Biomedical subjects

S Wang

Publications and source records attributed to S Wang.

At least 235 records · Page 13Linked to original sources

PubMed2001

A role for the helix-loop-helix protein Id2 in the control of oligodendrocyte development.

Compared to neurons, the intracellular mechanisms that control glial differentiation are still poorly understood. We show here that oligodendrocyte lineage cells express the helix-loop-helix proteins Mash1 and Id2. Although Mash1 has been found to regulate neuronal development, we found that in the absence of Mash1 oligodendrocyte differentiation occurs normally. In contrast, we found that overexpression of Id2 powerfully inhibits oligodendrocyte differentiation, that Id2 normally translocates out of the nucleus at the onset of differentiation, and that absence of Id2 induces premature oligodendrocyte differentiation in vitro. These findings demonstrate that Id2 is a component of the intracellular mechanism that times oligodendrocyte differentiation and point to the existence of an as yet unidentified MyoD-like bHLH protein necessary for oligodendrocyte differentiation.

Animals↗

PubMed2001

Molecular modeling, structure--activity relationships and functional antagonism studies of 4-hydroxy-1-methyl-4-(4-methylphenyl)-3-piperidyl 4-methylphenyl ketones as a novel class of dopamine transporter inhibitors.

We previously disclosed the discovery of 4-hydroxy-1-methyl-4-(4-methylphenyl)-3-piperidyl 4-methylphenyl ketone (3) as a novel class of dopamine transporter (DAT) inhibitors and showed that (+/-)-3 has a significant functional antagonism against cocaine in vitro. Our previous preliminary structure-activity relationship study led to identification of a more potent DAT inhibitor [(+/-)-4] but this compound failed to show any significant functional antagonism. To search for more potent analogues than 3 but still displaying significant functional antagonism, further SARs, molecular modeling studies and in vitro pharmacological evaluation of this novel class of DAT inhibitors were performed. Sixteen new analogues were synthesized in racemic form and evaluated as DAT inhibitors. It was found that seven new analogues are reasonably potent DAT inhibitors with K(i) values of 0.041--0.30 and 0.052--0.16 microM in [(3)H]mazindol binding and inhibition of DA reuptake. Chiral isomers of several potent DAT inhibitors were obtained through chiral HPLC separation and evaluated as inhibitors at all the three monoamine transporter sites. In general, the (-)-isomer is more active than the (+)-isomer in inhibition of DA reuptake and all the (-)-isomers are selective inhibitors at the DAT site. Evaluation of cocaine's effect on dopamine uptake in the presence and absence of (+)-3 and (-)-3 showed that (-)-3 is responsible for the functional antagonism obtained with the original lead (+/-)-3. Out of the new compounds synthesized, analogue (+/-)-20, which is 8- and 3-fold more potent than (+/-)-3 in binding and inhibition of DA reuptake, appeared to have improved functional antagonism as compared to (+/-)-3.

Animals↗

PubMed2001

Effective control of asthma with hydrofluoroalkane flunisolide delivered as an extrafine aerosol in asthma patients.

BACKGROUND: Inhaled corticosteroids are established as maintenance therapy for persistent asthma. A new aerosol formulation of flunisolide delivers a small particle size by using a hydrofluoroalkane (HFA) propellant with a built-in spacer. OBJECTIVE: To compare efficacy and safety of two different flunisolide formulations, HFA and chlorofluorocarbon (CFC), with placebo treatment over a range of doses. METHODS: The multicenter, randomized, double-blind, placebo-controlled trial consisted of a 2-week, active run-in phase with CFC flunisolide 500 microg, twice daily, followed by 12 weeks of double-blind treatment with placebo, HFA flunisolide (85, 170, or 340 microg, twice daily), or CFC flunisolide (250, 500, or 1,000 microg, twice daily). Patients (N = 669) were nonsmokers, at least 12 years of age, with mild to moderate asthma who were being treated with inhaled corticosteroids. Outcome measures were change from baseline in forced expiratory volume in 1 second (FEV1), peak expiratory flow rate, as needed albuterol use, nocturnal awakenings, and asthma symptoms. RESULTS: After 12 weeks of treatment, patients receiving 170 microg, twice daily, and 340 microg, twice daily, of HFA flunisolide showed a significant (P < 0.01) improvement in percentage increase in FEV1 (12.22% at 170 microg, twice daily, and 14.69% at 340 microg, twice daily) compared with the placebo group (5.35%). At one-third the dose of CFC flunisolide, HFA flunisolide provided similar improvement in pulmonary function versus placebo. Both formulations demonstrated comparable linear dose dependency for the change from baseline in FEV1 without any evidence of cortisol suppression. Outcome values for all seven secondary efficacy measures were numerically superior in patients receiving HFA flunisolide compared with the CFC formulation. Both formulations seemed to be safe and well tolerated. CONCLUSIONS: HFA flunisolide provides comparable efficacy and safety at one-third the dose of CFC flunisolide.

Administration, Inhalation↗

PubMed2001

Comparison of the hammerhead cleavage reactions stimulated by monovalent and divalent cations.

Although the hammerhead reaction proceeds most efficiently in divalent cations, cleavage in 4 M LiCl is only approximately 10-fold slower than under standard conditions of 10 mM MgCl2 (Murray et al., Chem Biol, 1998, 5:587-595; Curtis & Bartel, RNA, 2001, this issue, pp. 546-552). To determine if the catalytic mechanism with high concentrations of monovalent cations is similar to that with divalent cations, we compared the activities of a series of modified hammerhead ribozymes in the two ionic conditions. Nearly all of the modifications have similar deleterious effects under both reaction conditions, suggesting that the hammerhead adopts the same general catalytic structure with both monovalent and divalent cations. However, modification of three ligands previously implicated in the binding of a functional divalent metal ion have substantially smaller effects on the cleavage rate in Li+ than in Mg2+. This result suggests that an interaction analogous to the interaction made by this divalent metal ion is absent in the monovalent reaction. Although the contribution of this divalent metal ion to the overall reaction rate is relatively modest, its presence is needed to achieve the full catalytic rate. The role of this ion appears to be in facilitating formation of the active structure, and any direct chemical role of metal ions in hammerhead catalysis is small.

Base Sequence↗

PubMed2001

Depletion-induced demixing in aqueous protein-polysaccharide solutions.

We explore the separation of aqueous protein-polysaccharide solutions into two liquid phases. In particular, we have studied the combinations beta-lactoglobulin/pullulan, alpha-lactalbumin/pullulan, and other examples from the literature under a variety of conditions such as varying salt content, pH (in most cases at the isoelectric point), and protein radius. We restrict ourselves to relatively small proteins (globular) and long polysaccharide chains. The mechanism behind the phase separation is explained in terms of the depletion interaction (i.e., the cross-interaction) in a suspension of small spheres (proteins) immersed in a semidilute solution of coils (polysaccharide) forming an entangled network. Weak attractions between the spheres have been taken into account by assuming the formation of small clusters. As a general rule, we find that the depletion free energy per protein particle governing the protein partitioning in the phase equilibrium is linear in the polysaccharide concentration over the whole range of experimentally accessible coexistence curves. Furthermore, the proportionality constant is shown to be a very useful quantity to understand the characteristics of the coexistence curves. The linearity thus found is supported by theoretical arguments developed by de Gennes and Odijk.

Animals↗

PubMed2001

Five new diprenylated flavonols from the leaves of Broussonetia kazinoki.

Five new diprenylated flavonols, broussonol A (1), broussonol B (2), broussonol C (3), broussonol D (4), and broussonol E (5), along with two known compounds, were isolated from an ethanolic extract of the leaves of Broussonetia kazinoki. Their structures were elucidated by chemical and spectral methods. Cytotoxic activities were evaluated against several different cell lines.

Dose-Response Relationship, Drug↗

PubMed2001

Cloning and characterization of KLHL5, a novel human gene encoding a kelch-related protein with a BTB domain.

Most kelch family proteins contain two conserved domains, the BTB domain and the kelch repeat domain. Here we describe the cloning and characterization of a novel human KLHL5 gene. The 3488 bp cDNA encodes a kelch family protein homologous to the Drosophila kelch protein. It also contains the two conserved domains. Northern blot analysis reveals a single transcript. It is abundantly expressed in ovary, adrenal gland, and thyroid and less abundantly expressed in trachea, prostate, testis, lymph node, and spinal cord. KLHL5 was mapped to 4p13-4p15.1.

Amino Acid Sequence↗

PubMed2001

Functional preference of the constituent amino acid residues in a phage-library-based nonphosphorylated inhibitor of the Grb2-SH2 domain.

A nonphosphorylated disulfide-bridged peptide, cyclo(Cys-Glu1-Leu-Tyr-Glu-Asn-Val-Gly-Met-Tyr9-Cys)-amide (termed G1) has been identified, by phage library, that binds to the Grb2-SH2 domain but not the src SH2 domain. Synthetic G1 blocks the Grb2-SH2 domain association (IC50 of 15.5 microM) with natural phosphopeptide ligands. As a new structural motif that binds to the Grb2-SH2 domain in a pTyr-independent manner, the binding affinity of G1 is contributed by the highly favored interactions of its structural elements interacting with the binding pocket of the protein. These interactions involve side-chains of amino acids Glu1, Tyr3, Glu4, Asn5, and Met8. Also a specific conformation is required for the cyclic peptide when bound to the protein. Ala scanning within G1 and molecular modeling analysis suggest a promising model in which G1 peptide binds in the phosphotyrosine binding site of the Grb2-SH2 domain in a beta-turn-like conformation. Replacement of Tyr3 or Asn5 with Ala abrogates the inhibitory activity of the peptide, indicating that G1 requires a Y-X-N consensus sequence similar to that found in natural pTyr-containing ligands, but without Tyr phosphorylation. Significantly, the Ala mutant of Glu1, i.e. the amino acid N-terminal to Y3, remarkably reduces the binding affinity. The position of the Glu1 side-chain is confirmed to provide a complementary role for pTyr3, as demonstrated by the low micromolar inhibitory activity (IC50 = 1.02 microM) of the nonphosphorylated peptide 11, G1(Gla1), in which Glu1 was replaced by gamma-carboxy-glutamic acid (Gla).

Adaptor Proteins, Signal Transducing↗

PubMed2001

Caspase-2 deficiency prevents programmed germ cell death resulting from cytokine insufficiency but not meiotic defects caused by loss of ataxia telangiectasia-mutated (Atm) gene function.

It is well established that programmed cell death claims up to two-thirds of the oocytes produced during gametogenesis in the developing fetal ovaries. However, the mechanisms underlying prenatal germ cell loss in females remain poorly understood. Herein we report that caspase-11 null female mice are born with a reduced number of oocyte-containing primordial follicles. This phenotype is likely due to failed cytokine processing known to occur in caspase-11 mutants since neonatal female mice lacking both interleukin (IL)-1alpha and IL-1beta also exhibit a reduced endowment of primordial follicles. In addition, germ cell death in wild-type fetal ovaries cultured ex vivo is suppressed by either cytokine, likely via ligand activation of type 1 IL-1 receptors expressed in fetal germ cells. Normal oocyte endowment can be restored in caspase-11 null female mice by simultaneous inactivation of the gene encoding the cell death executioner enzyme, caspase-2. However, caspase-2 deficiency cannot overcome gametogenic failure resulting from meiotic recombination defects in ataxia telangiectasia-mutated (Atm) null female mice. Thus, genetically distinct mechanisms exist for developmental deletion of oocytes via programmed cell death, one of which probably functions as a meiotic quality-control checkpoint that cannot be overridden.

Animals↗

PubMed2001

A new siglec family member, siglec-10, is expressed in cells of the immune system and has signaling properties similar to CD33.

The siglecs (sialic acid-binding Ig-like lectins) are a distinct subset of the Ig superfamily with adhesion-molecule-like structure. We describe here a novel member of the siglec protein family that shares a similar structure including five Ig-like domains, a transmembrane domain, and a cytoplasmic tail containing two ITIM-signaling motifs. Siglec-10 was identified through database mining of an asthmatic eosinophil EST library. Using the Stanford G3 radiation hybrid panel we were able to localize the genomic sequence of siglec-10 within the cluster of genes on chromosome 19q13.3-4 that encode other siglec family members. We have demonstrated that siglec-10 is an immune system-restricted membrane-bound protein that is highly expressed in peripheral blood leukocytes as demonstrated by Northern, RT-PCR and flow cytometry. Binding assays determined that the extracellular domain of siglec-10 was capable of binding to peripheral blood leukocytes. The cytoplasmic tail of siglec-10 contains four tyrosines, two of which are embedded in ITIM-signaling motifs (Y597 and Y667) and are likely involved in intracellular signaling. The ability of tyrosine kinases to phosphorylate the cytoplasmic tyrosines was evaluated by kinase assay using wild-type siglec-10 cytoplasmic domain and Y-->F mutants. The majority of the phosphorylation could be attributed to Y597 andY667. Further experiments with cell extracts suggest that SHP-1 interacts with Y667 and SHP-2 interacts with Y667 in addition to another tyrosine. This is very similar to CD33, which also binds the phosphatases SHP-1 and SHP-2, therefore siglec-10, as CD33, may be characterized as an inhibitory receptor.

Amino Acid Motifs↗

PubMed2001

Establishment of a tetraploid Meth-A cell line through polyploidization by demecolcine but not by staurosporine, K-252A and paclitaxel.

Polyploid cells are made by DNA reduplication without cell division, however, it is not easy to establish polyploid mammalian cell lines. It is worth studying the difference in cell character between hyperploid and parent cell lines. Meth-A cells were polyploidized by demecolcine, K-252a, staurosporine and paclitaxel. The cell-cycle responses of highly polyploid Meth-A cells after the removal of the drugs were examined by flow cytometry (FCM). Meth-A cells were highly polyploidized by these drugs. The polyploid Meth-A cells gradually decreased in ploidy after the drug release. A tetraploid Meth-A cell line was established only from the demecolcine-induced polyploid Meth-A cells. The duration of G1, S and G2/M phases of the tetraploid cell line were mostly the same as those of the parent diploid cells, except that the G2/M phase was 1.5 h longer. The chromosome number of tetraploid Meth-A cell line was about twice of the diploidy. A tetraploid Meth-A cell line was established.

Animals↗

PubMed2001

Antisense inhibition of BCL-2 expression induces retinoic acid-mediated cell death during differentiation of human NT2N neurons.

Changes in expression of the proto-oncogene Bcl-2 are well known in the developing brain, with a high expression level in young post-mitotic neurons that are beginning the outgrowth of processes. The physiological significance of the Bcl-2 up-regulation in these neurons is not fully understood. We used a differentiation model for human CNS neurons to study the expression and function of Bcl-2. NT2/D1 human neuronal precursor cells differentiated into a neuronal phenotype in the presence of 10 microM retinoic acid for 3-5 weeks. This concentration of retinoic acid was not toxic to undifferentiated NT2/D1 cells but was sufficient to up-regulate the BCL-2 protein in 6 days. The BCL-2 levels increased further after 3 weeks, i.e. when the cells started to show neuronal morphology. Inhibition of the accumulation of endogenous BCL-2 with vectors expressing the antisense mRNA of Bcl-2 caused extensive apoptosis after 3 weeks of the retinoic acid treatment. The loss of neuron-like cells from differentiating cultures indicated that the dead cells were those committed to neuronal differentiation. Death was related to the presence of retinoic acid since withdrawal of retinoic acid after 16 days of treatment dramatically increased cell surviving. The ability of BCL-2 to prevent retinoic acid-induced cell death was also confirmed in undifferentiated NT2/D1 cells that were transfected with a vector containing Bcl-2 cDNA in sense orientation and exposed to toxic doses (40-80 microM) of retinoic acid. Furthermore, down-regulation of BCL-2 levels by an antisense oligonucleotide in neuronally differentiated NT2/D1 cells increased their susceptibility to retinoic acid-induced apoptosis. These results indicate that one function of the up-regulation of endogenous BCL-2 during neuronal differentiation is to regulate the sensitivity of young post-mitotic neurons to retinoic acid-mediated apoptosis.

Apoptosis↗

PubMed2001

Connective tissue growth factor in tubulointerstitial injury of diabetic nephropathy.

BACKGROUND: Chronic interstitial fibrosis, which follows the onset of glomerular proteinuria, importantly contributes to progressive renal failure in diabetic nephropathy. The present studies examine the potential role of tubular connective tissue growth factor (CTGF). METHODS: The expression of CTGF was examined in rats with diabetic nephropathy. Regulation and actions of CTGF were studied in in vitro cell culture models. RESULTS: CTGF mRNA levels were increased in the renal cortex of rats with streptozotocin-induced diabetes compared with controls. Immunohistology indicated that CTGF was expressed in renal cortex of diabetic rats, in contrast to controls in some tubular cross-sections, particularly dilated-appearing proximal tubules, in which it tended to colocalize with insulin-like growth factor-I (IGF-I). Glomerular ultrafiltrate from diabetic rats, which contained bioactive transforming growth factor-beta (TGF-beta) and hepatocyte growth factor (HGF), induced increased CTGF expression in tubular cells. TGF-beta1 and, to a lesser extent, HGF also raised CTGF expression in cultured proximal tubular cells. In contrast, high glucose (25 mmol/L) did not increase the secretion of CTGF. In cultured tubular cells, rhCTGF moderately increased fibronectin but not collagen (Col) type I and type III expression. In NRK-49F renal interstitial fibroblasts, CTGF raised Col alpha1III and thrombospondin-1 levels. CTGF has an IGF-binding domain and binds to IGF-I. In NRK-49F cells, IGF-I increased the activity of CTGF towards the expression of Col alpha1III. CONCLUSIONS: CTGF is expressed and regulated downstream from TGF-beta and HGF in proximal tubular cells, is induced by diabetic rat glomerular ultrafiltrate, and has moderate profibrogenic activity in tubular cells and renal interstitial fibroblasts, where its activity is IGF-I dependent. By these means, CTGF may act downstream of TGF-beta and HGF and may contribute to chronic tubulointerstitial fibrosis in diabetic nephropathy.

Animals↗

PubMed2001

Pharmacokinetics in dogs after oral administration of two different forms of ascorbic acid.

The dog is able to synthesise ascorbic acid (AA), but is frequently given the vitamin in an attempt to improve health and performance. The pharmacokinetics of AA in this species, however, are not well studied. Using a selective analytical method and careful stability control, the pharmacokinetics of orally given AA was studied in 20 dogs, at two dosage levels (15 and 50 mg kg(-1)) and with two forms of supplement [crystalline AA and the vitamin C product Ester-C(Inter-Cal Corp., Prescott, AZ, USA)]. After oral administration, a rapid increase was found in the plasma level of AA, indicating a possible intestinal active transport mechanism in this species. The obtained C(max)and AUC values were found to increase in a non-linear fashion when the dose of AA was increased. The pharmacokinetic modeling of the elimination of AA was made difficult by a pronounced secondary peak appearing after about 9 hours. The comparison of crystalline AA and Ester-C did not indicate any significant differences in pharmacokinetic parameters between the two preparations of the vitamin.

Administration, Oral↗

PubMed2001

Plasma ascorbic acid concentrations in healthy dogs.

Using a highly sensitive and selective analytical method and careful stability control, plasma concentrations of ascorbic acid were determined in German Shepherd Dogs, Labrador Retrievers and Siberian Huskies, a total 99 animals. Mean concentration was 35.9 micromol l(-1)(range 18.2-50.7), and no significant variation was observed neither between breeds nor between females and males. These and previous reported data on plasma ascorbic acid levels in dogs are discussed in the light of methodological aspects.

Animals↗

PubMed2001

Automatic differentiation of melanoma from melanocytic nevi with multispectral digital dermoscopy: a feasibility study.

BACKGROUND: Differentiation of melanoma from melanocytic nevi is difficult even for skin cancer specialists. This motivates interest in computer-assisted analysis of lesion images. OBJECTIVE: Our purpose was to offer fully automatic differentiation of melanoma from dysplastic and other melanocytic nevi through multispectral digital dermoscopy. METHOD: At 4 clinical centers, images were taken of pigmented lesions suspected of being melanoma before biopsy. Ten gray-level (MelaFind) images of each lesion were acquired, each in a different portion of the visible and near-infrared spectrum. The images of 63 melanomas (33 invasive, 30 in situ) and 183 melanocytic nevi (of which 111 were dysplastic) were processed automatically through a computer expert system to separate melanomas from nevi. The expert system used either a linear or a nonlinear classifier. The "gold standard" for training and testing these classifiers was concordant diagnosis by two dermatopathologists. RESULTS: On resubstitution, 100% sensitivity was achieved at 85% specificity with a 13-parameter linear classifier and 100%/73% with a 12-parameter nonlinear classifier. Under leave-one-out cross-validation, the linear classifier gave 100%/84% (sensitivity/specificity), whereas the nonlinear classifier gave 95%/68%. Infrared image features were significant, as were features based on wavelet analysis. CONCLUSION: Automatic differentiation of invasive and in situ melanomas from melanocytic nevi is feasible, through multispectral digital dermoscopy.

Diagnosis, Differential↗

PubMed2001

Role of placental tissues in the intrauterine transmission of hepatitis B virus.

OBJECTIVE: The purpose of this study was to determine the mechanism of intrauterine transmission of hepatitis B virus. STUDY DESIGN: Placental tissues from 158 pregnant women who tested positive for hepatitis B surface antigen were examined for hepatitis B virus markers, Fc gamma receptors, and hepatitis B surface antigen-anti-hepatitis B surface antigen in different layers of cells. RESULTS: It was shown that the hepatitis B virus infection rate among different layers of placental cells gradually decreased from the maternal side to the fetal side. Furthermore, the closer the infected cell layer was to the fetal side, the higher the risk of intrauterine hepatitis B virus infection. Fc gamma receptors were found on cells of both hepatitis B surface antigen positive and negative placentas; Fc gamma receptors III were found on trophoblastic cells and villous mesenchymal cells, and Fc gamma receptors II were found on only villous mesenchymal cells. Hepatitis B surface antigen-antibodies to hepatitis B surface antigen was detected in the cytoplasm and on the membrane of trophoblastic cells and villous mesenchymal cells in 2 hepatitis B surface antigen-positive placentas. CONCLUSION: The results support the hypothesis that intrauterine hepatitis B virus transmission could be caused through "cellular transfer" in the placenta. One of the means of cellular transfer could be through Fc gamma receptor III-mediated entry of hepatitis B surface antigen-antibodies to hepatitis B surface antigen into cells.

Adolescent↗

PubMed2001

Expression of aquaporin-8 in human placenta and chorioamniotic membranes: evidence of molecular mechanism for intramembranous amniotic fluid resorption.

OBJECTIVE: The intramembranous pathway of amniotic fluid absorption has recently been recognized as a critical regulatory path for amniotic fluid resorption, which contributes importantly to amniotic fluid volume homeostasis. Yet, the underlying molecular and cellular mechanisms for water absorption across the amniotic membranes remain unknown. We hypothesize that amniotic fluid water is absorbed across aquaporin water channels in the chorioamniotic membranes. Recently the water channel aquaporin-8 complementary DNAs for mice, rats, and humans have been cloned; and aquaporin-8 has been found to be expressed in the placenta and in other organs. The objective of the present study was to determine whether aquaporin-8 is expressed in human chorioamniotic membranes. STUDY DESIGN: Freshly frozen human amnion, chorion, placenta, and umbilical cord of a normal term pregnancy were used in this study. Reverse transcriptase-polymerase chain reaction was used to determine the aquaporin-8 gene expression. In situ hybridization with biotin-labeled aquaporin-8 riboprobe was undertaken to localize the cellular expression of aquaporin-8 gene in these human fetal membranes. RESULTS: Using reverse transcriptase-polymerase chain reaction, we have demonstrated that aquaporin-8 is expressed in human amnion, chorion, and placenta. Further studies of in situ hybridization of aquaporin-8 identified aquaporin-8 gene expression in epithelial cells of chorion and amnion and of the syncytiotrophoblasts and outer layer trophoblasts of placenta. CONCLUSION: This is the first study that demonstrates the expression of aquaporin-8 water channel in human chorioamniotic membranes. These results suggest that aquaporin-8 may be a water channel that mediates amniotic fluid resorption by way of the intramembranous pathway.

Amnion↗