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Biomedical subjects

J Wen

Publications and source records attributed to J Wen.

At least 19 recordsLinked to original sources
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

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↗
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

Elastoderma.

Elastoderma is an exceedingly rare condition that is characterized clinically as an acquired localized laxity of skin resembling cutis laxa and histologically as an excessive accumulation of pleomorphic elastic structures within the dermis. We report the case of a 27-year-old white man with a 2-year history of lax, extensible, wrinkled skin with areas of protrusion and ulceration localized to the anterior aspect and back of the neck. Histologic examination of specimens from the affected area revealed increased masses of intertwined thin, elastic fibers without calcification in the papillary and upper reticular dermis. Transmission and scanning electron microscopy demonstrated irregular deposition of elastic material at the periphery of elastic tissue fibers, with grapelike globular structures. To our knowledge, this is the second reported case of elastoderma.

Adult↗
PubMed1995

Cultivation of Ehrlichia chaffeensis in mouse embryo, Vero, BGM, and L929 cells and study of Ehrlichia-induced cytopathic effect and plaque formation.

We successfully propagated Ehrlichia chaffeensis in mouse embryo, Vero, BGM, and L929 cells inoculated with host cell-free ehrlichiae, indicating that E. chaffeensis is capable of entry, survival, and growth in a relatively wide range of cell types derived from different species. We demonstrated rapid adaptation of E. chaffeensis in these cell lines, so that typical morulae could be detected as early as 5 days after inoculation. E. chaffeensis-induced cytopathic effect with different morphological characteristics in mouse embryo, Vero, and L929 cells. The earliest cytopathic effect appeared in untreated and irradiated mouse embryo cells at 4 days postinoculation. As the infected foci gradually expanded, the center of the foci showed necrotic cells with pyknotic nuclei and degraded morulae. E. chaffeensis caused cell lysis in untreated and irradiated L929 cells, with formation of distinct, round macroscopic plaques at 18 days postinoculation. In untreated and irradiated Vero cells, E. chaffeensis produced infected foci composed of loosely interwoven necrotic cells, spaces of detached cells, cells filled with morulae, and uninfected cells, resulting in characteristic reticular foci. Irradiated cells generally contained many large morulae and presented larger cytopathic foci. DH82 and BGM cells did not develop obvious cytopathic foci under the conditions employed. The findings reported herein offer the opportunity to study the pathogenic mechanism of cell injury by E. chaffeensis, the basis for quantification of infectious E. chaffeensis, improved approaches for recovery of ehrlichiae from human patients and tick hosts, and additional methods for cultivation of E. chaffeensis for molecular analysis.

Animals↗
PubMed1995

Interaction of human gallbladder mucin with calcium hydroxyapatite: binding studies and the effect on hydroxyapatite formation.

Calcium hydroxyapatite (HAP) crystals formed in vitro in the presence of polymeric human gallbladder mucin (1.0 mg/mL) were smaller (0.75 +/- 0.39 microns) than control crystals (7.86 +/- 2.76 microns), but the mucin did not affect the kinetics of crystal formation or alter the amount of mineral phase present at equilibrium. In contrast, glycopeptide subunits produced by proteolysis of the native mucin had no effect on HAP crystal size. Both native mucin and glycopeptides bound to mature HAP crystals, but the glycopeptides were much more readily displaced by phosphate ions. Therefore, in experiments where HAP was being formed, the phosphate ions inhibited the interaction of glycopeptides with the nascent HAP. These results indicate that gallbladder mucin may modulate HAP formation in vivo, and that this ability may be altered during pathological states, such as neutrophil infiltration or bacterial colonization, that may cause the release of proteinases capable of digesting mucin.

Amino Acids↗
PubMed1995

Serum activity of angiotensin converting enzyme (ACE) is not affected by hepatitis B viral infection.

Activity of angiotensin converting enzyme (ACE) in the serum from healthy and hepatitis B-infected subjects were examined. Preservation of the serum samples for 32 days at -80 degrees C did not significantly alter the activity of ACE. We have determined the ACE activity in three groups: healthy subjects with negative hepatitis B surface antigen (HBsAg) and normal liver function or the (-,N) group, subjects exhibiting positive HBsAg and normal liver function or the (+,N) group, as well as subject with positive HBsAg and abnormal liver function or the (+,Ab) group. The healthy group (-,N) exhibited serum activity of ACE of 31.5 +/- 1.2(25) nmoles/min/ml serum. There was no difference in ACE activity whether surface antigen was present, i.e., between (-,N) and (+,N) groups. Although a slight (about 10%) elevation of ACE activity was observed in the third group (+,Ab), it was not significantly different from either (-,N) or (+,N) group. These results suggest the lack of association between the serum activity of ACE and hepatis B, however, further studies are required to clarify whether correlation exists between serum activity and liver function.

Adult↗
PubMed1995

Features of cholesterol structure that regulate the clearance of chylomicron-like lipid emulsions.

Cholesterol is an absolute requirement for the clearance from plasma of the remnants of triglyceride-rich lipoproteins. Our laboratory previously established that cholesterol was essential for the hepatic uptake of remnant particles after intravenous injection of chylomicron-like lipid emulsions (1). The aim of the present study was to determine the structural features of the cholesterol molecule that regulate the metabolism of chylomicrons. Chylomicron-like lipid emulsions, which reflect the size and composition and mimic the physiology of lymph chylomicrons, were prepared with tracer amounts of labeled triolein ([14C]TO) and cholesteryl oleate ([3H]CO) to follow the hydrolysis of triglyceride and the uptake of chylomicron remnant particles by the liver. Sterols selected as cholesterol congeners with functional group variations were incorporated into the emulsions in place of cholesterol and injected intravenously in rats. Control emulsions contained either no cholesterol or approximately 1% (by weight) cholesterol. The effects of the different sterol structures on lipolysis and hepatic remnant uptake were compared with controls to determine the significance of various functional groups. Clearance of emulsion CO was impaired when cholesterol was absent or replaced by cholesteryl chloride, cholesteryl formate, or 3-keto-cholesterol. Clearance of emulsions containing epicholesterol, where the OH group at the 3-position is in the alpha configuration, was similar to control emulsions containing cholesterol. Congeners with an additional hydroxyl group, viz. 7 alpha-hydroxycholesterol, 7 beta-hydroxycholesterol, or 25-hydroxycholesterol, reduced CO clearance. Androstenol, which lacks the side chain at the C17-position, also retarded CO clearance from plasma. In contrast, emulsions incorporating congeners with side chain variations such as campesterol, beta-sitosterol, stigmasterol, or saturated congeners of cholesterol such as cholestanol, coprostanol and its epimer, epicoprostanol, all were cleared similarly to emulsions containing cholesterol. In conclusion, for physiological clearance of a chylomicron-like emulsion, the presence of a hydroxyl (-OH) group at the 3-position and an alkyl side chain at the C17-position of cholesterol are essential, while the structure of the side chain and the saturation of the ring structure are not critical. The mechanism of the specificity of sterols on the metabolism of protein-free emulsions is unclear, but does not relate to changes in microfluidity of the surface lipids, nor to the amount or isoform of associated apolipoproteins.

Animals↗
PubMed1994

Interactions of neurotrophin-3 (NT-3), brain-derived neurotrophic factor (BDNF), and the NT-3.BDNF heterodimer with the extracellular domains of the TrkB and TrkC receptors.

Interactions of three neurotrophin dimers, brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and a NT-3.BDNF heterodimer with extracellular, soluble TrkB and TrkC receptors were studied using native gels, light scattering, and sedimentation equilibrium. These three neurotrophins showed binding of two TrkB receptors per neurotrophin dimer, with a tendency to dissociate into one TrkB per dimer for NT-3 and the heterodimer, as determined by native gels, light scattering, and sedimentation equilibrium. For TrkC, native gels suggested binding of NT-3, heterodimer, and BDNF but not of nerve growth factor. Sedimentation equilibrium revealed that all three neurotrophin molecules bind to TrkC at two receptors per dimer but that BDNF binds much more weakly and that the heterodimer has an intermediate binding strength. Light scattering/size exclusion chromatography showed complexes with two TrkC receptors per NT-3 dimer and one TrkC per heterodimer but did not detect binding of BDNF to TrkC. This latter result is not inconsistent with the sedimentation data, because the weak binding of BDNF to TrkC may be easily dissociated by nonspecific interactions of BDNF with the size exclusion column. The relative binding constants for these neurotrophins and the soluble receptor extracellular domains, as determined by sedimentation equilibrium, are correlated with their biological activity. However, the magnitude of these binding constants is insufficient by approximately 3 orders of magnitude to promote receptor dimerization at physiologically active concentrations.

Animals↗
PubMed1994

The effect of mutant beta 2-microglobulins on the conformation of HLA-B27 detected by antibody and by CTL.

The arthritis-predisposing HLA-B27 consists of a heavy chain, a small peptide, and the monomorphic beta 2-microglobulin (beta 2-m). CTLs and a mAb, Ye-2, which recognize the complex with specificities both for the heavy chain and for the peptide, are available. The beta 2-m is in noncovalent association with the heavy chain at multiple points and is exchangeable with free beta 2-m outside of the complex. The purpose of our experiments was to test whether mutant beta 2-m capable of modulating HLA-B27 activity could be created. Eighteen recombinant mutants of the human beta 2-m were experimentally generated. In 14 of these, mutations were at or near residues that are either contact residues or interface residues with the heavy chain. Relative to the parent beta 2-m, two-thirds of the mutants showed reduced ability to exchange into HLA-B27 complexes. However, at least four of them induced more than 80% decrease in Ye-2 Ab reactivity. Two mutants were able to induce a minor decrease in susceptibility to lysis by four CTL clones. One of the CTL clones was autoreactive. Two of the CTL clones were specific for HLA-B27 cells experimentally infected with arthritis-causing Yersinia enterocolitica. These results indicate that certain beta 2-m residues play an indirect role in peptide presentation, although they are not directly associated with the peptide residues.

Amino Acid Sequence↗
PubMed1994

Testing the importance of each residue in a HLA-B27-binding peptide using monoclonal antibodies.

When a peptide derived from histone 3.3 was incubated with mouse L cells transfected with HLA-B27, the cells became highly reactive with Ye-2, an anti-HLA-B27 mAb. The critical residues were analyzed by testing analogues in which each of the nine residues in the peptide was consecutively substituted by 19 other amino acids. The conclusions were separately verified using a different HLA-B27-positive cell line. The ability of some of these peptides to bind to HLA-B27 was also assayed by their ability to stabilize HLA-B27 in a mutant cell line which required HLA-B27-binding peptides to express HLA-B27 at 37 degrees C. These experiments showed that in P4, P5, P6, P7, P8, and P9, all 20 different amino acids could be substituted without eliminating the ability of the analogues to bind to HLA-B27. The residues which were responsible for the HLA-B27-peptide complex reacting with the Ye-2 antibody were P8 and P9. The latter might mediate its effect by altering either the surface conformation of the closely associated HLA-B27 heavy chain or the conformation of the peptide itself.

Amino Acid Sequence↗
PubMed1994

A monoclonal antibody that recognizes HLA-B27 in the context of peptides.

The T2 mutant cell line is unable to load peptides into the MHC class I Ags inside the cells. These "empty" MHC class I Ags are not expressed on the cell surface unless the cells are cultured at low temperatures. Expression will occur at 37 degrees C only in the presence of peptides that bind to and stabilize the class I Ags. T2 cells transfected with the B*2705 gene were tested with a panel of anti-HLA-B27 mAb. Two of the antibodies, ME1 and KS3, reacted with the "empty" HLA-B27 expressed at low culture temperatures. Three antibodies, B27.M1, B27.M2, and Ye-2, were unreactive with these "empty" HLA-B27. The cells were then incubated with a panel of HLA-B27-binding peptides. One of the antibodies, Ye-2, became reactive when the cells were incubated with a peptide derived from HIV gp120 and to a less degree with a peptide derived from histone H3.3. Mouse L cells transfected with the B*2705 and the human beta 2m genes also reacted very poorly with B27.M1, B27.M2, and Ye-2. Those two peptides were also able to induce high increase in Ye-2 reactivity. Alternately, increase in Ye-2 reactivity was also observed when the L cells were incubated with IFN-gamma or TNF-alpha. These experiments indicate that the Ye-2 anti-HLA-B27 mAb recognizes HLA-B27 in the context of certain residing peptides either added exogenously or expressed endogenously. The B27.M1 and B27.M2 antibodies might share similar characteristics.

Amino Acid Sequence↗
PubMed1994

Application of magnetic sector thermal ionization mass spectrometry to studies of erythrocyte iron incorporation in small children.

The optimal evaluation of iron metabolism requires the administration of two isotopes of iron. However, high-precision measurement of isotopic ratios from blood samples obtained after administration of two stable isotopes of iron to human subjects has not previously been reported. Using a cation-exchange system to isolate iron from blood samples, we found that high-precision (< 0.2%) measurements of 58Fe/56Fe and 57Fe/56Fe could be performed using magnetic sector thermal ionization mass spectrometry. Clinical studies in four 1-year-old infants showed that this technique could be used to demonstrate a lower rate of iron absorption in small children given an iron supplement (57Fe) with milk compared to those given iron (58Fe and ferrous sulfate) with ascorbic acid. This technique will enable the evaluation of iron metabolism in populations in whom the use of radioactive iron tracers is not appropriate.

Adolescent↗
PubMed1994

Stoichiometry of heparin binding to basic fibroblast growth factor.

Fibroblast growth factors (FGFs) strongly bind to heparin and are thereby stabilized against deactivation and proteolytic cleavage. We have investigated the interactions of basic fibroblast growth factor (bFGF) with low- and high-molecular-weight heparin using size exclusion chromatography with on-line light scattering, absorbance, and refractive index detection. When heparin-bFGF mixtures with excess heparin are chromatographed using eluant that does not contain heparin, essentially all the protein is seen to elute as a complex with the heparin, indicating strong binding such that the complex does not dissociate significantly during chromatography (approximately 20 min). Combining the data from the light scattering, absorbance, and refractive index chromatograms allows us to determine the molecular weight of the protein component of the complex, and therefore to measure the number of bFGF molecules bound per heparin. A series of samples were prepared with a constant concentration of bFGF and variable amounts of a low-molecular-weight heparin (LMWH, M(r) = approximately 5000). At bFGF: heparin ratios above 1.5, a mix of complexes containing 3, 2, and 1 bFGF molecules is observed, with an average of 2.2 bFGF molecules per complex. Since the amount of bFGF incorporated into complexes implies an average of 2.5 +/- 0.3 bFGF molecules per heparin, there is only one heparin molecule per complex. The coexistence of complexes of different size when bFGF is in excess implies that the LMWH molecules are heterogeneous with respect to their ability to bind bFGF. When a high-molecular-weight heparin (HMWH, M(r) = 15,000) is used, complexes averaging 6.3 bFGF molecules per HMWH molecule are seen, while the overall amount of bFGF appearing in complexes implies six to seven sites per HMWH. These data show that the protein molecules can be packed very closely together. Both types of heparin give a heparin mass of 2300 Da per bFGF binding site, which corresponds approximately to an octasaccharide.

Chromatography, Gel↗