PubMed Health⌕ Search

Biomedical subjects

J Wen

Publications and source records attributed to J Wen.

At least 19 recordsLinked to original sources
PubMed1999

A well-collimated quasi-continuous atom laser

Extraction of sodium atoms from a trapped Bose-Einstein condensate (BEC) by a coherent, stimulated Raman process is demonstrated. Optical Raman pulses drive transitions between trapped and untrapped magnetic sublevels, giving the output-coupled BEC fraction a well-defined momentum. The pulsed output coupling can be run at such a rate that the extracted atomic wave packets strongly overlap, forming a highly directional, quasi-continuous matter wave.

Journal Article↗
PubMed1999

The human telomerase catalytic subunit hTERT: organization of the gene and characterization of the promoter.

Telomerase, the enzyme that synthesizes telomeric DNA, is not expressed in most human somatic cells but is activated with in vitro immortalization and during tumorigenesis, and repressed by cell differentiation. Of the two components of the core enzyme, the catalytic protein hTERT is limiting for activity. To investigate mechanisms of hTERT gene regulation, we have cloned genomic sequences encompassing the complete hTERT transcription unit. The hTERT gene consists of 16 exons and 15 introns spanning approximately 35 kb. Transient transfections of immortal human cells with potential regulatory 5' sequences linked to a reporter, combined with deletion analysis of these sequences, indicated that elements responsible for promoter activity are contained within a region extending from 330 bp upstream of the ATG to the second exon of the gene. Assays in different cell types have shown that the hTERT promoter is inactive in normal and in transformed pre-immortal cells, but, like telomerase, it is activated with cell immortalization. Sequence analysis revealed that the hTERT promoter is GC-rich, lacks TATA and CAAT boxes but contains binding sites for several transcription factors that may be involved in its regulation. The abundance of these sites suggests the possibility that hTERT expression may be subject to multiple levels of control and be regulated by different factors in different cellular contexts.

Amino Acid Sequence↗
PubMed1998

Localization of the binding site for transforming growth factor-beta in human alpha2-macroglobulin to a 20-kDa peptide that also contains the bait region.

alpha2-Macroglobulin (alpha2M) functions as a major carrier of transforming growth factor-beta (TGF-beta) in vivo. The goal of this investigation was to characterize the TGF-beta-binding site in alpha2M. Human alpha2M, which was reduced and denatured to generate 180-kDa subunits, bound TGF-beta1, TGF-beta2, and NGF-beta in ligand blotting experiments. Cytokine binding was not detected with bovine serum albumin that had been reduced and alkylated, and only minimal binding was detected with purified murinoglobulin. To localize the TGF-beta-binding site in alpha2M, five cDNA fragments, collectively encoding amino acids 122-1302, were expressed as glutathione S-transferase (GST) fusion proteins. In ligand blotting experiments, TGF-beta2 bound only to the fusion protein (FP3) that includes amino acids 614-797. FP3 bound 125I-TGF-beta1 and 125I-TGF-beta2 in solution, preventing the binding of these growth factors to immobilized alpha2M-methylamine (alpha2M-MA). The IC50 values were 33 +/- 5 and 26 +/- 6 nM for TGF-beta1 and TGF-beta2, respectively; these values were comparable with or lower than those determined with native alpha2M or alpha2M-MA. A GST fusion protein that includes amino acids 798-1082 of alpha2M (FP4) and purified GST did not inhibit the binding of TGF-beta to immobilized alpha2M-MA. FP3 (0.2 microM) neutralized the activity of TGF-beta1 and TGF-beta2 in fetal bovine heart endothelial (FBHE) cell proliferation assays; FP4 was inactive in this assay. FP3 also increased NO synthesis by RAW 264.7 cells, mimicking an alpha2M activity that has been attributed to the neutralization of endogenously synthesized TGF-beta. Thus, we have isolated a peptide corresponding to 13% of the alpha2M sequence that binds TGF-beta and neutralizes the activity of TGF-beta in two separate biological assays.

Animals↗
PubMed1998

CRP interacts with promoter-bound sigma54 RNA polymerase and blocks transcriptional activation of the dctA promoter.

The cAMP receptor protein (CRP) is an activator of sigma70-dependent transcription. Analysis of the sigma54-dependent dctA promoter reveals a novel negative regulatory function for CRP. CRP can bind to two distant sites of the dctA promoter, sites which overlap the upstream activator sequences for the DctD activator. CRP interacts with Esigma54 bound at the dctA promoter via DNA loop formation. When the CRP-binding sites are deleted, CRP still interacts in a cAMP-dependent manner with the stable Esigma54 closed complex via protein-protein contacts. CRP is able to repress activation of the dctA promoter, even in the absence of specific CRP-binding sites. CRP affects both the final level and the kinetics of activation. The establishment of the repression and its release by the NtrC activator proceed via slow processes. The kinetics suggest that CRP favours a new form of closed complex which interconverts slowly with the classical closed intermediate. Only the latter is capable of interacting with an activator to form an open promoter complex. Thus, Esigma54 promoters are responsive to CRP, a protein unrelated to sigma54 activators, and the repression exerted is the direct result of an interaction between Esigma54 and the CRP-cAMP complex.

Bacterial Proteins↗
PubMed1998

Coexpression of G-CSF with an unglycosylated G-CSF receptor mutant results in secretion of a stable complex.

Previously, we have shown that the entire extracellular domain of the granulocyte-colony stimulating factor receptor (sG-CSFr) produced in Chinese hamster ovary (CHO) cells forms a stable complex with its ligand G-CSF, at a stoichiometry of 2:2. A truncated receptor molecule consisting of the cytokine receptor homology domain and N-terminus Ig-like domain (Ig CRH) behaves quite similarly. Both of these forms of the receptor are highly glycosylated. To address the importance of glycosylation toward receptor activity and stability, and possibly obtain nonglycosylated receptor for crystallization, mutations were made to replace four Asn residues which are N-glycosylated in the truncated receptor. Virtually no receptor was recovered from conditioned media of CHO cells transfected with this mutant construct, although a high-level of mRNA coding for receptor was detected; this mRNA was translated as determined by Western blots of cell lysates. These results indicate that the translated product is apparently not secreted from these cells. Cells transfected with mutant receptor cDNA were cotransfected with a cDNA construct expressing G-CSF in which the single O-glycosylation site was eliminated by mutation. Upon fermentation of the cotransfectants, we observed a large amount of receptor-ligand complex in the conditioned media. The purified unglycosylated complex appeared to be of the same binding stoichiometry and approximate binding affinity as that of complex formed by addition of purified ligand and unmutated receptor. These results show that while glycosylation of sG-CSFr is not necessary for ligand binding, it appears to be crucial in folding and export from the cell.

Animals↗
PubMed1998

Interactions between NFkappaB and its inhibitor ikappaB: biophysical characterization of a NFkappaB/ikappaB-alpha complex.

The N-terminal domain (1-318 amino acids) of mouse NFkappaB (p65) has been purified to homogeneity from the soluble fraction of Escherichia coli cells expressing this protein. Its complex with a full-length ikappaB-alpha (MAD3, 1-317 amino acids) molecule was generated by binding the E. coli-derived ikappaB-alpha to the purified NFkappaB and purifying the complex by sequential chromatography. The stoichiometry of NFkappaB to ikappaB in the complex was determined to be 2 to 1 by light scattering and SDS-polyacrylamide gel electrophoresis. The secondary structure of the NFkappaB (p65) determined by Fourier-transform infrared (FTIR) spectroscopy is in good agreement with that of the p50 in the crystal structure of the p50/DNA complex, indicating that no significant structural change in NFkappaB occurs upon binding of DNA. The FTIR spectrum of the NFkappaB/ikappaB complex indicates that its secondary structure is composed of 17% alpha-helix, 39% beta-strand, 18% irregular structures, and 26% beta-turns and loops. By comparing these data to the FTIR data for NFkappaB alone, it is concluded that the ikappaB (MAD3) in the complex contains 35% alpha-helix, 27% beta-strand, 22% irregular structures, and 16% beta-turns and loops. Circular dichroism (CD) analysis of a shorter form of ikappaB (pp40) indicates that it contains at least 20% alpha-helix and that the ikappaB subunit accounts for nearly all of the alpha-helix present in the NFkappaB/ikappaB complex, consistent with the FTIR results. The stabilities of NFkappaB, ikappaB, and their complex against heat-induced denaturation were investigated by following changes in CD signal. The results indicate that the thermal stability of ikappaB is enhanced upon the formation of the NFkappaB/ikappaB complex.

Animals↗
PubMed1998

Reconstitution of wild-type or mutant telomerase activity in telomerase-negative immortal human cells.

Telomere shortening in human somatic cells and telomere maintenance in most human immortal cell lines and tumours correlate respectively with the absence and presence of telomerase, the enzyme that synthesizes telomeric DNA de novo . However, approximately 30% of in vitro immortalized human cell lines do not express this enzyme and maintain telomeres by an alternative pathway (ALT) that may also operate in some tumours. Human telomerase is a reverse transcriptase comprising minimally an RNA subunit (hTER) and a catalytic protein moiety (hTERT). Normal somatic cells retain expression of hTER but not of hTERT, and can be converted to a telomerase-positive phenotype by ectopic expression of the catalytic protein. We similarly have restored enzymatic activity to those ALT cell lines that retain hTER expression. We also report that in those ALT cells that are hTER negative, reintroduction of both hTER and hTERT is necessary and sufficient for conversion to telomerase positivity. Moreover, transfection of these cells with hTERT in conjunction with hTERs with a mutated template results in the expression of an enzyme with altered specificity. Reconstitution of telomerase activity in ALT cells, particularly an activity capable of synthesizing mutant telomeric DNA, may be exploited for the study of the ALT mechanism and its interaction with the telomerase-dependent pathway, and for assessing the effects of mutant telomeres on cell viability.

Cell Line, Transformed↗
PubMed1998

Duodenal motility in fasting dogs: humoral and neural pathways mediating the colonic brake.

We have previously described a negative feedback loop that inhibits duodenal motility when nutrients are infused into the ileum and colon. In the present study, we examined the role of extrinsic innervation and plasma levels of peptide YY (PYY) in mediating this phenomenon. We perfused neurally intact (n = 5 dogs) or extrinsically denervated (n = 6 dogs) isolated loops of proximal colon with isomolar NaCl or a mixed-nutrient solution at 2 and 6 ml/min for 4 h during fasting or for 2 h beginning 15 min after a meal. Both rates of infusion with NaCl prolonged the cycle length of the duodenal migrating motor complex (MMC) in the group with neurally intact loops but not in the group with extrinsically denervated loops. Nutrient infusions increased the MMC cycle length in both groups. Integrated plasma concentrations of PYY were increased by nutrients but not by NaCl in both groups. These data suggest that increased volumes and unabsorbed nutrients in the proximal colon alter proximal small bowel motility. Volume-induced effects are mediated via extrinsic nerves, whereas nutrient-induced effects may be mediated by humoral factors, such as plasma PYY.

Animals↗
PubMed1998

Rat serum induces a differentiated phenotype in a rat parotid acinar cell line.

To establish a continuous cell line, freshly prepared rat parotid acinar cells were stably transfected with a plasmid vector containing the SV40 large T antigen. The acinar origin of these cells was confirmed by Western blotting, enzyme analysis, and morphological analysis. Transformed cells grown in 10% rat serum showed a modest reduction in cell number after 7 days and a concentration- and time-dependent increase in amylase levels approximately 16 times greater than those observed in fetal bovine serum-treated cells. Ultrastructural analysis revealed that cells grown in rat serum harbored protein-filled secretory granules localized adjacent to the endoplasmic reticulum, and punctate amylase-specific immunofluorescence distributed throughout the cytoplasm was consistent with the presence of amylase in secretory organelles. Clonal cells express tissue-specific proline-rich proteins and the four protein kinase C isozymes present in primary culture. Carbachol and isoproterenol stimulated [3H]protein secretion and isoproterenol enhanced amylase secretion from cells grown in rat serum. Moreover, norepinephrine, carbachol, and substance P produced a time- and concentration-dependent rise in cytoplasmic Ca2+. This continuous cell line of parotid acinar cells, which after treatment with rat serum retains the basic structural and functional properties of primary culture cells, will be utilized as a model system for studying long-term biological processes that regulate parotid cell function.

Animals↗
PubMed1998

Scintigraphic measurement of regional gastrointestinal transit in the dog.

Scintigraphic techniques can measure sequentially gastric emptying, small bowel transit, and colonic transit in humans, and comparable methods for experimental studies in animals would be useful. We developed such a method in dogs and examined the effects of prokinetic drugs on regional transit. Two isotopes were given to fasting dogs. Polystyrene pellets labeled with 99mTc were mixed in a can of dog food and 111In- labeled pellets were given in a gelatin capsule coated with a pH-sensitive polymer, designed to dissolve in the distal bowel. Gamma camera images were obtained for up to 24 h. Prokinetic drugs were given by intravenous injection. Duplicate baseline studies showed good agreement in seven dogs. In a second group (n = 4), intra- and interanimal variabilities were established. Two novel prokinetic drugs (AU-116 and AU-130) accelerated small bowel and colonic transit. A simple noninvasive method for measuring whole gut transit in dogs was developed and validated. Two new prokinetics accelerated small bowel and colonic transit.

Administration, Oral↗
PubMed1997

Genomic characterization of members of the Bet v 1 family: genes coding for allergens and pathogenesis-related proteins share intron positions.

Bet v 1, the major birch pollen allergen, is a member of a multigene family; a number of isoforms and homologous proteins from closely related species (alder, hazel and hornbeam) has been isolated and their cDNAs cloned and characterized. Genomic clones coding for Bet v 1 and homologues from apple and hazel were isolated and sequenced. Some of these clones contained intervening sequences. The exon-intron formation is highly conserved throughout this family of pathogenesis-related proteins in dicot plants and is also found in Aopr1 (Asparagus officinalis), a monocol species. Phylogenetic analysis suggested a possible common origin of the intron position in these homologous proteins at codon 62 in various families of flowering plants, including Fagaceae, Rosaceae and Apiaceae. This conserved 'proto-splice site' may point to a structure/function relationship. A conserved sequence motif (P-loop) was also found in all members of this protein family. Moreover, there is a certain degree of sequence similarity among the proteins derived from various species throughout the dicots and the only monocot examined. This fact is reflected by cross-reactivity from monoclonal and polyclonal antibodies raised against Bet v 1.

Allergens↗
PubMed1997

Inhibition of expression of PKC-alpha by antisense mRNA is associated with diminished cell growth and inhibition of amylase secretion by AR4-2J cells.

AR4-2J pancreatoma cells were stably transfected with an expression vector containing the cDNA for PKC-alpha in the antisense orientation. Transfectants designated antisense-alpha AA1, AA2, and AA3 exhibited marked reductions in PKC-alpha expression and decrements in cell growth. The magnitude of the decrement in cell growth paralleled the reduction in PKC-alpha expression, i.e., AA3 > AA1 > AA2. The ability of dexamethasone to induce cell differentiation as assessed by a rise in cellular amylase levels was not markedly affected by the reduction in PKC-alpha expression. Unstimulated amylase release was attenuated in AA1 cells and almost completely blocked in AA2 transfectants. The AA2 transfectant cell line failed to elicit a secretory response to caerulein, and the AA1 transfectant exhibited a lack of the secondary phase of stimulated amylase secretion. These findings demonstrate that PKC-alpha is involved in the mechanisms regulating growth and secretion in AR4-2J cells, but is not necessary for the induction of amylase stores following differentiation.

Amylases↗
PubMed1997

A receptor binding site on intrinsic factor is located between amino acids 25-44 and interacts with other parts of the protein.

A receptor-binding region of intrinsic factor (IF) has been localized to amino acid residues 25-44 by in vitro transcription/translation studies. To further define sites within the region that are necessary for binding, the effects on binding activity of brush border membranes were tested when peptides corresponding with residues 1-44 were added to normal IF and when point mutations were made between residues 25 and 44. Both human IF and IF-cobalamin (cbl) complex bound equally to membranes. None of the peptides tested inhibited human IF or IF-cbl complex binding. Both control rat IF and rat IF that was mutated to be like human IF in residues 25-44 bound to guinea pig and rat membranes; the mutant with altered charge showed 50- to 100-fold decreased affinity. Thus, the putative receptor binding region is important, but cannot alone account for all the physiologic parameters of IF binding. Conformational changes in IF are additionally important.

Amino Acids↗
PubMed1997

Delivery of liposome-sequestered hydrophobic proteins to lysosomes of normal and Batten disease cells.

We have developed a method to deliver hydrophobic proteins such as ATP synthase subunit c and ubiquitin to lysosomes of PMN (polymorphonucleocytes) and fibroblasts. ATP synthase subunit c is stored in the lysosomes of various tissues in late infantile and juvenile forms of neuronal ceriod lipofuscinosis, also called Batten disease (BD). Whether this protein storage is due to an abbreviation in protein or in the lysosomal hydrolases of BD is still not clear. We have sequestered this protein and ubiquitin in the lipid membrane of liposomes. The liposomes coated with autologous heat-aggregated IgG or apolipoprotein E when presented to the PMN and fibroblasts, respectively, accumulated in the lysosomes. Both normal and BD PMN degraded 125I-ubiquitin; the rate of degradation was, however, slower by Batten PMN. These studies indicate that a hydrophobic molecule such as subunit c can be delivered to PMN and fibroblasts, and the sequestered proteins are accessible to lysosomal hydrolases. Therefore, this technique can be used to study the metabolism of highly hydrophobic proteins by lysosomes, especially the biochemical mechanism(s) of subunit c storage in BD.

Apolipoproteins E↗
PubMed1997

The potential of Betv1 homologues, a nuclear multigene family, as phylogenetic markers in flowering plants.

Betv1 homologues are a ubiquitous group of genes in flowering plants encoding a class of highly conserved defense-related proteins and containing open reading frames from 465 to 480 bp. Betv1-like genes consist of two exons interrupted by an intron of 76-359 bp, with the intron position highly conserved. The pairwise p distance ranged from 0 to 0.583 among flowering plants. Within plant families, the ranges of the p distance were 0-0.403, 0-0.253, and 0.011-0.369, for Apiaceae, Betulaceae, and Fabaceae, respectively. The most striking feature of the betv1 gene phylogeny was that the multiple sequences from each plant family formed a monophyletic group and sequences from each species were generally more similar to each other than those from other species. The almost exclusive paralogous relationships of genes from the same species suggested that the genes of the multigene family underwent strong concerted evolution. Phylogenies of Betulaceae and Fabaceae inferred from betv1 gene trees were generally congruent with those based on morphology and other molecules. Betv1 homologues constitute potential phylogenetic markers at the intrafamilial level or among closely related families in flowering plants.

Base Sequence↗
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↗