Routine dilated fundus examination diagnostic yield.
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Fundus Oculi↗
Biomedical subjects
S Wang
Publications and source records attributed to S Wang.
Production of a biologically active novel goldfish growth hormone in Escherichia coli.
Goldfish pituitary contains two types of growth hormones. One with five cysteine residues (type-I) similar to other Cyprinid GHs, and the other with four Cys residues (type-II) similar to those of other fish and tertapod species. Recombinant goldfish type II GH (gfGH-II) was produced in Escherichia coli using the pRSETB expression vector. The gfGH-II was produced fused to a leader sequence, which sequestered into inclusion bodies after expression. The inclusion bodies were solubilized using sodium hydroxide and the fusion protein purified by chelating affinity chromatography. Subsequently, gfGH-II was cleaved and analyzed by Western blotting, using a specific antiserum. For comparison we also produced recombinant common carp GH (cGH) which has 95% similarity to gfGH-II, and tested their growth promoting activity in goldfish. Both forms of GH significantly increased the growth rate of goldfish (P < 0.05), although cGH was found to have a somewhat higher potency than gfGH-II.
Amino Acid Sequence↗
Effects of early stress on adult affiliative behavior.
The recently evolved mammalian species preservative behavior as opposed to the ancient self preservative behavior involves parental care, nursing, social interaction, pair bonding and mutual defense. Gonadal steroids together with oxytocin are critical for this affiliative, attachment behavior. When there is stressful loss of control, gonadotrophins are diminished, and the self preservative, fight-flight catecholamine coping response takes priority. It is suggested that self preservation is associated with left hemispheric brain function and that species preservation is associated with right hemispheric function. Stress during infancy that is severe enough to create insecure attachment has a dissociative effect, disrupting right hemispheric emotional functioning and species preservative behavior, and a permanent bias towards self preservation can become an adult trait. In such a person with impaired affiliation, corticoid responses may be deficient. The coronary type A behavior pattern common in our society exhibits some of this deficiency in species preservative activity.
Animals↗
Notch receptor activation inhibits oligodendrocyte differentiation.
In this study, we show that oligodendrocyte differentiation is powerfully inhibited by activation of the Notch pathway. Oligodendrocytes and their precursors in the developing rat optic nerve express Notch1 receptors and, at the same time, retinal ganglion cells express Jagged1, a ligand of the Notch1 receptor, along their axons. Jagged1 expression is developmentally regulated, decreasing with a time course that parallels myelination in the optic nerve. These results suggest that the timing of oligodendrocyte differentiation and myelination is controlled by the Notch pathway and raise the question of whether localization of myelination is controlled by this pathway.
Animals↗
Expression of p68 protein kinase and its prognostic significance during IFN-alpha therapy in patients with carcinoid tumours.
The aim of this study was to evaluate the antiproliferative effects of interferon alpha (IFN-alpha) on neuroendocrine differentiated cell lines and, retrospectively, to assess the prognostic significance of p68 protein kinase (PKR) induction in neuroendocrine gut and pancreatic tumour patients. Archive specimens from 56 patients were studied, 43 before IFN-alpha and 56 during therapy. The tissues were immunostained for p68 protein kinase (PKR) using the monoclonal antibody (MAb) TJ4C4. A significant increase in immunostaining after treatment with IFN-alpha compared with before treatment (3.47 +/- 0.12 versus 2.72 +/- 0.15, P < 0.001) was noted. The p68 score was significantly increased after treatment only in patients with stable disease before = 2.71 +/- 0.19, after = 3.40 +/- 0.14 (P < 0.001) or an objective response before 3.13 +/- 0.22, after = 4.00 +/- 0.24 (P < 0.05) but not in those with progressive disease (before = 2.32 +/- 0.24, after 2.86 +/- 0.26, NS). A low p68 score (< 3.0) during treatment was a predictor of shorter duration of response and overall survival (P = 0.0062 and P < 0.0001, respectively). Furthermore, IFN-alpha showed a significant antiproliferative effect (by [3H]thymidine incorporation) on two carcinoid tumour cell lines in a dose-dependent manner which correlated with a dose-dependent induction of p68 mRNA and protein expression (by Northern and Western blot analysis). We conclude that IFN-alpha can effectively inhibit the in vitro growth of carcinoid tumor cell lines and upregulates the expression of p68 at both mRNA and protein levels in carcinoid tumours. The induction of p68 could be a prognostic indicator of response in patients with carcinoid tumours during IFN-alpha treatment.
Antineoplastic Agents↗
Structure-based design and synthesis of small molecule protein-tyrosine phosphatase 1B inhibitors.
Protein-tyrosine phosphatase (PTP) inhibitors are attractive as potential signal transduction-directed therapeutics which may be useful in the treatment of a variety of diseases. We have previously reported the X-ray structure of 1,1-difluoro-1-(2-naphthalenyl)methyl] phosphonic acid (4) complexed with the human the protein-tyrosine phosphatase 1B (PTP1B) and its use in the design of an analogue which binds with higher affinity within the catalytic site (Burke, T. R., Jr. et al. Biochemistry 1996, 35, 15989). In the current study, new naphthyldifluoromethyl phosphonic acids were designed bearing acidic functionality intended to interact with the PTP1B Arg47, which is situated just outside the catalytic pocket. This residue has been shown previously to provide key interactions with acidic residues of phosphotyrosyl-containing peptide substrates. Consistent with trends predicted by molecular dynamics calculations, the new analogues bound with 7- to 14-fold higher affinity than the parent 4, in principal validating the design rationale.
Drug Design↗
Transcapillary transport of metaiodobenzylguanidine (MIBG) in isolated rat heart.
A better understanding of transcapillary transport for tracer metaiodobenzylguanidine (MIBG) is desirable for development of tracer kinetic models that yield meaningful estimates of neuronal uptake function from tissue radioactivity time courses. This study utilized a multiple-indicator approach in Langendorff-perfused rat hearts to define transport mechanisms and determine the capillary permeability-surface area (PSc) over a broad range of flow (F). Multiple injections within the same heart at different flows allowed characterization of the PSc/F relationship within the same heart. The coefficient of variation of E for multiple injections within the same hearts at constant flow was 6 +/- 2% (3 to 6 injections in 9 hearts). In 10 hearts (4 to 6 injections per heart), flow was varied between 2.0-16.5 mL/min. PSc was found to be nearly proportional to flow in each heart (r = 0.88 +/- 0.14; slope = 0.23 +/- 0.10; intercept = 11 +/- 7 mL/min/g dry). Tissue hypoxia at low flows, as evidenced by enhanced lactate production, did not appear to influence the PSc/F relationship. Pharmacologic blockade of uptake-1 and uptake-2 had negligible affect on E or PSc as compared with flow-matched controls, although tissue retention was markedly reduced. The results show PSc of MIBG to be nearly proportional to flow but independent of specific neuronal and extraneuronal transport mechanisms and tissue hypoxia. The results are consistent with a passive diffusion process across the capillary endothelial barrier. The increase in PSc with increasing flow could reflect capillary recruitment and/or enhanced capillary permeability.
3-Iodobenzylguanidine↗
Gland atrophy following retrograde injection of methyl violet as a treatment in chronic obstructive parotitis.
OBJECTIVE: The purpose of this study was to evaluate the clinical effectiveness of retrograde injection of 1% methyl violet as a treatment for chronic obstructive parotitis. STUDY DESIGN: Sixteen patients with chronic obstructive parotitis were treated with retrograde injection of 1% methyl violet. Pretreatment evaluation and assessment of the treatment effect were performed by clinical and imaging methods, including sialography and sonography of the treated glands. RESULTS: Clinical symptoms following treatment included initial acute swelling and then a decrease in parotid swellings; the final outcome was a clinical cure of the affected glands characterized by disappearance of all symptoms, absence of secretion, and complete obliteration of the main duct orifice. Posttreatment imaging showed total atrophy of the diseased glands. CONCLUSION: Retrograde injection of 1% methyl violet caused total atrophy of the diseased glands and brought about complete relief in all 16 patients, with no detectable side effects.
Adolescent↗
Structure-function relationships in the hammerhead ribozyme probed by base rescue.
We previously showed that the deleterious effects from introducing abasic nucleotides in the hammerhead ribozyme core can, in some instances, be relieved by exogenous addition of the ablated base and that the relative ability of different bases to rescue catalysis can be used to probe functional aspects of the ribozyme structure [Peracchi et al., Proc NatAcad Sci USA 93:11522]. Here we examine rescue at four additional positions, 3, 9, 12 and 13, to probe transition state interactions and to demonstrate the strengths and weaknesses of base rescue as a tool for structure-function studies. The results confirm functional roles for groups previously probed by mutagenesis, provide evidence that specific interactions observed in the ground-state X-ray structure are maintained in the transition state, and suggest formation in the transition state of other interactions that are absent in the ground state. In addition, the results suggest transition state roles for some groups that did not emerge as important in previous mutagenesis studies, presumably because base rescue has the ability to reveal interactions that are obscured by local structural redundancy in traditional mutagenesis. The base rescue results are complemented by comparing the effects of the abasic and phenyl nucleotide substitutions. The results together suggest that stacking of the bases at positions 9, 13 and 14 observed in the ground state is important for orienting other groups in the transition state. These findings add to our understanding of structure-function relationships in the hammerhead ribozyme and help delineate positions that may undergo rearrangements in the active hammerhead structure relative to the ground-state structure. Finally, the particularly efficient rescue by 2-methyladenine at position 13 relative to adenine and other bases suggests that natural base modifications may, in some instance, provide additional stability by taking advantage of hydrophobic interactions in folded RNAs.
Adenine↗
Human 15-lipoxygenase gene promoter: analysis and identification of DNA binding sites for IL-13-induced regulatory factors in monocytes.
In order to study the transcriptional control of 15-LO expression, we have cloned and sequenced the human 15-LO promoter region. The 15-LO promoter is associated with a CpG island at the 5'-end of the gene, and sequence analysis reveals putative Sp1 and Ap2 binding site/s and absence of TATA or CAAT motifs. Transcription is initiated at one major site. Using deletion constructs, we have defined an active promoter region of 1056 bp. Gel-shift assays revealed that transcriptional factor(s) induced only in response to IL-13 treatment of human peripheral blood monocytes bind to the 15-LO promoter DNA. Two regions, DP1 (-140 to -92 bp) and DP2 (-353 to -304 bp) of the promoter were essential for transcription in HeLa cells and human peripheral monocytes. Hela nuclear extracts contained a specific nuclear factor(s) binding to 15-LO promoter DNA which are distinct from those derived from IL-13-treated human peripheral monocyte nuclear extracts. In addition, fluorescent in situ hybridization (FISH) results refined the previous localization of 15-LO to human chromosome 17p13.3.
Animals↗
A molecular basis for retinol stimulation of vesicle budding in vivo and in vitro.
Retinol stimulates the formation of transition vesicles in situ and in all free systems based on rat liver. The stimulation is on vesicle formation from transitional endoplasmic reticulum and not on vesicle fusion with donor membranes. Vesicle budding in the cell free system requires a nucleoside triphosphate and is sensitive to inhibition by thiol reagents. In this report we develop and test a model whereby a retinol-modulated NADH:protein disulfide reductase (NADH oxidase) with protein disulfide-thiol interchange activity is implicated in the vesicle budding mechanism. The protein has the ability to restore activity to scrambled, inactive RNase A and is stimulated or inhibited by retinol depending on the redox environment. Under reducing conditions and in the presence of a chemical reductant such as GSH, the partial reaction stimulated by retinol appears to be the oxidation of membrane thiols. This is the first report of an enzymatic mechanism to explain specific retinol effects both in vivo and in vitro on membrane trafficking not given by retinoic acid.
Animals↗
The effects of pH and intraliposomal buffer strength on the rate of liposome content release and intracellular drug delivery.
Targeted liposomal drug formulations may enter cells by receptor-mediated endocytosis and then traffick by membrane flow into acidic intracellular compartments. In order to understand the impact of these intracellular pH changes on liposomal drug unloading, the effect of pH on the release from folate-targeted liposomes of three model compounds with distinct pH dependencies was examined. 5(6)-carboxyfluorescein, which titrates from its anionic to uncharged form following internalization by KB cells, displays strong endocytosis-dependent release, since only its uncharged (endosomal) form is membrane permeable. Endocytosis-triggered unloading of drugs of this sort is enhanced by encapsulating the drug in a weak buffer at neutral pH, so that acidification of the intraliposomal compartment following cellular uptake can occur rapidly. Sulforhodamine B, in contrast, retains both anionic and cationic charges at endosomal pH (approximately pH 5), and consequently, escapes the endosomes only very slowly. Doxorubicin, which is commonly loaded into liposomes in its membrane-impermeable (cationic) form using an acidic buffer, still displays endocytosis-triggered unloading, since sufficient uncharged doxorubicin remains at endosomal pHs to allow rapid re-equilibration of the drug according to the new proton gradient across the membrane. In this case, when the extraliposomal [H+] increases 250-fold from 4 x 10(-8) M (pH 7.4, outside the cell) to 10(-5) M (pH 5, inside the endosome), the ratio of doxorubicin inside to outside the liposome must decrease by a factor of 250. Therefore, the collapse of the transliposomal pH gradient indirectly drives an efflux of the drug molecule from the liposome. Since a change in intraliposomal pH is not required to unload drugs of this type, the intraliposomal compartment can be buffered strongly at acidic pH to prevent premature release of the drug outside the cell. In summary, pH triggered release of liposome-encapsulated drugs can be achieved both with drugs that increase as well as decrease their membrane permeabilities upon acidification, as long as the intraliposomal buffer strength and pH is rationally selected.
Antineoplastic Agents↗
NGF gene expression in dividing and non-dividing cells from AAV-derived constructs.
NGF expression in COS cells when driven by pTR.NGF (CMV promoter, AAV TRs) was more effective than either pc.NGF (CMV promoter, no AAV TRs) or dlk.NGF (AAV promoters and TRs). This NGF was able to differentiate PC12 cells. Differentiated PC12 cells transfected with pTR.NGF released NGF into medium. The fraction of pTR.NGF transfected PC12 cells that extended neurite-like processes 7 days post-transfection was similar to the transfection efficiency, suggesting that transfected cells were selectively differentiated by locally released NGF. pTR.NGF-transfected primary cultures of either neurons or glia did not express exogenous NGF. These results indicate that NGF can be released by dividing and non-dividing cells, but not neonatally derived brain cells.
Animals↗
Isolation of neuronal precursors by sorting embryonic forebrain transfected with GFP regulated by the T alpha 1 tubulin promoter.
Neuronal precursor cells are widespread in the forebrain ventricular/subventricular zone, and may provide a cellular substrate for brain repair. Clonal lines derived from single progenitors can become progressively less representative of their parental precursors with time and passage in vitro. We have developed an alternative strategy for the isolation and enrichment of precursor cells, by fluorescence-activated cell sorting of forebrain cells transfected with the gene for green fluorescent protein, driven by the neuronal T alpha 1 tubulin promoter. Using this approach, neural precursors and young neurons can be identified and selectively harvested from a variety of samples, including both avian and mammalian forebrains at different developmental stages.
Animals↗
Crystal structure of calsequestrin from rabbit skeletal muscle sarcoplasmic reticulum.
Calsequestrin, the major Ca2+ storage protein of muscle, coordinately binds and releases 40-50 Ca2+ ions per molecule for each contraction-relaxation cycle by an uncertain mechanism. We have determined the structure of rabbit skeletal muscle calsequestrin. Three very negative thioredoxin-like domains surround a hydrophilic center. Each monomer makes two extensive dimerization contacts, both of which involve the approach of many negative groups. This structure suggests a mechanism by which calsequestrin may achieve high capacity Ca2+ binding. The suggested mechanism involves Ca2+-induced collapse of the three domains and polymerization of calsequestrin monomers arising from three factors: N-terminal arm exchange, helix-helix contacts and Ca2+ cross bridges. This proposed structure-based mechanism accounts for the observed coupling of high capacity Ca2+ binding with protein precipitation.
Animals↗
Adenovirus-mediated expression of melanoma antigen gp75 as immunotherapy for metastatic melanoma.
Melanocyte differentiation antigens, such as the brown locus protein gp75, are potential biological targets for immunotherapy. We investigated whether expression of the murine gp75 cDNA mediated by an adenovirus (Ad) vector could induce melanoma rejection using this model self antigen that usually induces tolerance, and whether Ad vector-directed production of interleukin-2 (IL2) might augment this response. To evaluate this approach, Ad vectors were constructed containing the murine gp75 cDNA (Ad.gp75) and the human IL2 cDNA (Ad.IL2). Efficacy was evaluated in C57BI/6 mice challenged i.v. with 10(5) B16 cells, using the number of lung metastases as the efficacy parameter. Naive control mice developed 175 +/- 12 metastases by day 14. Controls receiving intranasal Ad.IL2 1 day after B16 cell injection, intraperitoneal (i.p.) mitomycin-C-treated B16 cells +/- i.p. Ad.IL2 before B16 cell challenge and Ad.beta gal-treated mice had similar numbers of metastases as controls (P > 0.1). In marked contrast, preimmunization with intradermal Ad.gp75 provided dramatic reduction in the number of lung metastases (52 +/- 7, 29% of control). Addition of regional (intranasal delivery to the lung) Ad.IL2 to intradermal Ad.gp75 preimmunization 1 day following tumor challenge provided further protection (18 +/- 6, 10% of control). Depletion of CD4+ and CD8+ T-cell subsets effectively blocked the protective effect seen following immunization. Adoptive transfer of macrophage-depleted splenocytes from Ad.gp75-immunized mice similarly afforded significant protection against B16 tumor cell challenge. Further, serum obtained 21 days following Ad.gp75 immunization showed no detectable anti-gp75 antibody by immunoprecipitation. These results suggest that immunization with Ad.gp75 induces cellular immune responses that are capable of rejecting B16 melanoma in a host that is usually tolerant to gp75 antigen.
Adenoviridae↗
The effects of ceftiofur sodium (Naxcel) on bovine oocyte and preimplantation embryonic development assessed by in vitro embryo production techniques.
Ceftiofur sodium is a third-generation cephalosporin antibiotic with broad spectrum bactericidal activity against Gram-positive and Gram-negative bacteria including the beta-lactamase producing strains. In this study, we use in vitro techniques to examine the effects of low and high levels of ceftiofur sodium on the development of bovine oocytes/embryos during oocyte maturation, oocyte fertilization and embryo culture. A total of 8590 oocytes was used in six independent experiments, each in a randomized complete block design. Each replication within each experiment consisted of oocytes from the same abattoir collection of ovaries. There was no difference in embryo development when oocytes were exposed to ceftiofur sodium during oocyte maturation or fertilization at low (10 and 50 micrograms/mL) or high (100 and 200 micrograms/mL) concentrations. However, when fertilized oocytes were exposed to concentrations > or = 50 micrograms/mL during culture, ceftiofur sodium significantly retarded embryo development (e.g. the numbers of ova developing to the morula and blastocyst stages were reduced, and a large proportion of embryos were blocked at the 8-cell stage). We conclude that ceftiofur sodium does not appear to have detrimental effects on oocyte maturation and fertilization. However, long term exposure to high dosages of ceftiofur sodium during post-fertilization culture adversely affects embryo development in vitro.
Animals↗
The mouse GalR2 galanin receptor: genomic organization, cDNA cloning, and functional characterization.
The diverse physiological actions of galanin are thought to be mediated through activation of galanin receptors (GalRs). We report the genomic and cDNA cloning of a mouse GalR that possesses a genomic structure distinct from that of GalR1 and encodes a functional galanin receptor. The mouse GalR gene consists of two exons separated by a single intron within the protein-coding region. The splicing site for the intron is located at the junction between the third transmembrane domain and the second intracellular loop. The cDNA encodes a 370-amino acid putative G protein-coupled receptor that is markedly different from human GalR1 and rat GalR3 (38 and 57%) but shares high homology with rat GalR2 (94%). In binding studies utilizing membranes from COS-7 cells transfected with mouse GalR2 cDNA, the receptor displayed high affinity (K(D) = 0.47 nM) and saturable binding with 125I-galanin (Bmax = 670 fmol/mg). The radioligand binding can be displaced by galanin and its analogues in a rank order: galanin approximately = M40 approximately = M15 approximately = M35 approximately = C7 approximately = galanin(2-29) approximately = galanin(1-16) >> galanin(10-29) approximately = galanin(3-29), which resembles the pharmacological profile of the rat GalR2. Receptor activation by galanin in COS-7 cells stimulated phosphoinositide metabolism, which was not reversed by pertussis toxin. Thus, the galanin receptor encoded in the cloned mouse GalR gene is the type 2 galanin receptor and is active in both ligand binding and signaling assays.
Amino Acid Sequence↗