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Biomedical subjects

B R Huang

Publications and source records attributed to B R Huang.

17 recordsLinked to original sources
PubMed2001

Crystal structure of human epidermal growth factor and its dimerization.

Epidermal growth factor (EGF) is a typical growth-stimulating peptide and functions by binding to specific cell-surface receptors and inducing dimerization of the receptors. Little is known about the molecular mechanism of EGF-induced dimerization of EGF receptors. The crystal structure of human EGF has been determined at pH 8.1. There are two human EGF molecules A and B in the asymmetric unit of the crystals, which form a potential dimer. Importantly, a number of residues known to be indispensable for EGF binding to its receptor are involved in the interface between the two EGF molecules, suggesting a crucial role of EGF dimerization in the EGF-induced dimerization of receptors. In addition, the crystal structure of EGF shares the main features of the NMR structure of mouse EGF determined at pH 2.0, but structural comparisons between different models have revealed new detailed features and properties of the EGF structure.

Crystallization↗
PubMed2001

Regulating colored textile wastewater by 3/31 wavelength ADMI methods in Taiwan.

The wastewater from textile dyeing facilities is difficult to treat satisfactorily because of high compositional variability and high color intensity. To reduce colored effluents discharged into watercourses, the government of Taiwan adopted the Effluent True Color Standard in 1998. The true color discharge limit is 400 American Dye Manufactures Institute (ADMI) units. The adopted analytical method is the ADMI Tristimulus Filter Method (3 wavelength (WL) method), and the 31 WL ADMI method might be also adopted as an alternative for color value measurement. The refractory nature of textile dyes and the introduction of this new regulation present an environmental challenge to the Taiwanese textile industry. The main objectives of this study were to (1) evaluate the efficacy of current wastewater treatment systems for controlling the colored textile wastewater discharges, and (2) evaluate the correlations between 3 and 31 WL ADMI methods. Ten representative textile wastewater treatment facilities employing biological and chemical coagulation treatment technologies were selected to perform a 10-consecutive-day effluent sampling and analysis. Results show that a significant difference between 3 and 31 ADMI methods was observed. These two ADMI methods cannot be substituted for each other, and the discharge standard should be determined based on the selected testing method. Investigation results also suggest that the commonly used wastewater treatment technology (biological + chemical coagulation) fails to effectively remove dye from the colored textile wastewater. Sodium hypochlorite (NaOCl) addition was applied by most facilities as the temporary post-polishment step to comply with the color discharge standard.

Chemistry Techniques, Analytical↗
PubMed2001

[Purification of recombinant hEGF expressed in yeast Pichia pastoris and the study on its characters].

OBJECTIVE: To obtain recombinant human epidermal growth factor(hEGF) that can be used in animal experiments and clinical trial. METHOD: Chemically synthesized hEGF gene was expressed in Yeast Pichia pastoris and the secretory hEGF was purified by Phenysepharose 6 Fast Flow(high sub), Q-sepharose High Performance, and Superdex 30 chromatography, and its characters were studied by respective methods. RESULTS: The purified hEGF doesn't contain pyrogen, endotoxin, or yeast chromosome DNA and the purity reached 98%. The recombinant human EGF has correct molecular weight, pI, N-terminal amino acids sequences, peptide map, ultraviolet spectrum and well-biological activity. CONCLUSION: The purified hEGF is in accord with the requirements for animal experiments and clinical trial which provides the basis of preparing EGF agents for clinical test.

Animals↗
PubMed2001

[Hunting for novel protein factors in G-protein pathway with yeast two-hybrid system].

OBJECTIVE: To explore the protein factors that could interact with G beta subunit within the G protein signal transducing pathway. METHODS: The highly sensitive protein-protein interaction system--Yeast Two-hybrid System was applied to screen the human cDNA library with constructed "Bite plasmid" containing G beta subunit gene fragment. And then the false positive test was adapted. RESULTS: Three positive gene fragments were obtained. One codes for "Actin bundling protein". The other two are new ones and their Gene Bank accession numbers are AF288405 and AF288406 respectively. CONCLUSIONS: It is the first time to find that among human brain tissue, G beta subunit becomes a structural or functional unit interacts with actin bundling protein and the other two unknown protein factors to activate the following pathway. This result may be important to understand the relationship between the actin cytoskeleton and G proteins.

Actins↗
PubMed2001

[Roles of G-protein beta and gamma subunits in the interaction of G beta gamma with adenylyl cyclases II].

OBJECTIVE: To explore the individual roles of G protein beta and gamma subunit in the interactions with effectors. METHODS: We investigated the interactions of G beta 1 and G beta 1 gamma 2 with adenylyl cyclase II(AC II) using the yeast two-hybrid and three-hybrid systems. RESULTS: When assayed for the abilities to activate the reporter gene, the interactions among AD-beta 1, gamma 2 and BD-AC IIQ in the three-hybrid system were more potent than the interactions between AD-beta 1 and BD-AC II Q in the two-hybrid system. The expressions of BD-AC IIQ and AD-beta 1 in transformants coexpressed AD-beta 1 and BD-AC IIQ, and transformants coexpressed AD-beta 1, gamma 2 and BD-AC IIQ were respectively detected. The comparisons between the reporter activity and the expression levels of BD-AC IIQ and AD-beta 1 in the yeast cells show there was no correlations, i.e. The difference in the reporter activity was not a reflection of differential expression level of the hybrid proteins. CONCLUSIONS: All these results suggest that G protein beta 1 subunit is sufficient to maintain the basic interaction between G beta 1 gamma 2 and AC IIQ, and gamma 2 subunit plays an important role in the high affinity interaction of G beta 1 gamma 2 with AC IIQ.

Adenylyl Cyclases↗
PubMed2001

[Toxicity study of epidermal growth factor eye medicine liquid].

OBJECTIVE: To apply for a new medicine certificate, the experiments of acute, long toxicity and side effects of EGF eye medicine liquid were investigated in animals. METHODS: All of examinations were done according to the instructions of "Requirements of the Toxicity Study of New Medicine" RESULTS: No toxicity or side-effect of EGF eye medicine liquid given by i.v., i.p., s.c. or eye-drop were been found. CONCLUSION: For the results of whole experiments are in complete accord with "the requirements of the toxicity study of new medicine", EGF eye medicine liquid will be safety in clinical application.

Animals↗
PubMed2001

[Expression of neurotrophin-4(NT-4) gene in baculovirus expression vector system].

OBJECTIVE: We selected baculovirus expression vector system to express human NT-4 with biological activity. METHODS: The hNT-4 mature peptide-coding sequence is amplified by polymerase chain reaction (PCR), ligated to baculovirus expression vector PacGP67B, and expressed in the insert sf9 cell line. RESULTS: The protein molecular weight of the expressed hNT-4 was about 15,000 by SDS-PAGE, and the hNT-4 antibody could react with this protein in the infected supernatant and total cell by western-blot. The activity of hNT-4 determined by PC12 cell line was definite. CONCLUSIONS: The results may aid for studying the effect of the hNT-4 on basic medical research and clinical application.

Animals↗
PubMed2001

[The basic and applied study on the epidermal growth factor].

This article reviews the results of the basic research about epidermal growth factor and its receptor, and the development of the novel drug, EGF eyedrop, that containing chemically synthesized EGF gene, the construction of EGF expression vector, the transformation of the host cells, the purification of the recombinant protein EGF, the preparation of three batches of the EGF product and identification, the preclinical and clinical trials. Relevant studies show that recombinant EGF consisting of 51 amino acids can be secreted into the medium under the control of the alpha factor leading sequence in the yeast cells. The EGF can accelerate the growth of corneal-limbal epithelial cells and the healing of an alkali burned corneal. The EGF can be used in curing oral cavity ulcer and skin burned wound. And it has the preventive effects on experimental duodenal ulcer of rat. The antiserum was made for test of the concentration of blood EGF and urine EGF by RIA. Data from studies demonstrate the inhibition effect of EGF on the growth of tumor cells, such as A431 and BT325 cells in the presence of high EGF concentration (> 10 ng/ml). The expression of EGFR and DNA ploidy in renal carcinoma has clinical significance. Crystallization and preliminary x-ray diffraction studies of the EGF has been made. The MW of the EGF product is 6000, and the pI is about 4.6 and it has correct N-terminal amino acids sequences, immunogenicity and biological activity. There is no vestige of the DNA of the yeast cells. Animal experiments reveal that there is no cumulation of the EGF in the body, and EGF can promote corneal epithelial healing. There is no toxicological effect during cornea wound healing of rabbit. A randomized, double-blind, placebo-controlled, multi-center clinical trial was conducted in four hospitals to assess safety, ocular tolerance and efficacy of an ophthalmic solution of EGF for 200 cases of cornea transplantation and 247 cases of nebulae. Unequivocal results were obtained as the eyedrop really accelerate the wounded cornea healing. So, the EGF eyedrop as a novel drug of class I is approved by the National Drug Administration, and this is the first gene engineering drug that come from yeast expression system in China.

Animals↗
PubMed2000

Differential actions of neurotrophins on apoptosis mediated by the low affinity neurotrophin receptor p75NTR in immortalised neuronal cell lines.

The low affinity neurotrophin receptor (p75NTR) mediates apoptosis of a number of neuronal and non-neuronal cells but the signals leading to the apoptosis remain obscure. To reveal the mechanism of p75NTR-mediated apoptosis, a neural cell line expressing human p75NTR was established. The human cDNA fragment encoding for p75NTR was PCR-amplified, cloned into the retrovirus expression vector pXT-1 and transfected into the rat cerebellum cell line R2. The expression of p75NTR in the R2 cell line was demonstrated by both Northern blotting analysis and immunocytochemistry. Serum withdrawal induced dramatic apoptosis in p75NTR-expressing R2 cells (R2L1) but not in pXT-1 transfected control R2 cells (R2P). Reverse transcription polymerase chain reaction (RT-PCR) revealed that these cell lines express trkA and trkB but not trkC. The apoptosis of R2L1 cells triggered by the serum deprivation for 48 h was completely prevented by neurotrophin-3 and the antibody to p75NTR but only partially prevented by the nerve growth factor and brain derived neurotrophic factor. We conclude that the p75NTR mediates apoptosis of R2L1 cells by its intrinsic receptor effects requiring an unbound status of this receptor and that the apoptosis is prevented by neurotrophins or the antibody to p75NTR through distinct mechanisms.

Animals↗
PubMed2000

Crystallization and preliminary X-ray diffraction studies of human epidermal growth factor.

Human epidermal growth factor (hEGF), a 6.2 kDa protein of 53 amino acids with three internal disulfide bridges, has been crystallized by the hanging-drop method. hEGF crystallizes in space group P3(1)21 (or P3(2)21) using MgCl(2) as precipitant, with unit-cell parameters a = b = 61.4, c = 87.0 A. Another type of crystal, obtained using NaCl as precipitant, belongs to a tetragonal point group and has unit-cell dimensions a = b = 102.5, c = 166.6 A. The trigonal crystals with the smaller unit cell diffract X-rays better and a native data set from a single crystal has been collected to 3.0 A resolution.

Crystallization↗
PubMed1999

Distribution of neurturin mRNA and immunoreactivity in the peripheral tissues of adult rats.

Neurturin (NTN) is a recently discovered neurotrophic factor related to glial cell line-derived neurotrophic factor (GDNF) and has a wide spectrum of biological roles in different types of neurons in the central and peripheral nervous systems. However, information on its expression in peripheral tissues has been limited, and there is no information on its peptide distribution. As a step to examine its role and action mechanisms in neuronal and non-neuronal cells in the periphery, the present study investigated the distribution patterns of its mRNA and peptide in some major peripheral organs of adult rats by in situ hybridization and immunohistochemistry. A widespread expression of NTN mRNA was found in the selected organs of various systems, with a high level in pituitary intermediate lobe, intestine, salivary gland, and testis, and a moderate level in ovary, adrenal gland, kidney, thyroid, and spleen. NTN peptide was also present in the peripheral organs studied, with its distribution corresponding to that of mRNA. In conclusion, NTN is expressed widely in many regionally well-defined cellular systems in various peripheral tissues, suggesting that NTN may act as a target-derived neurotrophic factor for innervating neurons and may have maintenance functions in non-neuronal cells of these adult organs.

Adrenal Glands↗
PubMed1999

Synthesis and anticancer evaluation of certain alpha-methylene-gamma-(4-substituted phenyl)-gamma-butyrolactone bearing thymine, uracil, and 5-bromouracil.

Certain alpha-methylene-gamma-(4-substituted phenyl)-gamma-butyrolactone bearing thymine, uracil, and 5-bromouracil were synthesized and evaluated for their anticancer activity. These compounds demonstrated a strong growth inhibitory activity against leukemia cell lines. The anticancer potency for the substituents of the lactone C(gamma)-phenyl is in an order of 4-Ph > 4-Cl, 4-Br > 4-Me, 4-NO2 > 4-F. For the pyrimidine portion, 5-bromouracil is more potent than uracil and thymine.

4-Butyrolactone↗
PubMed1996

Cloning strategies using a third irrelevant enzyme site (TIES) to overcome certain cloning problems.

We describe a cloning strategy, third irrelevant enzyme site (TIES), based on three-component assembly ligation reactions to overcome seven types of problematical cloning reactions. Implementation of the TIES strategy has made it possible to obtain the desired recombinant where the corresponding approach using conventional recombinant DNA methodologies and two-component ligations failed to give the desired product.

Binding Sites↗
PubMed1995

[Progress in the study of nerve growth factor family and its receptors].

There were several noticeable progress in the study of the neurotrophic factors, their receptors and the neuronal apoptosis. Firstly, several members of the nerve growth factor family have been identified: NGF, BDNF, NT-3 and NT-4/5. Secondly, the relationship between the three-dimensional structure of the NGF and its function and evolution has been revealed. Thirdly, two kinds of the neurotrophin family receptors were characterized, they are low-affinity NGFR (p75 NGFR) and the trk proto-oncogene product such as p140trkA, p145trkB and p145trkC. Induction of apoptosis by the low-affinity NGFR was also found. Finaly it is possible to use the neurotrophin for treating the degenerative neurological illnesses.

Amino Acid Sequence↗
PubMed1993

Synthetic and cytotoxic studies of alpha-methylene-gamma-butyrolactone bearing pyrimidines.

A total of ten pyrimidine alpha-methylene-gamma-butyrolactones were synthesized as potential antitumor agents on the basis of their possible action as Michael acceptors for DNA/RNA and cellular enzymes. The synthesis of these heterocycles involved a convenient Reformatsky-type reaction of pyrimidinyl ketones with ethyl alpha-(bromomethyl)acrylate. The preliminary in vitro cytotoxic assay indicated that these synthetic compounds were essentially active against the growth of KB, Hep-2, HeLa and Colo-205 cells. Among them, 5'-biphenyl-5'-(uracil-1-ylmethyl)-2'-oxo-3'-methylenetetrahydr ofuran (5f) demonstrated to be the most potent antileukemic agent.

4-Butyrolactone↗