Biomedical subjects

L Wu

Publications and source records attributed to L Wu.

At least 433 records · Page 24Linked to original sources

PubMed1997

CCR5 levels and expression pattern correlate with infectability by macrophage-tropic HIV-1, in vitro.

Chemokine receptors serve as coreceptors for HIV entry into CD4+ cells. Their expression is thought to determine the tropism of viral strains for different cell types, and also to influence susceptibility to infection and rates of disease progression. Of the chemokine receptors, CCR5 is the most important for viral transmission, since CCR5 is the principal receptor for primary, macrophage-tropic viruses, and individuals homozygous for a defective CCR5 allele (delta32/delta32) are highly resistant to infection with HIV-1. In this study, CCR5-specific mAbs were generated using transfectants expressing high levels of CCR5. The specificity of these mAbs was confirmed using a broad panel of chemokine receptor transfectants, and by their non-reactivity with T cells from delta32/delta32 individuals. CCR5 showed a distinct pattern of expression, being abundant on long-term activated, IL-2-stimulated T cells, on a subset of effector/memory T cells in blood, and on tissue macrophages. A comparison of normal and CCR5 delta32 heterozygotes revealed markedly reduced expression of CCR5 on T cells from the heterozygotes. There was considerable individual to individual variability in the expression of CCR5 on blood T cells, that related to factors other than CCR5 genotype. Low expression of CCR5 correlated with the reduced infectability of T cells with macrophage-tropic HIV-1, in vitro. Anti-CCR5 mAbs inhibited the infection of PBMC by macrophage-tropic HIV-1 in vitro, but did not inhibit infection by T cell-tropic virus. Anti-CCR5 mAbs were poor inhibitors of chemokine binding, indicating that HIV-1 and ligands bind to separate, but overlapping regions of CCR5. These results illustrate many of the important biological features of CCR5, and demonstrate the feasibility of blocking macrophage-tropic HIV-1 entry into cells with an anti-CCR5 reagent.

Alleles↗

PubMed1997

The chemical modification of KCa channels by carbon monoxide in vascular smooth muscle cells.

The chemical modification of big conductance calcium-activated potassium (KCa) channels in rat tail artery smooth muscle cells by carbon monoxide (CO) was investigated using the cell-free single channel recording technique. Exposure of the internal surface of cell membranes to diethyl pyrocarbonate (DEPC) neither affected the characteristics of KCa channels nor modified the stimulatory effect of CO on KCa channels. However, when DEPC was applied to the external surface of cell membranes, the open probability of KCa channels was reduced. The pH and concentration dependence of the effect of DEPC indicated the specific modification of histidine residues. Kinetic analysis suggested that one externally located histidine residue was modified by DEPC. Treatment of the external surface of cell membranes with DEPC abolished the CO-induced increase in the open probability of KCa channels. Likewise, the presence of CO partially protected KCa channels from inhibition by DEPC. Moreover, photooxidation of the histidine residue located on the external membrane surface abolished the CO-induced activation of KCa channels. Our study demonstrates that the CO-induced increase in the open probability of KCa channels may rely specifically on the structure and topological locations of histidine residues.

Animals↗

PubMed1997

Nif1, a novel mitotic inhibitor in Schizosaccharomyces pombe.

In Schizosaccharomyces pombe, the activity of the M-phase-inducing Cdc2/Cdc13 cyclin-dependent kinase is inhibited by Wee1 and Mik1 tyrosine kinases, and activated by Cdc25 and Pyp3 tyrosine phosphatases. Cdc2/Cdc13 activity is also indirectly regulated by the approximately 70 kDa Nim1 (Cdrl) serine/threonine kinase, which promotes mitosis by inhibiting Wee1 via direct phosphorylation. To understand better the function and regulation of Nim1, the yeast two-hybrid system was used to isolate S.pombe cDNA clones encoding proteins that interact with Nim1. Sixteen of the 17 cDNA clones were derived from the same gene, named nif1 + (nim1 interacting factor-1). Nif1 is a novel approximately 75 kDa protein containing a leucine zipper motif. The Nif1-Nim1 interaction requires a small region of Nim1 that immediately follows the N-terminal catalytic domain. This region is required for Nim1 activity both in vivo and in vitro. delta nif1 mutants are approximately 10% smaller than wild type, indicating that Nif1 is involved in inhibiting the onset of mitosis. Consistent with this proposal, overproduction of Nif1 was found to cause a cell elongation phenotype that is very similar to delta nim1 mutants. Nif1 overproduction causes cell cycle arrest in cells that are partly defective for Cdc25 activity, but has no effect in delta nim1 or delta wee1 mutants. Nif1 also inhibits Nim1-mediated phosphorylation of Wee1 in an insect cell expression system. These observations strongly suggest that Nif1 negatively regulates the onset of mitosis by a novel mechanism, namely inhibiting Nim1 kinase.

Amino Acid Sequence↗

PubMed1997

Comparative kinetic analysis and substrate specificity of the tandem catalytic domains of the receptor-like protein-tyrosine phosphatase alpha.

The catalytic activity and substrate specificity of protein-tyrosine phosphatase alpha (PTPalpha) is primarily controlled by the membrane proximal catalytic domain (D1). The membrane distal (D2) domain of PTPalpha by itself is a genuine PTPase, possessing catalytic activity comparable to that of D1 using aryl phosphates as substrates. Surprisingly, kcat and kcat/Km for the D2-catalyzed hydrolysis of phosphotyrosine-containing peptides are several orders of magnitude reduced in comparison with those of D1. Substitution of the putative general acid/base Glu-690 in D2 by an Asp, which is invariably found in the WPD motifs in all cytoplasmic PTPases and all the D1 domains of receptor-like PTPases, only increases the kcat for D2 by 4-fold. Thus the much reduced D2 activity toward peptide substrates may be due to structural differences in the active sites other than the general acid/base. Alternatively, the D2 domain may have a functional active site with a highly stringent substrate specificity. PTPalpha display modest peptide substrate selectivity and are sensitive to charges adjacent to phosphotyrosine. In the sequence context of DADEpYLIPQQG (where pY stands for phosphotyrosine), the minimal sizes recognized by PTPalpha are either ADEpYLI or DADEpY-NH2.

Amino Acid Sequence↗

PubMed1997

The HIV coreceptors CXCR4 and CCR5 are differentially expressed and regulated on human T lymphocytes.

The chemokine receptors CXCR4 and CCR5 function as coreceptors for HIV-1 entry into CD4+ cells. During the early stages of HIV infection, viral isolates tend to use CCR5 for viral entry, while later isolates tend to use CXCR4. The pattern of expression of these chemokine receptors on T cell subsets and their regulation has important implications for AIDS pathogenesis and lymphocyte recirculation. A mAb to CXCR4, 12G5, showed partial inhibition of chemotaxis and calcium influx induced by SDF-1, the natural ligand of CXCR4. 12G5 stained predominantly the naive, unactivated CD26(low) CD45RA+ CD45R0- T lymphocyte subset of peripheral blood lymphocytes. In contrast, a mAb specific for CCR5, 5C7, stained CD26(high) CD45RA(low) CD45R0+ T lymphocytes, a subset thought to represent previously activated/memory cells. CXCR4 expression was rapidly up-regulated on peripheral blood mononuclear cells during phytohemagglutinin stimulation and interleukin 2 priming, and responsiveness to SDF-1 increased simultaneously. CCR5 expression, however, showed only a gradual increase over 12 days of culture with interleukin 2, while T cell activation with phytohemagglutinin was ineffective. Taken together, the data suggest distinct functions for the two receptors and their ligands in the migration of lymphocyte subsets through lymphoid and nonlymphoid tissues. Furthermore, the largely reciprocal expression of CXCR4 and CCR5 among peripheral blood T cells implies distinct susceptibility of T cell subsets to viral entry by T cell line-tropic versus macrophage-tropic strains during the course of HIV infection.

Acquired Immunodeficiency Syndrome↗

PubMed1997

Vascular hypertrophy in experimental diabetes. Role of advanced glycation end products.

The accelerated formation of advanced glycation end products (AGEs) and the overexpression of transforming growth factor beta (TGF-beta) have both been implicated in the pathogenesis of diabetic microvascular and macrovascular complications. Previous studies in our laboratory have demonstrated that the vascular changes in diabetes include hypertrophy of the mesenteric vasculature. To examine the role of AGEs in this process, streptozotocin-induced diabetic rats and control animals were randomized to receive aminoguanidine, an inhibitor of AGE formation, or no treatment. Animals were studied at 7 d, 3 wk, and 8 mo after induction of diabetes. When compared with control animals, diabetes was associated with an increase in mesenteric vascular weight and an increase in media wall/lumen area. By Northern analysis, TGF-beta1 gene expression was increased 100-150% (P < 0.01) and alpha1 (IV) collagen gene expression was similarly elevated to 30-110% compared to controls (P < 0.05). AGEs and extracellular matrix were present in abundance in diabetic but not in control vessels. Treatment of diabetic rats with aminoguanidine resulted in significant amelioration of the described pathological changes including overexpression of TGF-beta1 and alpha1 (IV) collagen. These data implicate the formation of AGEs in TGF-beta overexpression and tissue changes which accompany the diabetic state.

Animals↗

PubMed1997

Rapid loop dynamics of Yersinia protein tyrosine phosphatases.

The Yersinia protein tyrosine phosphatases (PTPase) contain a single and invariant tryptophan (W354) located at one of the hinge positions of the flexible loop (WpD loop), which is essential for catalysis. The wild-type Yersinia PTPase and an active site mutant in which the esential Cys 403 has been replaced by serine (C403S) have been examined using both time-resolved fluorescence anisotropy and steady-state UV resonance Raman (UVRR) spectroscopies. Both enzymes were examined with and without the bound inhibitor arsenate. The UVRR spectra indicate that in solution the ligand-free, wild-type PTPase exists as an equilibrium mixture of two tryptophan rotamer structures with chi2,1 dihedral angles of -4 degrees and -90 degrees. The two rotamers have been attributed to the presence of both "closed" and "open" WpD loop conformers of the ligand-free enzyme. Conversely, the UVRR spectra of the arsenate-ligated, wild-type PTPase and of ligand-free and arsenate-ligated C403S PTPase contain a single W3 band which is correlated to the -4 degrees rotamer of W354, indicating a predominance of the closed WpD loop conformer. The tryptophan fluorescence anisotropy decay measurements of the ligand-bound, wild-type Yersinia PTPase and of both ligation states of the C403S PTPase reveal a single correlation time of 30-48 ns due to the rotational motion of the protein, while the ligand-free, wild-type PTPase is found to have two correlation times of 31 and 3.8 ns. The 3.8 ns correlation time of the ligand-free enzyme is attributed to the hinged movement of the WpD loop which contains W354. These results indicate that under physiological conditions, the nonligated, wild-type Yersinia PTPase alternates between an open WpD loop and a closed loop form with a rate constant of approximately 2.6 x 10(8) s(-1). We conclude that the rate of WpD loop closure of the wild-type Yersinia PTPase is thus independent of the presence of ligand, whereas in the presence of ligand the rate of opening is dramatically reduced resulting in a closed conformation on ligand binding. In contrast, the ligand-free and ligated C403S PTPase remain in the loop closed configuration over the time course of our dynamic measurements. The lack of WpD loop motion in the C403S PTPase is believed to be due to either a loss of repulsive potential between the anionic thiolate and Asp 356 of the WpD loop and/or the formation of a hydrogen bond or water bridged hydrogen bond between Ser 403 and Asp 356.

Protein Conformation↗

PubMed1997

The single sulfur to oxygen substitution in the active site nucleophile of the Yersinia protein-tyrosine phosphatase leads to substantial structural and functional perturbations.

Protein-tyrosine phosphatases (PTPases) feature an essential nucleophilic thiol group which attacks the phosphorus atom in a substrate. A single S to O atom substitution in the nucleophile (via Cys to Ser mutation) renders PTPases catalytically inactive. We suggest that the lack of activity in the Cys to Ser mutant may be caused by structural and/or conformational perturbations in the active site. Yersinia PTPase contains a single tryptophan residue, Trp354, which is invariant among all PTPases and is located in the vicinity of the active site nucleophile Cys403. Thus, Trp354 serves as an intrinsic probe of the PTPase active site conformation. We show that although C403S displays a nearly identical circular dichroism spectrum to that of the wild type enzyme, its ultraviolet spectrum in the region attributed to Trp is significantly different from that of the wild-type enzyme. In addition, the intrinsic fluorescence intensity of C403S is enhanced 3-fold and exhibits different ionic strength dependency from that of the wild-type enzyme. Trp354 also has different accessibilities to quenchers in the wild-type and the C403S mutant PTPases. Furthermore, unfolding experiments demonstrate that the structure of C403S is significantly less stable than the wild-type PTPase and displays a different sensitivity to urea and guanidine hydrochloride. Finally, binding of tungstate enhances the fluorescence of the wild-type Yersinia PTPase with a Kd of 55 microM, whereas binding of tungstate quenches the fluorescence of the C403S mutant with a Kd of 690 microM. Collectively, these results indicate that the single sulfur to oxygen change in the active site nucleophile leads to substantial structural/conformational and functional alterations in the Yersinia PTPase.

Binding Sites↗

PubMed1997

Chemokine receptor usage by human eosinophils. The importance of CCR3 demonstrated using an antagonistic monoclonal antibody.

Chemokines bind and signal through G-protein coupled seven transmembrane receptors. Various chemokine receptors are expressed on leukocytes, and these may impart selective homing of leukocyte subsets to sites of inflammation. Human eosinophils express the eotaxin receptor, CCR3, but respond to a variety of CC chemokines apart from eotaxin, including RANTES, monocyte chemotactic protein (MCP)-2, MCP-3, and MCP-4. Here we describe a mAb, 7B11, that is selective for CCR3 and has the properties of a true receptor antagonist. 7B11 blocked binding of various radiolabeled chemokines to either CCR3 transfectants, or eosinophils. Pretreatment of eosinophils with this mAb blocked chemotaxis and calcium flux induced by all CCR3 ligands. In all individuals examined, including allergic and eosinophilic donors, > 95% of the response of eosinophils to eotaxin, RANTES, MCP-2, MCP-3, and MCP-4 was shown to be mediated through CCR3. The IL-8 receptors, particularly CXCR2, were induced on IL-5 primed eosinophils, however these eosinophils responded to CC chemokines in the same manner as unprimed eosinophils. These results demonstrate the importance of CCR3 for eosinophil responses, and the feasibility of completely antagonizing this receptor.

Antibodies, Monoclonal↗

PubMed1997

Artificial neural networks: application to electrical stimulation of the human nervous system.

Artificial neural networks are used increasingly in applications such as graphic pattern recognition, which are difficult to address with conventional statistical methods. In the management of chronic pain, graphic methods are used routinely; patients describe their patterns of pain using "pain drawings." The authors have previously reported an automated, computerized pain drawing methodology, which has been used by patients with implanted spinal cord stimulators to represent a technical goal of the procedure, the overlap of pain by stimulation paresthesias. Standard linear discriminant statistical methods have shown associations between stimulation parameters and electrode positions as independent variables and technical outcome and relief of pain as dependent variables. The authors have applied artificial neural networks to the problem of optimizing implanted stimulator adjustment. A data set of 3000 electrode combinations obtained in 41 patients was used to develop a linear discriminant statistical model on a mainframe computer and to train artificial neural networks on a personal computer. The performance of these two systems on a new data set obtained in 10 patients was compared with that of human "experts." The best neural network model was marginally better than the linear discriminant model; the variance in patient ratings was predicted by these models to a degree that the human experts were unable to predict. The authors anticipate expanding the role of these models and incorporating them into expert systems for clinical use.

Journal Article↗

PubMed1997

Transforming growth factor beta 3 (TGF beta 3) accelerates wound healing without alteration of scar prominence. Histologic and competitive reverse-transcription-polymerase chain reaction studies.

BACKGROUND: Transforming growth factor (TGF) beta 3 is a new isoform of the TGF beta superfamily and is presumed to play an important role in wound repair and scarring. OBJECTIVE: To examine the effects of TGF beta 3 on wound healing and on reducing scarring. DESIGN AND INTERVENTIONS: Dermal ulcers were created on the ears of 75 anesthetized young female rabbits. Either TGF beta 3 or vehicle was applied topically to the wounds. Wounds were bisected and analyzed histologically at postwounding day 7. A second group of wounds was treated with topical TGF beta 3 and TGF beta 2 or vehicle at days 0 and 3 and harvested at days 21 through 42 as an excessive scarring model. The third group of wounds was treated with TGF beta 1, TGF beta 2, and TGF beta 3 and vehicle. The granulation tissue was harvested at day 7, and cellular RNA was extracted for performing competitive reverse-transcription polymerase chain reaction. MAIN OUTCOME MEASUREMENT: The amount of new epithelium and granulation tissue was measured in TGF beta 3- and vehicle-treated wounds. The hypertrophic index was calculated for scarring wounds treated with TGF beta 2 and TGF beta 3 or vehicle. Levels of TGF beta 1 messenger RNA were measured in those wounds that were treated with TGF beta 1, TGF beta 2, and TGF beta 3 and in their controls. RESULTS: The use of TGF beta 3 (0.3-0.75 microgram per wound) increased granulation tissue formation by more than 100% (P < .005). Epithelialization showed a biphase, either increasing 30% (P < .04) or decreasing 25% (P < .001) dependent on dose. No significant difference in the hypertrophic index was noted in TGF beta 3-treated wounds compared with controls. Levels of TGF beta 1 messenger RNA increased (7.1- to 14.9-fold) in those wounds treated with TGF beta s compared with controls at day 7. CONCLUSIONS: Exogenous TGF beta 3 displays substantial vulnerary properties in wound healing and may be useful in treating nonhealing wounds. However, the observation that TGF beta 3 can reduce scarring was not confirmed in this study, and the messenger RNA level in response to TGF beta 3 suggests that it behaves similarly to TGF beta 1.

Animals↗

PubMed1997

Dendritic cells and T lymphocytes: developmental and functional interactions.

Dendritic cells (DCs) are specialized for presentation of antigen to T cells and are essential for primary T cell activation. Although DCs are generally considered to be myeloid derived, we now have evidence that a subgroup are of lymphoid origin. In particular, the DCs of the adult mouse thymus appear to be derived from the same early, lymphoid-restricted precursor cells that generate T lymphocytes. Purified early thymic T precursors have the capacity to produce T cells, B cells, NK cells and DCs, but not myeloid cells, on transfer to irradiated recipients. They also produce thymic DCs on culture with a mix of cytokines; this mix does not include GM-CSF, needed to generate myeloid-derived DCs. A subgroup of DCs in other lymphoid organs, which like thymic DCs express CD8 as an alpha alpha homodimer, may likewise be of lymphoid origin. These CD8+ DCs in mouse spleen differ functionally from the conventional CD8+ DCs. CD8+ DCs efficiently activate CD4+ T cells but then kill them via Fas ligand on the DC surface. CD8+ DCs efficiently recruit CD8+ T cells into the cell cycle, but their proliferation is then restricted by an inadequate production of interleukin 2. This subgroup of CD8+ DCs therefore appears to have a regulatory role.

Animals↗

PubMed1997

Selenium-induced growth reduction in Brassica land races considered for phytoremediation.

Brassica species considered for use in selenium (Se) phytoremediation need to accumulate large amounts of Se to be successful. Retarded plant growth and impaired protein synthesis are common symptoms for plants grown under seleniferous soils. Selenium accumulation by different land races of Brassica juncea (L.) Czern and Coss and one land race of Brassica carinata was investigated in Se-enriched water and soil cultures containing 2 mg Se kg-1. Effects of Se concentration in the root environment on the assimilation of Se, leaf surface area, dry matter yield, total leaf protein concentration, and free seleno-amino acid concentrations were analyzed for plants grown in Se-laden media. In water culture, shoot Se concentrations among the land races ranged from 501 to 1017 mg Se kg-1 dry matter (DM), and in plants grown in Se-laden soil, concentrations ranged from 407 to 769 mg Se kg-1 DM. Land races grown with Se exhibited decreases in dry matter yields from 12 to 23% and in leaf surface area from 5 to 26% compared to the same land races grown without Se. Protein content was significantly correlated both with shoot Se concentrations (r = 0.746, P < 0.001) and with leaf surface area (r = 0.446, P < 0.01) for all land races grown in Se-enriched water culture. There was also a significant correlation (r = 0.767, P < 0.001) between total Se assimilation and shoot protein for all land races. Free selenomethionine was detected for plants grown with Se and ranged from 92 to 958 ng g-1 DM. Other seleno-amino acids, Se-methyl-selenocysteine and selenocysteine, were not consistently detected as free amino acids in the different land races. Although visual symptoms of Se toxicity were not observed in the Brassica species, dry matter yield, leaf surface, and total shoot protein decreased, depending on the land races tested.

Amino Acids↗

PubMed1997

Effects of elevated selenium and salinity concentrations in root zone on selenium and salt secretion in saltgrass (Distichlis spicata L.).

The effects of elevated selenium (Se) and salinity concentrations in the root zone on Se and salt secretion and accumulation were studied for an inland (Kesterson) and a coastal (Bodega Bay) saltgrass in sand culture and under greenhouse conditions. The results of this study indicate that the secretory mechanism of the saltgrass exhibits high ion specificity. Plants of both ecotypes were more efficient at Cl- and Na+ secretion than at SO4(2-) and Se secretion, suggesting that the saltgrass secretion mechanism is adapted primarily to saline environments high in NaCl. The saltgrass plants of the Kesterson ecotype secreted more Se than the Bodega Bay plants when treated with Se alone. However, the Bodega Bay plants secreted more Se than the Kesterson plants when the plants were treated with Se+NaCl. These differences reflect a ecotypical difference in which the Kesterson plants are more adaptive to the seleniferous soil, and the Bodega Bay plants are more adaptive to a coastal saline soil high in NaCl. Sulfate availability inhibited both Se accumulation and Se secretion in the plants of both ecotypes by approximately 98%. Ion secretion molar ratios of Na:Cl were calculated and the results suggest that Na+ secretion is dependent on the availability of Cl-. Selenium was taken up by plants with little discrimination, and thus it may be regarded as a master of chemical mimicry sharing similar physical and chemical properties with sulfur (S). Selenium and salt accumulation indices and secretion efficiency indices were calculated and found that the accumulation indices were higher for Se than for S, suggesting that Se uptake may be more passive and less regulated by active transport than S. Secretion efficiency was much higher for Na+ and Cl- than for Se and SO4(2-), but the efficiency indices between ecotypes were comparable, suggesting that the secretion mechanism in this species is designed mainly for adaptation of high NaCl concentrations. About 85% of the secreted Se was found in selenate form. This finding suggests that the mechanism of Se translocation and Se secretion in the saltgrass resembles the movement of selenate in xylem found in nonhalophytic plants. Less than 15% of organic Se was detected in the secretions and only trace amount of selenite was detected. Overall, the Se secretion mechanism in saltgrass does not seem to contribute substantial Se speciation in the plant soil system.

Poaceae↗

PubMed1997

Macrophage colony-stimulating factor accelerates wound healing and upregulates TGF-beta1 mRNA levels through tissue macrophages.

Macrophage colony-stimulating factor (M-CSF) is produced by many cell types involved in wound repair, yet it acts specifically on monocytes and macrophages. The monocyte-derived cell is thought to be important in wound healing, but the importance of the role of tissue macrophages in wound healing has not been well defined. Dermal ulcers were created in normal and ischemic ears of young rabbits. Either rhM-CSF (17 microg/wound) or buffer was applied to each wound. Wounds were bisected and analyzed histologically at Days 7 and 10 postwounding. The amounts of epithelial growth and granulation tissue deposition were measured in all wounds. The level of increase of TGF-beta1 mRNA level in M-CSF-treated wounds was examined using competitive RT-PCR. M-CSF increased new granulation tissue formation by 37% (N = 21, P < 0.01) and 50% (P < 0.01) after single and multiple treatments, respectively, in nonischemic wounds. TGF-beta1 mRNA levels in rhM-CSF-treated wounds increased 5.01-fold (N = 8) over vehicle-treated wounds under nonischemic conditions. In contrast, no effect could be detected in ischemic wounds treated with rhM-CSF, and these wounds only showed a 1.66-fold increase in TGF-beta1 mRNA levels when compared to ischemic wounds treated with vehicle alone. GAPDH, a housekeeping gene, showed no change. As mesenchymal cells lack receptors for M-CSF, the improved healing of wounds treated with topical rhM-CSF must reflect a generalized enhancement of activation and function of tissue macrophages, as demonstrated by upregulation of TGF-beta. The lack of effect under ischemic conditions suggests that either macrophage activity and/or response to M-CSF is adversely affected under those conditions; this may suggest the pathogenesis of impaired wound healing at the cellular level.

Animals↗

PubMed1997

The direct effect of carbon monoxide on KCa channels in vascular smooth muscle cells.

The vasorelaxation induced by carbon monoxide (CO) has been demonstrated previously. Both a guanosine cyclic monophosphate (cGMP) signalling pathway and cGMP-independent mechanisms have been proposed to be responsible for the vascular action of CO. A direct effect of CO on the activity of calcium-activated K (KCa) channels in vascular smooth muscle cells (SMCs) and the underlying mechanisms were investigated in the present study. It was found that CO hyperpolarized single SMCs isolated from rat tail arteries. The whole-cell outward K+ channel currents in vascular SMCs, but not in neuroblastoma cells, were enhanced by CO. Extracellularly or intracellularly applied CO increased the open probability of single high-conductance KCa channels concentration-dependently without affecting the single channel conductance. Although it did not increase the resting level of intracellular free calcium concentration, CO significantly enhanced the calcium sensitivity of single KCa channels in SMCs. Furthermore, the effect of CO on KCa channels was not mediated by cGMP or guanine nucleotide-binding proteins (G proteins, Gi/Go or Gs) in excised membrane patches. Our results suggest that the direct modulation of high-conductance KCa channels in vascular SMCs by CO may constitute a novel mechanism for the vascular effect of CO.

Animals↗

PubMed1997

Characterization of neutralizing domains on varicella-zoster virus glycoprotein E defined by monoclonal antibodies.

The genome of varicella-zoster virus (VZV), encodes at least six glycoproteins and they elicit the formation of complement-independent, complement-dependent, and non-neutralizing antibody responses. We have used our library of MAbs to VZV glycoprotein E (gE) to determine the neutralizing epitopes of gE, and shown that gE has 3 distinct neutralizing domains. In this report we have used the baculovirus expression system to identify the antigenic domains of gE. We have generated 3 recombinant baculoviruses, expressing the full-length gE and two overlapping truncated forms (the amino-terminal and the carboxy-terminal) of gE. By immuno-fluorescence and immunoblotting we have explored the physical interactions of Mabs to gE on these constructs. Our panel of MAbs revealed 3 district antigenic domains on gE. All MAbs reacted with the full-length gE; MAbs with high titered complement-dependent neutralizing activities reacted with the N-terminal truncated gE; MAbs with low titered or non-neutralizing activities reacted with the C-terminal truncated gE; MAbs with complement-enhanced neutralizing activities reacted with both truncated constructs. However, although the antibody binding in immunofluorescence and immunoblotting was carried out under denatured conditions, whereas the neutralization is under non-denatured conditions, still the antigenic mapping was similar in both conditions.

Antibodies, Viral↗

PubMed1997

Intracoronary adenovirus-mediated transfer of immunosuppressive cytokine genes prolongs allograft survival.

BACKGROUND: Intracoronary transfer and expression of recombinant genes in the intact heart is now feasible. In the transplant setting, local modulation of host immune responses by a genetically modified allograft may offer an attractive alternative to systemic immunosuppression. METHODS: We tested the efficacy and in vivo effect of intracoronary transfer of two immunosuppressive cytokine genes. First-generation E1-deleted adenoviral vectors expressing the Epstein-Barr virus interleukin-10 (AdSvIL10) or human transforming growth factor--beta 1 (AdCMVTGF-beta) were used. Rabbit cardiac allografts were transduced during cold preservation by slow (1 ml/min) intracoronary infusion of 10(10) pfu/gm diluted viral vectors and then implanted heterotopically. Controls included E1-deleted adenovirus (Ad5dI434) and AdCMVLacZ. Beating allografts were collected on day 4 for analysis of gene transfer efficacy and distribution. Additional grafts were used for evaluation of alloreactivity (n = 34). RESULTS: Mean allograft viral uptake was 81% (up to 91%). Polymerase chain reactions and reverse transcription-polymerase chain reactions confirmed the presence and expression of both genes in the grafts. beta-Galactosidase staining in AdCMVLacZ-infected grafts demonstrated efficient gene expression, which was highest (100%) in subepicardial regions. More homogeneous transmyocardial distribution of the transgene (in 25% to 40% of cells) could be achieved by pulsatile slow delivery. Allograft survival was 6.9 +/- 0.9 days in controls (n = 12), 11.1 +/- 1.7 days in AdCMVTGF-beta-infected grafts (n = 11, p < 10(-4)), and 11.2 +/- 3 days in AdSvIL10-infected grafts (n = 11, p < 10(-4)). Histologic scores (blinded) showed significantly (p < 0.005) higher regression coefficients for rejection in controls compared with both cytokine-transduced groups. Perioperative administration of cyclosporine A (INN: ciclosporin) to recipients had no effect on survival of AdCMVTGF-beta-infected grafts but reduced survival of AdSvIL10-infected grafts. CONCLUSIONS: Intracoronary gene transfer of immunosuppressive cytokines to cardiac allografts is efficient and effectively prolongs graft survival. Vectors that would induce long-term expression of such genes may make this approach clinically applicable.

Adenoviridae↗