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Biomedical subjects

Y Feng

Publications and source records attributed to Y Feng.

At least 217 records · Page 12Linked to original sources

Characterization of murine Girk2 transcript isoforms: structure and differential expression.

A mutation in the G-protein-linked inwardly rectifying K+ channel 2 gene (Girk2) is the cause of the weaver mouse phenotype. We determined that the originally published Girk2 transcript is composed of five exons. The primary coding exon (designated exon 4a in our system) encodes over two-thirds of the protein. Five different full-length Girk2 transcript isoforms (designated Girk2-1, Girk2A-1, Girk2A-2, Girk2B, and Girk2C) originating from different transcriptional start sites and/or alternative splicing were isolated by cDNA RACE. Several of the transcripts were predicted to encode truncated proteins that may lack some of the G-proteincoupling sequence. Northern blotting and in situ hybridization studies with transcript-specific probes indicated that the transcripts were differentially expressed in both normal and weaver mice. All transcripts tested were expressed in the three major targets of action of the weaver mutation: cerebellum, substantia nigra, and testis. Two of the transcripts, Girk2A-1 and Girk2A-2, encode identical proteins and have a distinct pattern of expression in testis, which suggests that they are associated with specific stages of spermatogenesis. An additional transcript, Girk2D, appears to be brain-specific, not polyadenylated, and highly expressed in cerebellar granule cells.

Animals↗

Expression and phosphorylation of fibroblast-growth-factor-inducible kinase (Fnk) during cell-cycle progression.

Fnk is a member of the polo family of cell-cycle-regulated serine/threonine kinases. We report here that it is present in serum-starved quiescent cells and that mitogenic stimulation of quiescent cells with calf serum results in the modification of a significant fraction of the Fnk pool. This modification results in a slower migrating form when analysed by SDS/PAGE. The modification is transient and by 9 h after stimulation all of the Fnk is again present as the faster migrating form. We also show that the Fnk protein increases in abundance as cells progress from G1 to mitosis and is post-translationally modified as cells enter and exit mitosis. The Fnk modification is again manifested as a slower migrating species by SDS/PAGE and is due to phosphorylation of the protein. The mitotic-specific phosphorylation of Fnk correlates with an increase in its kinase activity, and this activity is dramatically reduced by phosphatase treatment of mitotic Fnk immunoprecipitates. During the later stages of mitosis, Fnk is dephosphorylated such that, by the time the cells enter G1, it is all present as the dephosphorylated form. These results suggest that Fnk has two functions, one during the entry of cells into the cell cycle and a second during mitosis of cycling cells.

3T3 Cells↗

Purified recombinant Fmrp exhibits selective RNA binding as an intrinsic property of the fragile X mental retardation protein.

Fragile X syndrome is caused by the transcriptional silencing of the FMR1 gene due to a trinucleotide repeat expansion. The encoded protein, Fmrp, has been found to be a nucleocytoplasmic RNA-binding protein containing both KH domains and RGG boxes that associates with polyribosomes as a ribonucleoprotein particle. RNA binding has previously been demonstrated with in vitro-translated Fmrp; however, it remained uncertain whether the selective RNA binding observed was an intrinsic property of Fmrp or required an associated protein(s). Here, baculovirus-expressed and affinity-purified FLAG-tagged murine Fmrp was shown to bind directly to both ribonucleotide homopolymers and human brain mRNA. FLAG-Fmrp exhibited selectivity for binding poly(G) > poly(U) >> poly(C) or poly(A). Moreover, purified FLAG-Fmrp bound to only a subset of brain mRNA, including the 3' untranslated regions of myelin basic protein message and its own message. Recombinant isoform 4, lacking the RGG boxes but maintaining both KH domains, was also purified and was found to only weakly interact with RNA. FLAG-purified I304N Fmrp, harboring the mutation of severe fragile X syndrome, demonstrated RNA binding, in contrast to previous suggestions. These data demonstrate the intrinsic property of Fmrp to selectively bind RNA and show FLAG-Fmrp as a suitable reagent for structural characterization and identification of cognate RNA ligands.

Animals↗

Direct binding to nucleic acids by Vpr of human immunodeficiency virus type 1.

Human immunodeficiency virus type 1 (HIV-1) viral protein R (Vpr) is a 15kDa regulatory protein packaged in the HIV-1 virion. Although the molecular mechanism of Vpr function during viral replication remains elusive, Vpr has been found to possess interesting biological activities, including cell-cycle arrest at the G2/M check point, promotion of the HIV-1 pre-integration complex for nuclear transport, and a low but significant level of transcriptional activation of a variety of viral and cellular promoters. We now present data suggesting that HIV-1 Vpr is a nucleic-acid-binding protein. This activity of Vpr was demonstrated by DNA-cellulose chromatography, antibody co-immunoprecipitation, and gel electrophoretic mobility shift assays. By mutational analysis, the C-terminal region of Vpr, which is rich in basic amino-acid residues, was shown to be critical for Vpr binding to nucleic acids. The nucleic-acid-binding activity of Vpr is consistent with several biological activities of Vpr and may provide an important clue for understanding the molecular interactions between HIV-1 and the host cells.

Amino Acid Sequence↗

Calcitonin gene-related peptide potentiates LPS-induced IL-6 release from mouse peritoneal macrophages.

The secretion of IL-6 after stimulation of macrophages has been found to play a central role in the regulation of defense mechanism, haematopoiesis, and acute phase reaction. It was reported that cAMP is involved in the regulation of IL-6 production. Since calcitonin gene-related peptide (CGRP) is known to increase cAMP accumulation in mouse macrophages, we examined whether CGRP would induce IL-6 release in macrophages. Macrophages were obtained from the peritoneal exudate of male Balb/c mouse. The cells were plated on culture dishes at a density of 2.5 x 10(5) cells per well and allowed to adhere for 2 h. After incubation for 48 h with two changes of PRMI-1640, the macrophages were cultured with CGRP and LPS 1 microg/ml for 12 h. The IL-6 level in medium was measured by ELISA kits. The results showed that CGRP had no direct effects on IL-6 production, but it potentiated LPS-induced IL-6 production in a concentration-dependent manner. When CGRP was at a concentration of 10(-10) M, the LPS-induced IL-6 production was increased from 5.16 +/- 0.48 to 8.88 +/- 0.48 ng/ml. The effect of CGRP 10(-10) M was reversed by hCGRP(8-37) 10(-8) M, an antagonist of CGRP1 receptor. The LPS-induced IL-6 production from macrophages was also potentiated by forskolin 5 microM, an activator of adenylate cyclase. Furthermore, pretreatment with H-89 1 microM or Rp-cAMPS 100 microM, the inhibitors of cAMP-dependent protein kinase, inhibited the effect of CGRP by 31% and 98%, respectively. These results demonstrate that the LPS-induced IL-6 release is potentiated by CGRP via the activation of cAMP pathway in mouse resident peritoneal macrophages.

Animals↗

A labeled guanidine ligand for studying sweet taste.

A synthesis of a biotinylated-, coumarin-substituted-N,N'-diarylguanidine 1 is reported. This ligand has structural features conducive to studying sweet taste including a fluorescent tag to facilitate spectroscopic studies of binding to protein-receptors for sweet ligands and an anchor for affinity purification.

Biotin↗

Mutant Rab7 causes the accumulation of cathepsin D and cation-independent mannose 6-phosphate receptor in an early endocytic compartment.

Stable BHK cell lines inducibly expressing wild-type or dominant negative mutant forms of the rab7 GTPase were isolated and used to analyze the role of a rab7-regulated pathway in lysosome biogenesis. Expression of mutant rab7N125I protein induced a dramatic redistribution of cation-independent mannose 6-phosphate receptor (CI-MPR) from its normal perinuclear localization to large peripheral endosomes. Under these circumstances approximately 50% of the total receptor and several lysosomal hydrolases cofractionated with light membranes containing early endosome and Golgi markers. Late endosomes and lysosomes were contained exclusively in well-separated, denser gradient fractions. Newly synthesized CI-MPR and cathepsin D were shown to traverse through an early endocytic compartment, and functional rab7 was crucial for delivery to later compartments. This observation was evidenced by the fact that 2 h after synthesis, both markers were more prevalent in fractions containing light membranes. In addition, both were sensitive to HRP-DAB- mediated cross-linking of early endosomal proteins, and the late endosomal processing of cathepsin D was impaired. Using similar criteria, the lysosomal membrane glycoprotein 120 was not found accumulated in an early endocytic compartment. The data are indicative of a post-Golgi divergence in the routes followed by different lysosome-directed molecules.

Animals↗

Peptides derived from the complementarity-determining regions of anti-Mac-1 antibodies block intercellular adhesion molecule-1 interaction with Mac-1.

Peptides or small molecules that can block the interaction of the integrin Mac-1 with its receptor, intercellular adhesion molecule-1 (ICAM-1), have not previously been developed. We studied this interaction by measuring the adherence of ICAM-1-expressing Chinese hamster ovary (CHO) cells to immobilized, purified Mac-1. Nucleotide sequence information was obtained for the complementarity determining regions (CDRs) of three antibodies (44aacb, MY904, and 118.1) shown to block Mac-1-mediated cell adherence. Peptides were synthesized based on the predicted amino acid sequences of the CDRs and tested for the ability to block cell adhesion to Mac-1. Peptides derived from CDR1 of 44aacb, CDR2 of 118.1, and CDRs 1 and 3 of MY904 heavy chains were found to possess blocking activity at 10-100 muM. This may indicate that one or two CDRs contribute disproportionately to the antibody binding affinity. The binding of ligands to Mac-1 has been shown to require a region of the alpha-chain known as the I- or A-domain. We have recombinantly produced Mac-1 I-domain, and show that it is also capable of supporting the adherence of ICAM-1-expressing CHO cells. The adherence of ICAM-1-CHO cells to the I-domain is inhibited by 44aacb and 118.1 and by the CDR peptides from 44aacb and 118.1. By using phage display of peptide libraries based on the 118.1 CDR peptide with five residues randomized, we were able to identify a novel peptide inhibitor of Mac-1 with substitutions at all five positions. These peptides provide lead structures for development of Mac-1 antagonists.

Amino Acid Sequence↗

Functional binding between Gbeta and the LIM domain of Ste5 is required to activate the MEKK Ste11.

BACKGROUND: In the budding yeast Saccharomyces cerevisiae, the pheromones that induce haploid cells of opposite cell types to mate activate the Gbeta and Ggamma subunits of a heterotrimeric G protein. These subunits signal through the PAK kinase Ste20 to activate a mitogen-activated protein (MAP) kinase cascade comprising the MEKK Ste11, the MEK Ste7 and two MAP kinases, Fus3 and Kss1. The pathway requires Ste5, a scaffold protein that tethers the MAP kinase cascade enzymes into a high molecular weight complex. Ste5 is thought to associate with Gbeta in a pheromone-independent manner, but it is not known if this interaction affects signaling. RESULTS: A ste5C180A mutant - which expresses Ste5 disrupted in the LIM domain, a putative metal-binding motif that has been proposed to be essential for Ste5 oligomerization - could not transmit the pheromone signal from Gbeta through Ste20 to Ste11. The Ste5C180A protein was impaired in binding Gbeta, although it could oligomerize, bind Ste11, Ste7 and Fus3, facilitate the basal activation of Ste11, and relay the Ste11 signal to MAP kinases. Ste5 bound to Gbeta in a pheromone-dependent manner and preferentially associated with a phosphorylated form of Gbeta in wild-type and ste20Delta, but not in ste5C180A, strains. CONCLUSIONS: Pheromone induces binding of Gbeta to Ste5 through its LIM domain. This binding is essential for activation of Ste11 and is distinct from the ability of Ste5 to oligomerize or to serve as a scaffold and relay the signal from Ste11 to the MAP kinases. Pheromone also induces Ste5-dependent phosphorylation of Gbeta.

Adaptor Proteins, Signal Transducing↗

The effect of neferine on foam cell formation by anti-low density lipoprotein oxidation.

Oxidatively modified low density lipoprtein (LDL) plays an important role in atheroslerosis (AS) development. To investigate the role of neferine (Nef) in anti-LDL oxidation and foam cell formation, the lipoprotein was derived and subjected to three different treatments: N-LDL (normal LDL), Cu2+ + LDL and Cu2+ + Nef + LDL. The LDLs were put at 25 degrees C for 24 h and the thiobarbituric acid reactive substance (TBARS) values were determined. They were 0.57 +/- 0.2, 6.01 +/- 0.22 and 2.26 +/- 0.13 nmol/mg protein, respectively. The difference was very significant (P < 0.01) for each two groups by t test. Mouse peritoneal macrophage (M phi) were exposed to 50 micrograms protein/ml of Cu2+ + LDL and Cu2+ + Nef + LDL at 37 degrees C for 60 h. The tryglyceride (TG) and total cholesterol (TC) content in M phi were assayed. The results showed that Cu2+ + LDL was more efficient than Cu2+ + Nef + LDL in stimulating lipid accumulation in M phi (P < 0.001). The study demonstrated that Nef could inhibit Cu(2+)-mediated LDL oxidation and thereby inhibiting macrophage-derived foam cell formation.

Alkaloids↗

Hepatic Helicobacter species identified in bile and gallbladder tissue from Chileans with chronic cholecystitis.

BACKGROUND & AIMS: Cancer of the gallbladder is the number one cause of cancer mortality in Chilean women. Incidence rates for this tumor vary widely on a worldwide basis, being approximately 30 times higher in high-risk than in low-risk populations, suggesting that environmental factors such as infectious microorganisms, carcinogens, and nutrition play a role in its pathogenesis. Because several Helicobacter sp. colonize the livers of animals and induce hepatitis, the aim of this study was to determine whether Helicobacter infection was associated with cholecystitis in humans. METHODS: Bile or resected gallbladder tissue from 46 Chileans with chronic cholecystitis undergoing cholecystectomy were cultured for Helicobacter sp. and subjected to polymerase chain reaction (PCR) analysis using Helicobacter-specific 16S ribosomal RNA primers. RESULTS: Recovery of Helicobacter sp. from frozen specimens was unsuccessful. However, by PCR analysis, 13 of 23 bile samples and 9 of 23 gallbladder tissues were positive for Helicobacter. Eight of the Helicobacter-specific PCR amplicons were sequenced and subjected to phylogenetic analysis. Five sequences represented strains of H. bilis, two strains of "Flexispira rappini" (ATCC 49317), and one strain of H. pullorum. CONCLUSIONS: These data support an association of bile-resistant Helicobacter sp. with gallbladder disease. Further studies are needed to ascertain whether similar Helicobacter sp. play a causative role in the development of gallbladder cancer.

Adult↗

Mutations in filamin 1 prevent migration of cerebral cortical neurons in human periventricular heterotopia.

Long-range, directed migration is particularly dramatic in the cerebral cortex, where postmitotic neurons generated deep in the brain migrate to form layers with distinct form and function. In the X-linked dominant human disorder periventricular heterotopia (PH), many neurons fail to migrate and persist as nodules lining the ventricular surface. Females with PH present with epilepsy and other signs, including patent ductus arteriosus and coagulopathy, while hemizygous males die embryonically. We have identified the PH gene as filamin 1 (FLN1), which encodes an actin-cross-linking phosphoprotein that transduces ligand-receptor binding into actin reorganization, and which is required for locomotion of many cell types. FLN1 shows previously unrecognized, high-level expression in the developing cortex, is required for neuronal migration to the cortex, and is essential for embryogenesis.

Abnormalities, Multiple↗

Mutations in the gene encoding gap junction protein beta-3 associated with autosomal dominant hearing impairment.

Hearing impairment is the most commonly occurring condition that affects the ability of humans to communicate. More than 50% of the cases of profound early-onset deafness are caused by genetic factors. Over 40 loci for non-syndromic deafness have been genetically mapped, and mutations in several genes have been shown to cause hearing loss. Mutations in the gene encoding connexin 26 (GJB2) cause both autosomal recessive and dominant forms of hearing impairment. To study the possible involvement of other members of the connexin family in hereditary hearing impairment, we cloned the gene (GJB3) encoding human gap junction protein beta-3 using homologous EST searching and nested PCR. GJB3 was mapped to human chromosome 1p33-p35. Mutation analysis revealed that a missense mutation and a nonsense mutation of GJB3 were associated with high-frequency hearing loss in two families. Moreover, expression of Gjb3 was identified in rat inner ear tissue by RT-PCR. These findings suggest that mutations in GJB3 may be responsible for bilateral high-frequency hearing impairment.

Adult↗

Rab11 is required for trans-golgi network-to-plasma membrane transport and a preferential target for GDP dissociation inhibitor.

The rab11 GTPase has been localized to both the Golgi and recycling endosomes; however, its Golgi-associated function has remained obscure. In this study, rab11 function in exocytic transport was analyzed by using two independent means to perturb its activity. First, expression of the dominant interfering rab11S25N mutant protein led to a significant inhibition of the cell surface transport of vesicular stomatitis virus (VSV) G protein and caused VSV G protein to accumulate in the Golgi. On the other hand, the expression of wild-type rab11 or the activating rab11Q70L mutant had no adverse effect on VSV G transport. Next, the membrane association of rab11, which is crucial for its function, was perturbed by modest increases in GDP dissociation inhibitor (GDI) levels. This led to selective inhibition of the trans-Golgi network to cell surface delivery, whereas endoplasmic reticulum-to-Golgi and intra-Golgi transport were largely unaffected. The transport inhibition was reversed specifically by coexpression of wild-type rab11 with GDI. Under the same conditions two other exocytic rab proteins, rab2 and rab8, remained membrane bound, and the transport steps regulated by these rab proteins were unaffected. Neither mutant rab11S25N nor GDI overexpression had any impact on the cell surface delivery of influenza hemagglutinin. These data show that functional rab11 is critical for the export of a basolateral marker but not an apical marker from the trans-Golgi network and pinpoint rab11 as a sensitive target for inhibition by excess GDI.

Animals↗

A study of multiple biomarkers in coke oven workers--a cross-sectional study in China.

We conducted a cross-sectional molecular epidemiological study of coke oven workers exposed to the established carcinogen polycyclic aromatic hydrocarbons (PAHs) to evaluate the relationships between both traditional 'exposure markers' and a series of biomarkers, including urinary 1-hydroxypyrene as a marker of internal dose, leukocyte aromatic DNA adducts as markers of biologically effective dose, serum p53 protein as a response marker and genetic polymorphisms of cytochrome P4501A1 and glutathione S-transferase MI as susceptibility markers. Twenty-five male subjects each were randomly selected from the top, middle and bottom work areas of the oven, and the control plant. They were matched for age and smoking status. The mean levels of PAH exposure, monitored by stationary and personal samplers, and of worker urinary 1-hydroxypyrene differed significantly between the top, middle and bottom of the oven and control work areas. The highest stationary and personal PAH concentrations and 1-hydroxypyrene levels were demonstrated at the top work area. Good correlations were found between the stationary PAH levels, personal PAH levels and urinary 1-hydroxypyrene levels. No positive correlations were demonstrated between aromatic DNA adduct levels and current or cumulative PAH exposure dose. In the presence of genetic polymorphisms of cytochrome P4501A1, a positive correlation was demonstrated between aromatic DNA adducts and urinary 1-hydroxypyrene levels. There was also a significant correlation between serum p53 protein levels and the cumulated benzo[a]pyrene exposure dose. Although these biomarkers have certain limitations, they are applicable to cancer epidemiology, and may contribute to our understanding of the mechanisms of carcinogenesis.

Adult↗

Epigastric antinociception by cervical dorsal column lesions in rats.

BACKGROUND: Previous clinical evidence and electrophysiologic studies in the authors' laboratory have implicated the dorsal column (DC) as an important pathway for the transmission of visceral colorectal pain. This study examined, behaviorally and electrophysiologically, the role of the DC in mediating epigastric nociception using a visceral pain model involving duodenal distension in rats. METHODS: For behavioral testing, the writhing-like responses produced in awake rats by graded intraduodenal balloon distension (0.1 to 0.7 ml) were tested. A DC mechanical lesion at the C2 level or a sham operation (SH, same spinal cord segment exposed but no DC lesion) was performed. The writhing-like responses to duodenal distension were tested again and the rats were compared with other rats with no lesions and with SH rats. For electrophysiologic testing, the extracellular activity of single neurons was recorded in the ventrobasal nucleus of the thalamus in anesthetized rats. The ventrobasal cells that responded to duodenal distension were tested further with this visceral stimulus before and after a lesion of the DC. RESULTS: The mechanical DC lesion significantly reduced the intensity of the writhing-like responses and increased the threshold volume that would elicit writhing-like responses compared with rats with no lesions and SH rats without any observable neurologic deficit. A lesion of the DC also significantly reduced the responses of ventrobasal cells to duodenal distension. CONCLUSIONS: The DC plays an important role in signaling epigastric nociception in this experimental model. A mechanical DC lesion can produce significant visceral antinociception in rats.

Abdominal Muscles↗

A role for the dorsal column in nociceptive visceral input into the thalamus of primates.

A possible role of the dorsal column (DC) in the processing of visceral pain has gained attention after studies in the rat have revealed that the DC transmits a major part of the pelvic visceral nociceptive input from the colon into the thalamus. Furthermore, clinical interventions aimed at interrupting ascending DC axons near the midline were successful in relieving the pain suffered by patients with cancer of the pelvic organs. The purpose of this study was to check whether a DC lesion in monkeys would reduce the responses of thalamic neurons to graded colorectal distension (CRD) as in rats. Experiments were done on anesthetized male monkeys (Macaca fascicularis). Extracellular single cell recordings were made in the ventrolateral complex of the thalamus, mainly the ventral posterolateral (VPL) nucleus, in response to visceral and cutaneous stimulation. Of 80 VPL cells isolated, CRD activated 25, inhibited 25, and had no effect on 30 neurons. The responses of six viscerosensitive VPL neurons were recorded before and after a lesion of the DC at or above the T10 spinal segment. Lesions of other spinal tracts were made after the DC lesion. The results show that the DC lesion significantly reduced the responses of the thalamic neurons tested with CRD by >50%. Lesions of other tracts did not have a consistent effect. These results corroborate findings in the rat and support the proposal that the DC plays an important role in transmitting nociceptive visceral input into the thalamus and subsequently in visceral pain.

Animals↗