renders the organ more resistant to subsequent high-dose irradiation. The increase of SOD activity and the decrease of lipid peroxidation levels induced by low-dose ionizing irradiation may be involved in this resistance. The effects with heavy ion irradiation were greater than with gamma-rays.

Animals↗

PubMed1998

Reconstruction of a large abdominal wall defect using combined free tensor fasciae latae musculocutaneous flap and anterolateral thigh flap.

A large abdominal wall defect was reconstructed with the use of a flap combining the tensor fasciae latae musculocutaneous flap and the anterolateral thigh flap in four individuals who had undergone extensive abdominal wall resection because of cancer. The flap was harvested as a single combined composite flap and was transferred to the recipient site by means of microvascular surgery. Morbidity was minimal and the outcome was satisfactory in all instances.

Abdominal Muscles↗