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Biomedical subjects

J Wen

Publications and source records attributed to J Wen.

At least 127 records · Page 7Linked to original sources
PubMed1996

Human stem cell factor dimer forms a complex with two molecules of the extracellular domain of its receptor, Kit.

Stem cell factor (SCF) is a cytokine that is active toward hematopoietic progenitor cells and other cell types, including germ cells, melanocytes, and mast cells, which express its receptor, the tyrosine kinase, Kit. SCF exists as noncovalently associated dimer at concentrations where it has been possible to study its quaternary structure; it stimulates dimerization and autophosphorylation of Kit at the cell surface. We have used recombinant versions of human SCF and human Kit extracellular domain (sKit) to study SCF-Kit interactions. By size exclusion chromatography, plus various physical chemical methods including light scattering, sedimentation equilibrium, and titration calorimetry, we demonstrate the formation of complexes containing a dimer of SCF (unglycosylated SCF1-165) plus two molecules of sKit. The concentrations of SCF and sKit in these studies were in the range of 0.35-16.2 microM. The data are analyzed and discussed in the context of several possible models for complex formation. In particular, the sedimentation data are not consistent with a model involving cooperative binding. The Kd estimate for SCF-sKit interaction, obtained by sedimentation equilibrium, is about 17 nm at 25 degrees C. With glycosylated SCF1-165, the Kd is considerably higher.

Animals↗
PubMed1996

Dimerization of the extracellular domain of the erythropoietin (EPO) receptor by EPO: one high-affinity and one low-affinity interaction.

Although there is considerable evidence that signaling by the erythropoietin (EPO) receptor is initiated when it is dimerized by binding EPO, it has been previously reported that the soluble extracellular domains of the EPO receptor (sEPOR) are not dimerized in the presence of EPO and are able to form only 1:1 complexes with EPO. We have now shown unambiguously by light scattering, sedimentation equilibrium, and titration calorimetry that two molecules of sEPOR can bind to a single EPO monomer but that the binding of the second sEPOR is approximately 1000-fold weaker than that of the first. Because this second binding interaction is quite weak (Kd of approximately 1 microM), the 2:1 sEPOR.EPO complexes are easily dissociated during chromatography (forming the 1:1 complexes reported previously) and cannot be isolated in pure form. Global analysis of the sedimentation equilibrium data has enabled us to determine the binding constants and is consistent with a model in which EPO has two independent binding sites for sEPOR but cannot exclude anticooperative or sequential binding models. The influence of glycosylation of EPO and/or sEPOR on the binding affinities has also been investigated. Titration calorimetry is consistent with the sedimentation data and shows that the weaker binding site has a more negative delta H. The relation of these results to the binding of EPO to membrane-bound receptors and to the phenomenon of apparent high-affinity and low-affinity classes of receptors is discussed.

Animals↗
PubMed1996

Phylogeny and biogeography of Panax L. (the ginseng genus, araliaceae): inferences from ITS sequences of nuclear ribosomal DNA.

Panax, the ginseng genus, is one of the most medicinally important genera in the Orient and demonstrates a classical eastern Asian and eastern North American disjunct distributional pattern. Sequences of the internal transcribed spacers (ITS) and the 5.8S coding region of the nuclear ribosomal DNA repeat were obtained for the 12 species of Panax to reconstruct phylogenetic relationships. Of the 2 eastern North American species, P. quinquefolius and P. trifolius, P. quinquefolius was suggested to be more closely related to the eastern Asian species in the ITS tree, while P. trifolius was phylogenetically isolated. Monophyly of the three medicinally most important species, P. ginseng, P. notoginseng, and P. quinquefolius, suggested by previous workers, was not supported by the ITS data. A close phylogenetic relationship between Panax and Aralia was supported. Several biogeographical implications were inferred: (1) two divergence events have produced the eastern Asian and eastern North American disjunct distribution in Panax, (2) no intercontinental species pairs are found in Panax; (3) a discrepancy between the sequence divergence pattern and the phylogenetic pattern was observed in Panax, suggesting the need for caution in using sequence divergence data alone in inferring biogeographical patterns; (4) the Himalayas and central and western China are the current centers of diversity of the ginseng genus; and (5) the low ITS sequence divergence and a close relationship among species in that region suggest that rapid evolutionary radiation may have created such a diversity of Panax in the Himalayas and in central and western China.

Asia↗
PubMed1996

Isolation of anti-leukemia compounds from Citrus reticulata.

In vitro effects of medicinal plant extracts from the pericarpium of Citrus reticulata (cv Jiao Gan) (PCRJ) on the growth and differentiation of a recently characterized murine myeloid leukemic cell clone WEHI 3B (JCS) were investigated. Extracts of PCRJ not only inhibited the proliferation of JCS cells in a dose dependent manner, but also induced differentiation of JCS cells into macrophages and granulocytes. Morphological differentiation of PCRJ treated JCS cells was associated with an increase in phagocytic activity of the cells. Furthermore, both in vitro clonogenicity and in vivo growth of PCRJ treated JCS leukemic cells in syngeneic BALB/c mice were significantly reduced. The survival rate of mice receiving PCRJ treated JCS tumour cells was also increased. Using 1H-NMR, 13C-NMR, and GC/MS, two active components isolated from PCRJ were identified as nobiletin and tangeretin.

Animals↗
PubMed1996

Exploring the allowed sequence space of a membrane protein.

We present a comprehensive view of the tolerance of a membrane protein to sequence substitution. We find that the protein, diacylglycerol kinase from Escherichia coli, is extremely tolerant to sequence changes with three-quarters of the residues tolerating non-conservative changes. The conserved residues are distributed with approximately the same frequency in the soluble and transmembrane portions of the protein, but the most critical active-site residues appear to residue in the second cytoplasmic domain. It is remarkable that a unique structure of the membrane embedded portion of the protein can be encoded by a sequence that is so tolerant to substitution.

Amino Acid Sequence↗
PubMed1996

Oxidative stability and alpha-tocopherol retention in turkey burgers during refrigerated and frozen storage as influenced by dietary alpha-tocopheryl acetate.

1. The effect of vitamin E (alpha-tocopheryl acetate) in turkey diets on the oxidative stability of raw and cooked turkey burgers and on the retention of alpha-tocopherol during refrigerated (4 degrees C) or frozen (-20 degrees C) storage was investigated. One hundred and two, one-day-old T-8s turkey poults were divided at random into 3 groups of 34 animals each and fed on either a basal diet (normal commercial turkey diet) supplemented with 20 mg alpha-tocopheryl acetate/kg (control) or fed an alpha-tocopherol supplemented diet containing 300 (E300) or 600 (E600) mg alpha-tocopheryl acetate/kg for 21 weeks. 2. Dietary supplementation with alpha-tocopheryl acetate significantly reduced TBARS numbers in both raw and cooked burgers during refrigerated and frozen storage. 3. The mean values of alpha-tocopherol in raw and cooked burgers stored at 4 degrees C did not change during storage. 4. In the case of both raw and cooked samples stored at -20 degrees C, the alpha-tocopherol values decreased from 5.67 to 3.54 and from 3.56 to 2.30 micrograms/g in the raw burgers from turkeys from the E600 and E300 treatments, respectively, after 4 months storage. The values decreased from 5.60 to 2.88 and from 3.29 to 1.85 micrograms/g in cooked burgers from turkeys from the E600 and E300 treatments, respectively, after 5 months storage.

Animals↗
PubMed1996

Complete sequence, subunit structure, and complexes with pancreatic alpha-amylase of an alpha-amylase inhibitor from Phaseolus vulgaris white kidney beans.

The complete amino acid sequence of a white kidney bean (Phaseolus vulgaris) alpha-amylase inhibitor (PHA-I), which is composed of two kinds of glycopolypeptide subunits, alpha and beta, was established by conventional methods. The polypeptide molecular weight of PHA-I determined by the light-scattering technique, considered together with the sequence molecular weights revealed for the subunits, indicated that PHA-I has the subunit stoichiometry of (alpha beta)2 complex. Inhibition test of PHA-I with increasing amounts of porcine pancreatic alpha-amylase (PPA) suggested that an inactive 2:1 complex is formed between PPA and PHA-I. In fact, two complexes differing from each other in the molar ratio of PPA to PHA-I were separated by gel filtration, and molecular weight estimation by the light-scattering technique confirmed that they are complexes of PHA-I with one or two PPA molecules. The binding of PPA to PHA-I appeared to follow simple binomial statistics, suggesting that two binding sites on PHA-I are independent and of high affinity for PPA.

Amino Acid Sequence↗
PubMed1996

A patient-derived cytotoxic T-lymphocyte clone and two peptide-dependent monoclonal antibodies recognize HLA-B27-peptide complexes with low stringency for peptide sequences.

HLA-B27 molecules expressed on the T2 mutant cell line do not have peptides. Such empty HLA-B27 molecules were not recognized by an HLA-B27-restricted cytotoxic T-lymphocyte (CTL) clone (auto-1) derived from synovial fluid. To test for peptide dependency of the clone, B27-T2 cells were incubated with a panel of 48 different peptides. This lack of stringency was compared with that of a peptide-dependent monoclonal antibody, B27.M2. Positive B27.M2 reactivity resulted when the B27-T2 cells were incubated with two peptides: RRKAMFEDI and RRMGPPVGHR, derived from Chlamydia HSP60 and human ribonucleoprotein, respectively. Because of the limited availability of CTL versus monoclonal antibody, the specificity of B27.M2 was studied in greater detail. The importance of the HLA-B27 heavy chain in antibody recognition of class I-peptide complexes was demonstrated by site-directed mutagenesis. The stringency of the peptide residues was tested by making analogs of each of the nine residues in RRKAMFEDI, creating a panel of 180 analogs. Although stringency was highest for the sixth position, as many as six different amino acids provided positive reactivity. These results indicate that immune recognition of HLA-B27-peptide complexes might have rather low stringency for the peptide sequences. In theory, then, pathogen-derived peptides which induce autoimmunity by generating autoreactive CTL might not share much sequence similarity with the responsible self peptides.

Amino Acid Sequence↗
PubMed1996

Absorption by 1-year-old children of an iron supplement given with cow's milk or juice.

A paucity of data are available on toddlers for the evaluation of optimal strategies of Fe supplementation. In this study, we used a two-tracer stable isotope technique to determine Fe absorption from a 5-mg dose of stable isotopically enriched (57Fe or 58Fe) ferrous sulfate given with cow's milk (CM) compared with the same dose given with apple juice. Ten children (age 13 +/- 1 mo, weight 10.8 +/- 1.1 kg) who had recently discontinued formula feeding and begun on CM were studied. Red blood cell (RBC) iron incorporation of the isotope was determined 14 d after dosing with 57Fe and 58Fe. Fe absorption was calculated based on the assumption that 90% of absorbed Fe is incorporated into RBC. Absorption of Fe was significantly greater (13.7 +/- 6.4%) when given with juice than with milk (5.7 +/- 4.0%), p < 0.01 by paired t tests. Fe absorption from the dose given with juice was significantly negatively correlated with serum ferritin (n = 9, r = -0.70, p < 0.05). These results indicate that 1) a small supplement of Fe is better absorbed when given with juice than with CM, and 2) a large variability in Fe absorption exists in healthy 1-y-old infants, which is related to their existing Fe stores.

Animals↗
PubMed1996

Pulmonary malakoplakia in acquired immunodeficiency syndrome: an ultrastructural study of morphogenesis of Michaelis-Gutmann bodies.

Malakoplakia is an unusual inflammatory reaction to a variety of infections, characterized by the accumulation of macrophages containing the target-like calcospherites, the Michaelis-Gutmann body (MGB). We report three patients with acquired immunodeficiency syndrome with pulmonary malakoplakia associated with Rhodococcus equi infection; two patients were diagnosed at autopsy and one by examination of a transbronchial biopsy specimen. All three patients had pulmonary bacterial cultures and light and electron microscopic examination. The patients were 33-, 41-, and 43-year old men, human immunodeficiency virus-positive for 2, 6, 8 years, respectively. The two patients diagnosed at autopsy had cavitary lesions, and the patient diagnosed by biopsy specimen had nodular lesions on chest radiographs. Histologically, the lungs had well-circumscribed areas of infiltration with benign macrophages with granular cytoplasm, scattered MGBs, and numerous gram-positive coccobacilli. Electron microscopic examination showed intracellular coccobacilli, from 990 X 702 to 972 X 648 nm in diameter, with thick, homogenous cell walls, trilaminar cytoplasmic membranes, and dense cytoplasm with from one to five vacuoles. Electron microscopic studies showed that the bacteria within the pulmonary macrophages had thicker cell walls, less prominent nucleoid areas, and more vacuoles than the bacteria in cultures from the sputum and blood. The mature MGB ultrastructurally had a concentric, trilaminate structure with central mineralized core and was without recognizable bacterial forms. Early MGBs, however, consisted of a circular, electron-dense core containing bacteria, ultrastructurally similar to the R. equi seen in the culture. Pulmonary malakoplakia in patients with the acquired immunodeficiency syndrome might thus represent an acquired macrophage dysfunction of the intracellular digestion of phagocytized bacteria. The bacteria within the macrophages, however, seemed to have thicker cell walls compared with those in culture, and thus might be protected from enzyme digestion. It seems that MGBs are formed around the undigested bacteria as an alternative pathway for bacterial destruction, because R. equi was identified within the cores of early MGBs but not the mature or late stage MGBs.

Acquired Immunodeficiency Syndrome↗
PubMed1996

Inguinal lymph node foreign body granulomas after placement of a silicone rubber (Silflex) implant of the first metatarsophalangeal joint.

We describe a woman who developed foreign body granulomas 8 years after metatarsophalangeal (MTP) joint silicone rubber implantation for hallux rigidus. She developed overnight swelling and tenderness in the inguinal region on the ipsilateral side. Histological evaluation of the lymph node showed foreign body granulomas. Electron microscopy and dispersion x-ray analysis identified the foreign body material in the inguinal lymph node and fibrous capsule surrounding the implant as silicone. Foreign body granulomas of surrounding tissues and regional lymph nodes are infrequently reported postoperative complications in patients with small joint silicone rubber implants. A fractured or eroded implant surface attributed to wear is usually noted at prosthesis excision. Granulomas in normal or enlarged lymph nodes have been described in both symptomatic and asymptomatic patients, with intact or fractured prostheses. Many related joint and lymph node symptoms resolve with removal of the silicone rubber prosthesis.

Adult↗
PubMed1995

Binding of Neu differentiation factor with the extracellular domain of Her2 and Her3.

The interaction of neu differentiation factor (NDF) with the extracellular domains of Her2 (sHer2) and Her3 (sHer3) have been studied using native gels, light scattering, and sedimentation equilibrium. The full-length NDF beta 2 was shown to bind sHer3 with a dissociation constant of 26 +/- 9 nM, while it showed a 1000-fold weaker binding to sHer2. Taken together, these results demonstrate that NDF is a high affinity ligand for Her3, but not for Her2. No increase in affinity of the NDF beta 2 for sHer3 was observed upon addition of sHer2 to the NDF beta 2-sHer3 mixture. Binding of NDF beta 2 to sHer3 did not induce receptor dimerization or oligomerization, the stoichiometry being one sHer3 per one NDF molecule. This finding suggests that transmembrane and/or intracellular domains of receptor family members or perhaps additional unidentified components may be involved in NDF induced dimerization and autophosphorylation, or alternatively, that dimerization is not the mechanism for Her3 autophosphorylation and signal transduction.

Animals↗
PubMed1995

Studies on the structure and function of glycosylated and nonglycosylated neu differentiation factors. Similarities and differences of the alpha and beta isoforms.

Comparative analyses of both glycosylated and nonglycosylated neu differentiation factor (NDF) isoforms revealed significant similarities and differences of their overall structures and functions. Biophysical analyses confirmed that all NDF isoforms are monomeric, but have an extended ellipsoidal shape in solution. All full-length NDFs are similar in secondary and tertiary structures and they contain no alpha-helix but are abundant in beta-strand structures. A small NDF fragment containing only the epidermal growth factor domain is also rich in beta-strand structures, but exhibits tertiary structure different from the long NDF forms. Monoclonal antibodies that selectively recognize epidermal growth factor domains of human NDF-alpha and -beta can specifically bind the respective NDF-alpha and -beta isoforms independent of NDF origins. Western blot analysis and quantitative binding assays further identify that an NDF preparation produced naturally from Rat1-EJ cells contains both alpha and beta isoforms in a 3 to 2 ratio. In receptor-binding competition experiments, human and rat NDF-beta isoforms have higher affinity than NDF-alpha isoforms. NDF-beta isoforms can dramatically enhance the stimulation of DNA synthesis for transfected NIH3T3 cells that overexpress HER-3 and HER-4 receptors, while NDF-alpha isoforms can only stimulate proliferation of HER-4-transfected cells with lower activity. Taken together, NDF-alpha and -beta isoforms share similar gross protein conformations but are biologically distinct.

3T3 Cells↗
PubMed1995

High level expression of human leukemia inhibitory factor (LIF) from a synthetic gene in Escherichia coli and the physical and biological characterization of the protein.

LIF is a multi-functional cytokine that elicits effects on a broad range of cell types. In this report, we present the high level expression of human LIF (hLIF) from a chemically synthesized gene template in Escherichia coli where it comprises up to 25% of the cellular protein. The recombinant hLIF, after purification and folding, was examined using CD, FTIR spectroscopy and light scattering. CD and FTIR spectra showed that the hLIF is an alpha-helical protein and has a distinct tertiary structure. The IFTR spectrum resembles that of other four helical bundle proteins including G-CSF and IL-6. Light scattering analysis indicated that it is a monomeric protein, distinguishing it from M-CSF and interferon gamma, which also belong to the class of four helical bundle proteins but are dimeric. Recombinant hLIF was assayed for its activity on the murine leukemic cell line, M-1 as well as on human leukemic cell line, ML-1. It inhibited the growth of M-1 cells and differentiated them towards macrophages. However, it did not have any differentiation inducing effect on human leukemic cell lines alone or in combination with other cytokines.

Amino Acid Sequence↗
PubMed1995

Expression of whey acidic protein (WAP) genes in tissues other than the mammary gland in normal and transgenic mice expressing mWAP/hGH fusion gene.

Whey acidic protein (WAP) is a major whey protein secreted in rodents' milk. Murine WAP (mWAP) genes have been assumed to be expressed solely in the mammary gland. However, several heterologous genes fused with the mWAP promoter and artificially introduced into animal genomes as transgene were expressed not only in the mammary gland but also in other tissues as well. In the present study, we investigated, by means of the reverse transcription polymerase chain reaction (RT-PCR), the patterns of expression of endogenous WAP genes in tissues of normal mice and in transgenic mice carrying hGH gene coupled to the mWAP promoter sequence. The results revealed that the genes driven by the mWAP promoter, regardless of whether they are endogenous genes or transgenes, were transcribed in a variety of tissues other than the mammary gland of lactating normal female mice, although the expression levels are generally low. The expression of WAP genes in the cerebrum and the liver is regulated, as in the mammary gland, according to the reproductive stages. However, the tissue distribution of endogenous WAP gene expression in mature virgin transgenic female mice was the same as that in lactating normal female mice.

Animals↗
PubMed1995

The importance of Arg40 and 45 in the mitogenic activity and structural stability of basic fibroblast growth factor: effects of acidic amino acid substitutions.

High-affinity binding of basic fibroblast growth factor (bFGF) to the tyrosine kinase receptor requires cell-surface heparan sulfate proteoglycan or exogenous addition of heparin. The crystal structure of bFGF shows Arg40 and 45 on the surface opposite to the heparin-binding region, suggesting that these charged residues may be involved in the receptor binding. Therefore, these amino acids were mutated to aspartic acid separately or simultaneously, and also a simultaneous mutation to glutamic acid was introduced. These mutants displayed a mitogenic activity decreased greater than tenfold compared to the wild-type protein. Addition of heparin had no effect on the activity, while these mutants showed heparin-binding characteristics resembling those of the native sequence protein. The mutants exhibited decreased stability compared to the native sequence protein. Gradual changes in conformation were observed by circular dichroic and infrared spectroscopy. Heparin chromatography also showed the presence of denatured form for these mutants. However, in the presence of multivalent anions such as citrate, sucrose octasulfate, and heparin, the conformation of the mutants resembled that of the wild-type protein, as revealed by X-ray crystallography and circular dichroism spectra of the mutant with a Arg40-->Asp substitution.

3T3 Cells↗