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Biomedical subjects

David L Williams

Publications and source records attributed to David L Williams.

At least 91 records · Page 5Linked to original sources
PubMed2003

Caveolin-1 does not affect SR-BI-mediated cholesterol efflux or selective uptake of cholesteryl ester in two cell lines.

Free cholesterol (FC) has been reported to efflux from cells through caveolae, which are 50-100 nm plasma membrane pits. The 22 kDa protein caveolin-1 is concentrated in caveolae and is required for their formation. The HDL scavenger receptor BI (SR-BI), which stimulates both FC efflux and selective uptake of HDL-derived cholesteryl ester (CE), has been reported to be concentrated in caveolae, suggesting that this localization facilitates flux of FC and CE across the membrane. However, we found that overexpression of caveolin-1 in Fischer rat thyroid (FRT) cells, which lack caveolin-1 and caveolae, or HEK 293 cells, which normally express very low levels of caveolin-1, did not affect FC efflux to HDL or liposomes. Transient expression of SR-B1 did not affect this result. Similarly, caveolin-1 expression did not affect selective uptake of CE from labeled HDL particles in FRT or HEK 293 cells transfected with SR-BI. We conclude that basal and SR-BI-stimulated FC efflux to HDL and liposomes and SR-BI-mediated selective uptake of HDL CE are not affected by caveolin-1 expression in HEK 293 or FRT cells.

Animals↗
PubMed2003

SR-BI and cholesterol uptake into steroidogenic cells.

Scavenger receptor class B, type I (SR-BI) is a receptor for high-density lipoprotein (HDL) that mediates cellular uptake of HDL cholesteryl ester (HDL CE) and is the major route for cholesterol delivery to the steroidogenic pathway. SR-BI is localized in specialized microvillar channels in the plasma membrane that retain HDL and are sites of selective uptake of HDL CE. The formation of microvillar channels in the adrenal gland requires SR-BI and is regulated by adrenocorticotropin hormone. SR-BI-mediated uptake of HDL CE is a two-step process that requires high-affinity binding of HDL followed by transfer of CE to the membrane. CE uptake is followed by hydrolysis to free cholesterol by a neutral CE hydrolase. In this review, we describe new information on the mechanism of transfer of cholesterol from plasma HDL to the steroidogenic pathway in endocrine cells.

Adrenal Glands↗
PubMed2003

Characterisation of a Rho homologue of Schistosoma mansoni.

The development and survival of the helminth parasite, Schistosoma mansoni, is dependent on its ability to interpret signals from its environment. Currently, little is known about signal transduction in schistosomes. Rho is a member of a super-family of small GTP-binding proteins. Rho is involved in a number of cell signalling pathways with effects on actin cytoskeleton organisation, gene transcription, cell cycle progression, and membrane trafficking. We have cloned an S. mansoni protein (Rho1) that has 71-75% identity and approximately 85% similarity with human Rho A, B, and C proteins. We have optimised expression of recombinant S. mansoni Rho1 protein in Escherichia coli by co-expression with rare tRNAs. Western blot analysis results showed expression of Rho1 protein in adult worm stages especially female worms. In vitro prenylation of recombinant S. mansoni Rho1 determined that, similar to Rho from other organisms, Rho1 is geranynlgeranylated but not farnesylated. A search of the gene database indicates that Rho GTPases exist as a small family in S. mansoni including orthologues of Rho, Cdc42, and Rac. These data suggest that S. mansoni Rho1 plays a role in signalling in adult worms, especially females.

Amino Acid Sequence↗
PubMed2003

Selecting selective suppressors of selective uptake.

Scavenger receptor BI (SR-BI) is a high-density lipoprotein (HDL) receptor that mediates the selective uptake of HDL cholesteryl ester (CE) and the bidirectional flux of free cholesterol (FC). The identification of selective uptake inhibitors holds promise for mechanistic studies of SR-BI and for discovery of pharmaceuticals useful in therapy of atherosclerosis.

Animals↗
PubMed2003

Biology and pathology of interactions between fungi and invertebrates.

Having started this review with Di Bassi's work on a fungus destroying the French silkworm industry 160 years ago, the article finishes with a fungus being rendered more pathogenic through genetic engineering to kill an invertebrate. Yet, even while using a fungus as a biological weapon, one needs to be careful not to gun down innocent civilian targets alongside the enemy! It may be argued that this article will have little or no immediate clinical significance for the veterinarian involved in exotic species. And yet it is hoped that it will have provided an interesting and informative (not to say exotic!) introduction to the important area of mycotic disease in exotic species.

Animals↗
PubMed2003

Oral biology and disease in invertebrates.

It is hoped that this article has shown some of the amazing breadth of research into invertebrate mouthpart anatomy, development and pathology, and its relation to important disease processes that impinge on fields as diverse as environmental toxicology and antimalarial therapeutics, while also highlighting the need for further study in this field.

Animals↗
PubMed2003

Differential production in vitro of antigen specific IgG1, IgG3 and IgA: a study in Schistosoma haematobium infected individuals.

This study has evaluated the individual control of isotype production and the influence of external signals that can be experimentally provided in vitro, in antibody responses to two different recombinant Schistosoma antigens (Sh28GST and TPx-1). Peripheral blood mononuclear cells or enriched B cell fractions obtained from S. haematobium infected Senegalese adults were induced to terminal differentiation in vitro. The production of antibody to either antigen was donor-dependent and for each donor it was antigen-dependent. Differentiation to IgG1 and IgG3 production, and possibly IgA, specific to these conserved parasite antigens could be regulated differentially in vitro. Exogenous IL-2 and IL-10 or IL-10 and TGF-beta led to the production of specific IgG3 or IgG1 and/or IgA, respectively. This is the first report on such experimentally induced differential regulation of antigen-specific IgG1 and IgG3. This may have implications in designing protocols for protein based-vaccinations aiming at eliciting antibody responses of certain protective-type isotypes.

Adolescent↗
PubMed2003

Symblepharon with aberrant protrusion of the nictitating membrane in the snowy owl (Nyctea scandiaca).

Two young snowy owl chicks were presented with aberrant protrusion of the nictitating membranes. This was caused by conjunctival adhesions causing symblepharon secondary to a previous septicemia episode. While symblepharon has been noted in birds before, this unusual presentation of the nictitating membrane has not been reported. Surgical intervention ameliorated the clinical signs, allowing vision in one bird by removal of the nictitating membranes, a technique which appeared to have no deleterious effects on the ocular surface.

Animals↗
PubMed2003

Modulation of tissue Toll-like receptor 2 and 4 during the early phases of polymicrobial sepsis correlates with mortality.

OBJECTIVE: To determine whether there was a correlation between induction of polymicrobial sepsis, modulation of tissue Toll-like receptor (TLR) gene, and protein expression and survival outcome. DESIGN: Prospective, randomized animal study. SETTING: University medical school research laboratory. SUBJECTS: Age- and weight-matched ICR/HSD mice. INTERVENTIONS: Sepsis was induced by cecal ligation and puncture (CLP). No-surgery and sham (laparotomy)-operated mice were controls. We also examined tissue TLR2 and TLR4 messenger RNA and TLR4 protein levels in mice treated with an immunomodulator that increases survival in polymicrobial sepsis. In the immunomodulator study, mice were treated with glucan phosphate (50 mg/kg, intraperitoneally) 1 hr before CLP. No-surgery, sham surgery, glucan + no-surgery, sham surgery + glucan, and CLP groups were employed as controls. MEASUREMENTS AND MAIN RESULTS: Total RNA was isolated from liver, lung, and spleen at 0, 1, 3, 6, 8, and 24 hrs after CLP. TLR gene expression was assessed by reverse transcription-polymerase chain reaction. Tissue TLR4 protein levels were evaluated at 24 hrs by Western blot and immunohistochemistry. CLP sepsis increased (p <.05) liver and lung TLR2 and TLR4 gene expression compared with controls. TLR4 protein concentrations also were increased. Increased TLR2/4 gene and TLR4 protein expression correlated with mortality. Immunoprophylaxis with glucan phosphate increased (p <.001) long-term survival (20% vs. 70%) but inhibited (p <.05) CLP-induced increases in tissue TLR2 and TLR4 messenger RNA expression as well as TLR4 protein expression. CONCLUSIONS: Early increases in TLR2/4 gene and TLR4 protein expression correlated with mortality, whereas blunting TLR gene and protein expression correlated with improved long-term survival. This suggests that early up-regulation of tissue TLR2/4 may play a role in the proinflammatory response and pathophysiology of polymicrobial sepsis.

Adjuvants, Immunologic↗
PubMed2003

A family of highly selective allosteric modulators of the metabotropic glutamate receptor subtype 5.

We have identified a family of highly selective allosteric modulators of the group I metabotropic glutamate receptor subtype 5 (mGluR5). This family of closely related analogs exerts a spectrum of effects, ranging from positive to negative allosteric modulation, and includes compounds that do not themselves modulate mGluR5 agonist activity but rather prevent other family members from exerting their modulatory effects. 3,3'-Difluorobenzaldazine (DFB) has no agonist activity, but it acts as a selective positive allosteric modulator of human and rat mGluR5. DFB potentiates threshold responses to glutamate, quisqualate, and 3,5-dihydroxyphenylglycine in fluorometric Ca2+ assays 3- to 6-fold, with EC50 values in the 2 to 5 microM range, and at 10 to 100 microM, it shifts mGluR5 agonist concentration-response curves approximately 2-fold to the left. The analog 3,3'-dimethoxybenzaldazine (DMeOB) acts as a negative modulator of mGluR5 agonist activity, with an IC50 of 3 microM in fluorometric Ca2+ assays, whereas the analog 3,3'-dichlorobenzaldazine (DCB) does not exert any apparent modulatory effect on mGluR5 activity. However, DCB seems to act as an allosteric ligand with neutral cooperativity, preventing the positive allosteric modulation of mGluRs by DFB as well as the negative modulatory effect of DMeOB. None of these analogs affects binding of [3H]quisqualate to the orthosteric (glutamate) site, but they do inhibit [3H]3-methoxy-5-(2-pyridinylethynyl)pyridine binding to the site for 2-methyl-6-(phenylethynyl)-pyridine, a previously identified negative allosteric modulator. With the use of these compounds, we provide evidence that allosteric sites on GPCRs can respond to closely related ligands with a range of pharmacological activities from positive to negative modulation as well as to neutral competition of this modulation.

Allosteric Regulation↗
PubMed2003

Boudicca, a retrovirus-like long terminal repeat retrotransposon from the genome of the human blood fluke Schistosoma mansoni.

The genome of Schistosoma mansoni contains a proviral form of a retrovirus-like long terminal repeat (LTR) retrotransposon, designated BOUDICCA: Sequence and structural characterization of the new mobile genetic element, which was found in bacterial artificial chromosomes prepared from S. mansoni genomic DNA, revealed the presence of three putative open reading frames (ORFs) bounded by direct LTRs of 328 bp in length. ORF1 encoded a retrovirus-like major homology region and a Cys/His box motif, also present in Gag polyproteins of related retrotransposons and retroviruses. ORF2 encoded enzymatic domains and motifs characteristic of a retrovirus-like polyprotein, including aspartic protease, reverse transcriptase, RNase H, and integrase, in that order, a domain order similar to that of the gypsy/Ty3 retrotransposons. An additional ORF at the 3' end of the retrotransposon may encode an envelope protein. Phylogenetic comparison based on the reverse transcriptase domain of ORF2 confirmed that Boudicca was a gypsy-like retrotransposon and showed that it was most closely related to CsRn1 from the Oriental liver fluke Clonorchis sinensis and to kabuki from Bombyx mori. Bioinformatics approaches together with Southern hybridization analysis of genomic DNA of S. mansoni and the screening of a bacterial artificial chromosome library representing approximately 8-fold coverage of the S. mansoni genome revealed that numerous copies of Boudicca were interspersed throughout the schistosome genome. By reverse transcription-PCR, mRNA transcripts were detected in the sporocyst, cercaria, and adult developmental stages of S. mansoni, indicating that Boudicca is actively transcribed in this trematode.

Amino Acid Sequence↗
PubMed2002

Enhancement of scavenger receptor class B type I-mediated selective cholesteryl ester uptake from apoA-I(-/-) high density lipoprotein (HDL) by apolipoprotein A-I requires HDL reorganization by lecithin cholesterol acyltransferase.

The severe depletion of cholesteryl ester (CE) in adrenocortical cells of apoA-I(-/-) mice suggests that apolipoprotein (apo) A-I plays an important role in the high density lipoprotein (HDL) CE selective uptake process mediated by scavenger receptor BI (SR-BI) in vivo. A recent study showed that apoA-I(-/-) HDL binds to SR-BI with the same affinity as apoA-I(+/+) HDL, but apoA-I(-/-) HDL has a decreased V(max) for CE transfer from the HDL particle to adrenal cells. The present study was designed to determine the basis for the reduced selective uptake of CE from apoA-I(-/-) HDL. Variations in apoA-I(-/-) HDL particle diameter, free cholesterol or phospholipid content, or the apoE or apoA-II content of apoA-I(-/-) HDL had little effect on HDL CE selective uptake into Y1-BS1 adrenal cells. Lecithin cholesterol acyltransferase treatment alone or addition of apoA-I to apoA-I(-/-) HDL alone also had little effect. However, addition of apoA-I to apoA-I(-/-) HDL in the presence of lecithin cholesterol acyltransferase reorganized the large heterogeneous apoA-I(-/-) HDL to a more discrete particle with enhanced CE selective uptake activity. These results show a unique role for apoA-I in HDL CE selective uptake that is distinct from its role as a ligand for HDL binding to SR-BI. These data suggest that the conformation of apoA-I at the HDL surface is important for the efficient transfer of CE to the cell.

Animals↗
PubMed2002

SR-BI-directed HDL-cholesteryl ester hydrolysis.

We have examined the metabolic fate of HDL cholesteryl ester (CE) delivered to cells expressing scavenger receptor class B type I (SR-BI). Comparison of SR-BI with a related class B scavenger receptor, CD36, showed a greater uptake and a more rapid and extensive hydrolysis of HDL-CE when delivered by SR-BI. In addition, hydrolysis of HDL-CE delivered by both receptors was via a neutral CE hydrolase. These data indicate that SR-BI, but not CD36, can efficiently direct HDL-CE to a neutral CE hydrolytic pathway. In contrast, LDL-CE was delivered and hydrolyzed equally well by SR-BI and CD36. Hydrolysis of LDL-CE delivered by SR-BI was via a neutral CE hydrolase but that delivered by CD36 occurred via an acidic CE hydrolase, indicating that SR-BI and CD36 deliver LDL-CE to different metabolic pathways. Comparison of inhibitor sensitivities in Y1-BS1 adrenal, Fu5AH hepatoma, and transfected cells suggests that hydrolysis of HDL-CE delivered by SR-BI occurs via cell type-specific neutral CE hydrolases. Furthermore, HDL-CE hydrolytic activity was recovered in a membrane fraction of Y1-BS1 cells. These findings suggest that SR-BI efficiently delivers HDL-CE to a metabolically active membrane compartment where CE is hydrolyzed by a neutral CE hydrolase.

Animals↗
PubMed2002

Stabilization of caveolin-1 by cellular cholesterol and scavenger receptor class B type I.

Caveolae are 50-100 nm plasma membrane invaginations, which function in cell signaling, in transcytosis, and in regulating cellular cholesterol homeostasis. These subcompartments of the plasma membrane are characterized by the presence of caveolin proteins. Recent studies have indicated that caveolae may be involved in the regulation of cellular cholesterol efflux to high-density lipoproteins (HDL), as well as selective cholesteryl ester uptake mediated by scavenger receptor class B type I (SR-BI). In the present studies, we show that caveolin-1 expression in HEK-293T cells has no effect on SR-BI-mediated cellular cholesterol efflux to reconstituted HDL. However, SR-BI-mediated selective cholesteryl ester uptake is significantly inhibited by caveolin-1. Interestingly, we also found that SR-BI, but not CD36, can induce the dramatic stabilization of the caveolin-1 protein, independently of its transcriptional control. On the other hand, caveolin-1 has little effect on SR-BI stability, but clearly increases CD36 stability. Since SR-BI expression has been shown to increase cellular cholesterol levels, we next examined the effect of cholesterol itself on caveolin-1 stabilization and localization. When cells were loaded with cholesterol, we observed the dramatic stabilization of caveolin-1 with significant clustering of caveolin-1 at the cell surface. In addition, a palmitoylation-deficient caveolin-1 mutant was still responsive to cholesterol-induced stabilization, indicating that palmitoylation of caveolin-1 is not required for the cholesterol-induced stabilization of caveolin-1. These results suggest an important role for cholesterol and SR-BI in the regulation of caveolin functioning, especially in cell types such as endothelial cells and macrophages, which can be dramatically affected by changes in their cholesterol content during the development of atherosclerosis.

Animals↗
PubMed2002

A nonpeptide oxytocin receptor antagonist radioligand highly selective for human receptors.

A novel, potent nonpeptide oxytocin receptor antagonist (1-(1-(2-(2,2,2-trifluoroethoxy)-4-(1-methylsulfonyl-4-piperidinyloxy) phenylacetyl)-4-piperidinyl)-3,4-dihydro-2(1H)-quinolinone) has been identified that can be labeled to high specific activity with [35S]. In binding studies, this compound exhibits sub-nanomolar affinity and a high degree of selectivity (900-1800-fold) for human oxytocin receptors compared to human vasopressin receptors. This compound appears suitable for studying the pharmacology of oxytocin receptors in human and nonhuman primate tissues, for which there is currently a paucity of highly selective tools. It may also be useful as a nonlabeled competitor or as a radioligand in autoradiographic studies of oxytocin receptor localization in these tissues.

Animals↗