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Biomedical subjects

J Wen

Publications and source records attributed to J Wen.

At least 109 records · Page 6Linked to original sources
PubMed1997

Structural characterization of an alpha-amylase inhibitor from a wild common bean (Phaseolus vulgaris): insight into the common structural features of leguminous alpha-amylase inhibitors.

The primary structures of two subunits of an alpha-amylase inhibitor (alpha AI-2) from a wild common bean (Phaseolus vulgaris) were revealed by a comparison of the amino acid sequence previously deduced from the nucleotide sequence with the amino- and carboxyl-terminal amino acid sequences determined by conventional methods. The polypeptide molecular weight of alpha AI-2 obtained by the light-scattering technique, considered together with the sequence molecular weights revealed for the subunits, indicated that alpha AI-2 has the subunit stoichiometry of an alpha 2 beta 2 complex. These structural features were closely similar to those recently elucidated for a white kidney bean (P. vulgaris) alpha-amylase inhibitor, which is quite different in the inhibitory specificity from alpha AI-2. The post-translational processing of the precursor glycoproteins to form the tetrameric structure appeared to require an Arg residue close to the processing site. Further, the proper associations of the subunits into the tetrameric structures seemed to be strictly controlled by a few amino acids on the subunit interfaces.

Amino Acid Sequence↗
PubMed1997

Comparison of solution properties of human and rat ciliary neurotrophic factor.

The solution structure and stability of rat and human ciliary neurotrophic factor (CNTF) were examined by circular dichroism (CD), Fourier transform infrared (FTIR) and fluorescence spectroscopy and sedimentation equilibrium analyses. The secondary structure of both proteins, as assessed by CD and FTIR, consists primarily of alpha-helix, consistent with CNTF being a member of the four-helical bundle family of cytokines and neurokines, with rat CNTF containing slightly less helix (about 10% less) and slightly more disordered structure. The environment of the tyrosine and tryptophan residues, assessed by intrinsic fluorescence emission spectroscopy, appears to be the same in both proteins. Binding of anilinonaphthalene sulfonate is identical for both proteins, indicating that these two proteins have similar surface hydrophobicities in the native state. The thermal stability of the human CNTF is significantly less than that of the rat CNTF, yet their stabilities to guanidine HCl-induced denaturation are equivalent. This apparent discrepancy in stability between the two proteins may be explained by solubility differences upon thermal unfolding. Although the human protein precipitates as it is denatured by heat, the rat protein does not. It thus appears that the unfolded state of human CNTF is less soluble and more prone to aggregation than that of the rat protein upon heating, although their conformational stability is similar. Both proteins remain largely folded at pH 3.0. Sedimentation equilibrium analysis demonstrates that both rat and human CNTF exist primarily as monomers; however, significant dimer formation occurs as the protein concentrations are increased to greater than 3 mg/mL, particularly in the presence of ammonium sulfate.

Anilino Naphthalenesulfonates↗
PubMed1997

Ryanodine receptor expression is associated with intracellular Ca2+ release in rat parotid acinar cells.

The ryanodine receptor mediates intracellular Ca2+ mobilization in muscle and nerve, but its physiological role in nonexcitable cells is less well defined. Like adenosine 3',5'-cyclic monophosphate and inositol 1,4,5-trisphosphate, cyclic ADP-ribose (0.3-5 microM) and ADP (1-25 microM) produced a concentration-dependent rise in cytosolic Ca2+ in permeabilized rat parotid acinar cells. Adenosine and AMP were less effective. Ryanodine markedly depressed the Ca2+-mobilizing action of the adenine nucleotides and forskolin in permeabilized cells and was likewise effective in depressing the action of forskolin in intact cells. Cyclic ADP-ribose-evoked Ca2+ release was enhanced by calmodulin and depressed by W-7, a calmodulin inhibitor. A fluorescently labeled ligand, 4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3,4-diaza-s-indac ene-3-propionic acid-glycyl ryanodine, was synthesized to detect the expression and distribution of ryanodine receptors. In addition, ryanodine receptor expression was detected in rat parotid cells with a sequence highly homologous to a rat skeletal muscle type 1 and a novel brain type 1 ryanodine receptor. These findings demonstrate the presence of a ryanodine-sensitive intracellular Ca2+ store in rat parotid cells that shares many of the characteristics of stores in muscle and nerve and may mediate Ca2+-induced Ca2+ release or a modified form of this process.

1-Methyl-3-isobutylxanthine↗
PubMed1997

Absorption of calcium, zinc, and iron from breast milk by five- to seven-month-old infants.

Data are scarce regarding mineral bioavailability from human milk in older infants who may also be receiving solid foods (beikost). We measured the absorption of Ca, Zn, and Fe in 14 healthy, nonanemic 5-7-mo-old breast-fed infants whose mothers milk was extrinsically labeled with stable isotopes (44Ca, 70Zn, and 58Fe) of these minerals. In addition, Ca and Zn stable isotopes (46Ca and 67Zn) were administered i.v., and a second isotope of Fe (57Fe) was given orally without food as a non-meal dose. Subjects were not receiving any artificial infant formula or cow's milk, but most (10/14) were receiving beikost. Ca and Zn absorption was calculated using the urinary excretion of the isotopes during the 24 h after dosing (Ca) or their urinary ratio 72 h after dosing (Zn). Fe absorption was calculated using the red blood cell incorporation at 14 d. Fe absorption averaged 20.7 +/- 14.8% from the 58Fe given with human milk (geometric mean, 14.8%) and 17.7 +/- 15.1% (geometric mean, 11.0%) from the 57Fe non-meal dose. Ca absorption averaged 61.3 +/- 22.7% and Zn absorption (n = 10) averaged 49.5 +/- 18.5%. Absorption of Fe (natural logarithm) from the non-meal Fe dose (57Fe) but not from the human milk (58Fe) was significantly negatively correlated to serum ferritin (r = -0.70, p = 0.007 versus r = -0.35, p = 0.24). At the intake levels in this study, total daily Fe, Ca, and Zn intakes from beikost were not significantly correlated to their fractional absorption from breast milk, but Fe intake from beikost was significantly negatively correlated to absorption of Fe from the non-meal dose (r = -0.61, p = 0.021). We conclude that minerals are well absorbed from human milk in older infants after the introduction of beikost to the diet.

Calcium↗
PubMed1997

[The effects of endothelin and bFGF on proliferation of MC and synergetic effect of insulin].

The effects of basic fibroblast growth factor (bFGF), insulin and endothelin-1 (ET-1) on proliferation of rat glomerular mesangial cells (MC) cultured in vitro and the synergetic effects of insulin and bFGF or ET-1 on MC proliferation were determined by H-TdR incorporation experiment. The results showed that bFGF at concentrations of 5-200 ng/ml and insulin at concentrations of 0.1-2.4 U/ml significantly increased the value of 3H-TdR incorporated into MC (the cpm value). The effect of ET-1 on the cpm value was different at different doses. At concentration ranging from 10(-9) mol/L to 10(-7) mol/L, the cpm value significantly increased with increasing the concentration of ET-1, and the highest value was at concentration of 10(-8) mol/L, However, the cpm value decreased at concentration of 10(-6) mol/L ET-1. When MCs were incubated simultaneously with insulin and bFGF or lower concentration of ET-1 (< 10(-8) mol/L), the cpm value was significantly higher than the sum of that induced independently by insulin and bFGF or ET-1. But the joint effect of insulin and higher concentration of ET-1 (> 10(-7) mol/L) on the cpm value was lower than the sum of that induced separately by insulin and ET-1. It was indicated that bFGF, insulin and ET-1 significantly stimulated MC proliferation; insulin exhibited positive synergetic effect with bFGF or lower concentration of ET-1 on MC proliferation, negative synergetic effect with higher concentration of ET-1 on MC proliferation.

Animals↗
PubMed1997

[Promotive effect of TNF-alpha and M-CSF on osteolysis induced by giant cell tumor of bone].

OBJECTIVE: To explore the effect of cell components in giant cell tumor of bone (GCT) and cytokines expressed by them on osteolysis. METHODS: Mononuclear stromal cells and multinucleated giant cells (MGCs) were isolated from 10 cases of GCT, and fibroblast-like stromal cells were obtained by long-term culture of mononuclear stromal cells. Osteolytic capability of above isolated cells were tested in an in vitro cell-bone resorption model. RESULTS: All cell components isolated from GCT had capability to resorb bone matrix directly. Exogenous tumore necrosis factor-alpha (TNF-alpha) could significantly increase the bone resorption induced by both kinds of stromal cell. There were higher level of TNF-alpha and greater expression rate of macrophage colony stimulating factor (M-CSF) in GCT than in the osteosarcoma tissues or the normal serum. CONCLUSIONS: The characteristic bone resorption behavior of GCT might be conducted by all it's three major cell components, and this bone resorption process could be promoted by both TNF-alpha and M-CSF they expressed.

Bone Neoplasms↗
PubMed1997

[Thermoregulation under simulated weightlessness].

The effect of simulated weightlessness on thermoregulation was studied in 5 subjects. The experiment consisted of 3d baseline measurements, 7d head-down bed rest and 2d recovery. Circadian rhythm was assessed by continuous measurements of rectal temperature and skin temperature with 2h intervals. Heat Stress Protein 70 (HSP70) was measured by Western-blot Dot method and the facial surface temperature distribution was measured by HR-2 infrared thermography. The results showed that rectal temperature keeps the wake-sleep variation, but the circadian rhythm changed during bed rest, and the change of rectum temperature rhythm appeared mainly in the early-days of bed rest; HSP70 and facial surface temperature increased during bed rest.

Bed Rest↗
PubMed1996

Sequential excision of internal eliminated DNA sequences in the differentiating macronucleus of the hypotrichous ciliate Stylonychia lemnae.

Elimination of internal eliminated sequences (IES) during macronuclear development of the hypotrichous ciliate Stylonychia lemnae was analyzed in one cluster of macronuclear precursor DNA sequences. The results indicate that IES elimination is a highly ordered process, it starts very early during macronuclear development and has only finished immediately before DNA fragmentation takes place. It occurs in distinct steps and the IES are eliminated in a specific order, where a defined IES is only removed after complete elimination of other IES. Transfection experiments clearly demonstrate that the structure of the IES itself is not sufficient for its correct excision but other cis-acting sequences or additional structural requirements are needed for IES elimination.

Animals↗
PubMed1996

Murine alpha-macroglobulins demonstrate divergent activities as neutralizers of transforming growth factor-beta and as inducers of nitric oxide synthesis. A possible mechanism for the endotoxin insensitivity of the alpha2-macroglobulin gene knock-out mouse.

alpha2-Macroglobulin null mice demonstrate increased resistance to endotoxin challenge (Umans, L., Serneels, L., Overbergh, L., Van Leuven, F., and Van den Berghe, H. (1995) J. Biol. Chem. 270, 19778-19785). We hypothesized that this phenotype might reflect the function of murine alpha2M (malpha2M) as a neutralizer of transforming growth factor-beta (TGF-beta) and inducer of nitric oxide synthesis in vivo. When incubated with wild-type mouse plasma, TGF-beta1 and TGF-beta2 bound only to malpha2M. Alternative TGF-beta-binding proteins were not detected in plasma from alpha2M(-/-) mice. Wild-type mouse plasma, but not plasma from alpha2M(-/-) mice, inhibited TGF-beta1 binding to TGF-beta receptors on fibroblasts. Purified malpha2M bound TGF-beta1 and TGF-beta2 with similar affinity; the KD values were 28 +/- 4 and 33 +/- 4 nM, respectively. Murinoglobulin, the second murine alpha-macroglobulin, bound both TGF-beta isoforms with 30-fold lower affinity. Malpha2M counteracted the activities of TGF-beta1 and TGF-beta2 in an endothelial cell growth assay. Malpha2M also induced NO synthesis when incubated with RAW 264.7 cells, an activity which probably results from the neutralization of autocrine TGF-beta activity. Human alpha2M induced NO synthesis comparably to malpha2M; however, MUG had no effect. These studies demonstrate that the ability to neutralize TGF-beta is a property of malpha2M, which is not redundant in the murine alpha-macroglobulin family or in murine plasma. Malpha2M is the only murine alpha-macroglobulin that promotes NO synthesis. The absence of malpha2M, in alpha2M(-/-) mice, may allow TGF-beta to more efficiently suppress excessive iNOS expression following endotoxin challenge.

Animals↗
PubMed1996

Human hepatic lipase subunit structure determination.

Chinese hamster ovary cells were stably transfected with a human hepatic lipase (HL) cDNA. The recombinant enzyme was purified from culture medium in milligram quantities and shown to have a molecular weight, specific activity, and heparin affinity equivalent to HL present in human post-heparin plasma. The techniques of intensity light scattering, sedimentation equilibrium, and radiation inactivation were employed to assess the subunit structure of HL. For intensity light scattering, purified enzyme was subjected to size exclusion chromatography coupled to three detectors in series: an ultraviolet absorbance monitor, a differential refractometer, and a light scattering photometer. The polypeptide molecular weight (without carbohydrate contributions) was calculated using the measurements from the three detectors combined with the extinction coefficient of human HL. A single protein peak containing HL activity was identified and calculated to have a molecular mass of 107,000 in excellent agreement with the expected value for a dimer of HL (106.8 kDa). In addition, sedimentation equilibrium studies revealed that HL had a molecular mass (with carbohydrate contributions) of 121 kDa. Finally, to determine the smallest structural unit required for lipolytic activity, HL was subjected to radiation inactivation. Purified HL was exposed to various doses of high energy electrons at -135 degrees C; lipase activity decreased as a single exponential function of the radiation dose to less than 0.01% remaining activity. The target size of functional HL was calculated to be 109 kDa, whereas the size of the structural unit was determined to be 63 kDa. These data indicate that two HL monomer subunits are required for lipolytic activity, consistent with an HL homodimer. A model for active dimeric hepatic lipase is presented with implications for physiological function.

Animals↗
PubMed1996

Size-exclusion chromatography with on-line light-scattering, absorbance, and refractive index detectors for studying proteins and their interactions.

Techniques of using size-exclusion chromatography (SEC) with on-line light-scattering, uv absorbance, and refractive index detectors to characterize the polypeptide molecular weights of simple proteins or glycoproteins or to determine the stoichiometry of protein complexes are described. Two unique advantages of this approach over conventional SEC are that the molecular weight measurement is independent of elution position and can exclude the contributions from carbohydrates. When a protein or complex contains no carbohydrates, a two-detector method, i.e., light scattering combined with refractive index, can be used to calculate the molecular weight. When a protein contains carbohydrates, a three-detector method is used to calculate the molecular weight of polypeptide alone. Finally, a self-consistent three-detector method is used to determine the stoichiometry of a protein complex containing carbohydrates. Example applications for all these methodologies are described.

Carbohydrates↗
PubMed1996

ErbB receptor activation, cell morphology changes, and apoptosis induced by anti-Her2 monoclonal antibodies.

A panel of mAbs were generated against the purified soluble form of erbB2/Her2 receptor, corresponding to the extracellular region of the receptor, and examined for their ability to mimic the receptor ligand. Some of the mAbs strongly induced tyrosine phosphorylation of 180-185 kDa proteins, including not only Her2 but also Her3 and Her4 receptors, when they were expressed on the surface of breast cancer cells. These mAbs do not cross-react with Her3 or Her4 as demonstrated by competition study. Receptor phosphorylation was also observed with the cell lines transfected with Her2 or a chimeric receptor consisting of the extracellular domain of Her2 and the transmembrane and cytoplasmic domains of epidermal growth factor receptor. Selected mAbs were tested for their ability to change cell morphology, and one specific mAb, mAb74, induced cell morphology changes and apoptosis.

Animals↗
PubMed1996

Characterization of keratinocyte growth factor binding to heparin and dextran sulfate.

Binding of keratinocyte growth factor (KGF) with heparin (molecular weight of 5000) and dextran sulfate (molecular weight of 8000) was studied using an online monitoring of size-exclusion chromatography with light scattering, refractive index, and uv absorbance detectors. This technique allows the determination of the molecular weight of KGF eluting as complexes with the above polymers. When mixtures of KGF with heparin were injected into the column, two peaks of heparin/KGF complexes were observed. The first peak corresponded to, on average, 3.4 KGF per complex and the second peak to an average of about 2 KGF per complex. These results suggest that the heparin/ KGF complex is heterogeneous, consisting of 1,2,3, and 4 KGF molecules per complex. To calculate the number of heparin molecules in these complexes, the rate of disappearance of free KGF was determined as heparin was added. The average number of KGF bound to 1 mol of heparin was calculated to be about 2 mol, suggesting that only one heparin molecule is present in these complexes. The heparin binding of two KGF mutants, i.e., (C1, 15S)KGF (with substitutions of serine for cysteines 1 and 15) and d28KGF (lacking 28 N-terminal amino acid residues), was essentially identical to that of the native sequence KGF. A similar experiment was carried out for KGF binding to dextran sulfate. The molecular weight of the complex corresponded to 2 to 2.6 KGF molecules per complex. The rate of disappearance of free KGF as the dextran sulfate added showed 2-3 mol of KGF bound to 1 mol of dextran sulfate, consistent with the idea that the complex contains only 1 dextran sulfate molecule.

Binding Sites↗
PubMed1996

Dimerization of the extracellular domain of granuloycte-colony stimulating factor receptor by ligand binding: a monovalent ligand induces 2:2 complexes.

Granulocyte-colony stimulating factor (G-CSF) binds to a specific cell surface receptor and induces signals for growth and differentiation in cells of granulocyte hematopoietic lineage. In order to understand how G-CSF binding initiates signals into these cells, we have studied its interactions with the entire extracellular domain of the receptor (sG-CSFR). The sG-CSFR was purified from CHO cell conditioned media with a G-CSF affinity column, resting in a preparation fully competent for ligand binding. However, when sG-CSFR was purified by conventional means, i.e., without affinity chromatography, only about half was competent. Therefore, all studies were carried out using affinity-purified material. The sG-CSFR exhibited a weak self-association into a dimer with a dissociation constant of 200microM in the absence of G-CSF. Addition of G-CSF dimerizes the receptor, with a preferred stoichiometry of 2 G-CSF molecules plus 2 receptors. Unexpectedly, receptor-receptor interactions rather than through two receptors binding to the same G-CSF molecule; i.e., G-CSF is a monovalent ligand. G-CSF binding to the receptor monomer occurs with high affinity. The binding of G-CSF also enhances the receptor-receptor dimerization; when G-CSF is bound to both receptors, dimerization is enhanced 2000-fold, while the interaction of a 1:1 receptor-ligand complex with a second ligand-free receptor is enhanced 80-fold. Thus, the mechanism of receptor dimerization is fundamentally different than that of related cytokine receptors such as growth hormone and erythropoietin receptors. Circular dichroic spectra showed a small but significant conformational change of receptor upon binding G-CSF. This is consistent with the idea that G-CSF binding alters the conformation of the receptor, resulting in an increase in receptor-receptor interactions.

Animals↗