MMo(3)Se(3) (M = Li(+), Na(+), Rb(+), Cs(+), NMe(4)(+)) nanowire formation via cation exchange in organic solution.
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Letter↗
Biomedical subjects
Y Wu
Publications and source records attributed to Y Wu.
Complete inhibition of vascular endothelial growth factor (VEGF) activities with a bifunctional diabody directed against both VEGF kinase receptors, fms-like tyrosine kinase receptor and kinase insert domain-containing receptor.
Vascular endothelial growth factor (VEGF) binds to and mediates its activity mainly through two tyrosine kinase receptors, VEGF receptor 1 [or fms-like tyrosine kinase receptor (Flt-1)] and VEGF receptor 2 [or kinase insert domain-containing receptor (KDR)]. Numerous studies have shown that overexpression of VEGF and its receptor plays an important role in tumor-associated angiogenesis and hence in both tumor growth and metastasis. We demonstrated previously that antagonistic antibodies to KDR specifically inhibited VEGF-stimulated receptor activation, cell migration, and endothelial cell mitogenesis. Here we constructed a recombinant bifunctional diabody that is capable of blocking both Flt-1 and KDR from binding to their ligands, including VEGF and placenta growth factor (PlGF). The diabody was expressed in Escherichia coli and purified by single-step affinity chromatography. The diabody retained the capacity to bind both KDR and Flt-1 and effectively blocked interaction between KDR and VEGF, Flt-1 and VEGF, and Flt-1 and PlGF. Furthermore, the diabody is a stronger inhibitor than its parent antibodies to VEGF-stimulated mitogenesis of human endothelial cells, as well as both VEGF- and PlGF-induced migration of human leukemia cells. Taken together, our results suggest that dual receptor blockade with the bifunctional diabody may prove to be a more efficient approach in inhibiting VEGF-stimulated angiogenesis.
3T3 Cells↗
p160 Bcr mediates platelet-derived growth factor activation of extracellular signal-regulated kinase in vascular smooth muscle cells.
BACKGROUND: The human Bcr gene was originally identified by its presence in the chimeric Bcr/Abl oncogene, which is causative for chronic myeloblastic leukemia. Because Bcr encodes a serine/threonine protein kinase, we studied its kinase activity and determined the role of Bcr in the PDGF signaling pathway to ERK1/2 activation and DNA synthesis in rat aortic smooth muscle cells (RASMCs). METHODS AND RESULTS: In RASMCs, platelet-derived growth factor-BB (PDGF) stimulated Bcr kinase activity, with a maximum at 1 minute. Because phosphatidylinositol 3'-kinase (PI3-K) is essential for Bcr/Abl leukemogenesis, we evaluated the role of mouse PDGF-beta-receptor binding sites for PI3-K (Y708, Y719) and for phospholipase C-gamma (Y977, Y989) in PDGF-mediated Bcr kinase activation. The mutant PDGF receptor Y708F/Y719F but not Y977F/Y989F showed significantly reduced Bcr kinase activity. To determine the role of Bcr in PDGF-mediated signal transduction events leading to ERK1/2 and its downstream Elk1 transcription activation, wild-type (WT) and kinase-negative (KN) Bcr were transiently expressed in RASMCs. Bcr WT enhanced, whereas Bcr KN inhibited, PDGF-stimulated ERK1/2 and Elk1 transcriptional activity. Overexpression of Bcr also enhanced PDGF-induced Ras/Raf-1 activity and DNA synthesis, but this regulation is independent of the kinase activity of Bcr. Finally, we found that Bcr expression was increased in the neointimal layer after balloon injury of rat carotid artery. CONCLUSIONS: These results demonstrated the importance of Bcr in PDGF-mediated events, such as activation of Ras, Raf-1, ERK1/2, and Elk1, and stimulation of DNA synthesis.
Animals↗
Calmodulin kinase and a calmodulin-binding 'IQ' domain facilitate L-type Ca2+ current in rabbit ventricular myocytes by a common mechanism.
1. Ca2+-calmodulin-dependent protein kinase II (CaMK) and a calmodulin (CaM)-binding 'IQ' domain (IQ) are both implicated in Ca2+-dependent regulation of L-type Ca2+ current (I(Ca)). We used an IQ-mimetic peptide (IQmp), under conditions in which CaMK activity was controlled, to test the relationship between these CaM-activated signalling elements in the regulation of L-type Ca2+ channels (LTCCs) and I(Ca) in rabbit ventricular myocytes. 2. A specific CaMK inhibitory peptide nearly abolished I(Ca) facilitation, but the facilitation was 'rescued' by cell dialysis with IQmp. 3. IQmp significantly enhanced I(Ca) facilitation and slowed the fast component of I(Ca) inactivation, compared with an inactive control peptide. Neither effect could be elicited by a more avid CaM-binding peptide, suggesting that generalized CaM buffering did not account for the effects of IQmp. 4. I(Ca) facilitation was abolished and the fast component of inactivation eliminated by ryanodine, caffeine or thapsigargin, suggesting that the sarcoplasmic reticulum (SR) is an important source of Ca2+ for I(Ca) facilitation and inactivation. IQmp did not restore I(Ca) facilitation under these conditions. 5. Engineered Ca2+-independent CaMK and IQmp each markedly increased LTCC open probability (P(o)) in excised cell membrane patches. The LTCC P(o) increases with CaMK and IQmp were non-additive, suggesting that CaMK and IQmp are components of a shared signalling pathway. 6. Both CaMK and IQmp induced a modal gating shift in LTCCs that favoured prolonged openings, indicating that CaMK and IQmp affect LTCCs through a common biophysical mechanism. 7. These findings support the hypothesis that CaMK is required for physiological I(Ca) facilitation in cardiac myocytes. Both CaMK and IQmp were able to induce a modal gating shift in LTCCs, suggesting that each of these signalling elements is important for Ca2+-CaM-dependent LTCC facilitation in cardiac myocytes.
Algorithms↗
Assessing the heritability of attentional networks.
BACKGROUND: Current efforts to study the genetics of higher functions have been lacking appropriate phenotypes to describe cognition. One of the problems is that many cognitive concepts for which there is a single word (e.g. attention) have been shown to be related to several anatomical networks. Recently we have developed an Attention Network Test (ANT) that provides a separate measure for each of three anatomically defined attention networks. In this small scale study, we ran 26 pairs of MZ and DZ twins in an effort to determine if any of these networks show sufficient evidence of heritability to warrant further exploration of their genetic basis. RESULTS: The efficiency of the executive attention network, that mediates stimulus and response conflict, shows sufficient heritability to warrant further study. Alerting and overall reaction time show some evidence for heritability and in our study the orienting network shows no evidence of heritability. CONCLUSIONS: These results suggest that genetic variation contributes to normal individual differences in higher order executive attention involving dopamine rich frontal areas including the anterior cingulate. At least the executive portion of the ANT may serve as a valid endophenotype for larger twin studies and subsequent molecular genetic analysis in normal subject populations.
Adolescent↗
CHIP is a U-box-dependent E3 ubiquitin ligase: identification of Hsc70 as a target for ubiquitylation.
Proper folding of proteins (either newly synthesized or damaged in response to a stressful event) occurs in a highly regulated fashion. Cytosolic chaperones such as Hsc/Hsp70 are assisted by cofactors that modulate the folding machinery in a positive or negative manner. CHIP (carboxyl terminus of Hsc70-interacting protein) is such a cofactor that interacts with Hsc70 and, in general, attenuates its most well characterized functions. In addition, CHIP accelerates ubiquitin-dependent degradation of chaperone substrates. Using an in vitro ubiquitylation assay with recombinant proteins, we demonstrate that CHIP possesses intrinsic E3 ubiquitin ligase activity and promotes ubiquitylation. This activity is dependent on the carboxyl-terminal U-box. CHIP interacts functionally and physically with the stress-responsive ubiquitin-conjugating enzyme family UBCH5. Surprisingly, a major target of the ubiquitin ligase activity of CHIP is Hsc70 itself. CHIP ubiquitylates Hsc70, primarily with short, noncanonical multiubiquitin chains but has no appreciable effect on steady-state levels or half-life of this protein. This effect may have heretofore unanticipated consequences with regard to the chaperoning activities of Hsc70 or its ability to deliver substrates to the proteasome. These studies demonstrate that CHIP is a bona fide ubiquitin ligase and indicate that U-box-containing proteins may comprise a new family of E3s.
Animals↗
Immunogene therapy of tumors with vaccine based on Xenopus homologous vascular endothelial growth factor as a model antigen.
Overcoming immune tolerance of the growth factors associated with tumor growth should be a useful approach to cancer therapy by active immunity. We used vascular endothelial growth factor (VEGF) as a model antigen to explore the feasibility of the immunogene tumor therapy with a vaccine based on a single xenogeneic homologous gene, targeting the growth factors associated with angiogenesis. To test this concept, we constructed a plasmid DNA encoding Xenopus homologous VEGF (XVEGF-p) and control vectors. We found that immunogene tumor therapy with a vaccine based on XVEGF was effective at both protective and therapeutic antitumor immunity in several tumor models in mice. VEGF-specific autoantibodies in sera of mice immunized with XVEGF-p could be found in Western blotting analysis and ELISA assay. The purified immunoglobulins were effective at the inhibition of VEGF-mediated endothelial cell proliferation in vitro, and at antitumor activity and the inhibition of angiogenesis by adoptive transfer in vivo. The elevation of VEGF in the sera of the tumor-bearing mice could be abrogated with XVEGF-p immunization. The antitumor activity and production of VEGF-specific autoantibodies, significantly elevated IgG1 and IgG2b, could be abrogated by the depletion of CD4(+) T lymphocytes. The observations may provide a vaccine strategy for cancer therapy through the induction of autoimmunity against the growth factors associated with tumor growth in a cross reaction with single xenogeneic homologous gene and may be of importance in the further exploration of the applications of other xenogeneic homologous genes identified in human and other animal genome sequence projects in cancer therapy.
Animals↗
Inhibition of both paracrine and autocrine VEGF/ VEGFR-2 signaling pathways is essential to induce long-term remission of xenotransplanted human leukemias.
Antiangiogenic agents block the effects of tumor-derived angiogenic factors (paracrine factors), such as vascular endothelial growth factor (VEGF), on endothelial cells (EC), inhibiting the growth of solid tumors. However, whether inhibition of angiogenesis also may play a role in liquid tumors is not well established. We recently have shown that certain leukemias not only produce VEGF but also selectively express functional VEGF receptors (VEGFRs), such as VEGFR-2 (Flk-1, KDR) and VEGFR1 (Flt1), resulting in the generation of an autocrine loop. Here, we examined the relative contribution of paracrine (EC-dependent) and autocrine (EC-independent) VEGF/VEGFR signaling pathways, by using a human leukemia model, where autocrine and paracrine VEGF/VEGFR loops could be selectively inhibited by neutralizing mAbs specific for murine EC (paracrine pathway) or human tumor (autocrine) VEGFRs. Blocking either the paracrine or the autocrine VEGF/VEGFR-2 pathway delayed leukemic growth and engraftment in vivo, but failed to cure inoculated mice. Long-term remission with no evidence of disease was achieved only if mice were treated with mAbs against both murine and human VEGFR-2, whereas mAbs against human or murine VEGFR-1 had no effect on mice survival. Therefore, effective antiangiogenic therapies to treat VEGF-producing, VEGFR-expressing leukemias may require blocking both paracrine and autocrine VEGF/VEGFR-2 angiogenic loops to achieve remission and long-term cure.
Animals↗
Expression and secretion of a larval-specific chitinase (family 18 glycosyl hydrolase) by the infective stages of the parasitic nematode, Onchocerca volvulus.
A recently reported chitinase gene, expressed in the infective, third-stage (L3) larvae of the human filarial parasite Onchocerca volvulus, belongs to the family 18 glycosyl hydrolases and has been designated Ov-chi-1. The gene product of Ov-chi-1 is chitinolytic. Allosamidin ablates activity of the native enzyme in a dose-dependent manner but did not significantly inhibit the moulting of L3 larvae. Mono-specific antibodies were used to characterize Ov-CHI-1 as a 60-kDa protein expressed almost exclusively in L3 stages. Immunoelectron microscopy showed that Ov-CHI-1 expression is initiated in late L2 larvae and increases markedly in infective, L3 larvae. It is synthesized exclusively in the glandular esophagus and stored within discrete secretory granules. Secretion occurs through de-granulation during post-infective development, and the primary route of transport appears to be via the pseudo-coelom. An orthologue of Ov-chi-1 was detected in Caenorhabditis elegans by BLAST analysis. It is constitutively expressed at a low level and is overexpressed in dauer larvae and embryonated eggs. It is chitinolytic. We conclude that Ov-CHI-1 is a highly stage-specific enzyme that may have a role in infectivity of the parasite, aiding escape from the vector or participating in early post-infective migration and/or development. The identification of an orthologue in C. elegans opens the way for further studies into the biological function(s) of this intriguing parasite product.
Amino Acid Sequence↗
Design and synthesis of glycolic and mandelic acid derivatives as factor Xa inhibitors.
A series of glycolic and mandelic acid derivatives was synthesized and investigated for their factor Xa inhibitory activity. These analogues are highly potent and selective inhibitors against fXa. In a rabbit deep vein thrombosis model, compound 26 showed significant antithrombotic effects (81% inhibition of thrombus formation) at 1.1 microM plasma concentration following intravenous administration.
Acetanilides↗
Delayed hypersensitivity skin testing to mumps and Candida albicans antigens is normal in middle-aged HTLV-I- and-II-infected U.S. cohorts.
It has been reported that human T cell lymphotropic virus (HTLV)-I-infected persons in Japan have decreased delayed hypersensitivity skin test reactivity to tuberculin purified protein derivative (PPD), but HTLV-I- or -II-infected persons do not generally develop opportunistic infections. We administered standardized intradermal testing with PPD, mumps, and Candida albicans antigens to 31 HTLV-I, 48 HTLV-II, and 143 seronegative subjects in the United States. Reactivity at 48 hr was compared among the three groups. Response rates to PPD were very low in all subjects. Fifty-five percent of seronegative subjects did not react to mumps antigen, compared with 55% of HTLV-I [adjusted odds ratio (OR) = 0.79, 95% confidence interval (CI) 0.27-2.33] and 38% of HTLV-II (OR = 0.73, 95% CI 0.33-1.64). Fifty-one percent of seronegatives did not react to Candida albicans antigen, compared with 34% of HTLV-I (OR = 0.37, 95% CI 0.15-0.93) and 46% of HTLV-II (OR = 0.71, 95% CI 0.34-1.52). Anergy was present in 33% of seronegatives, 28% of HTLV-I (OR = 0.60, 95% CI 0.20-1.78), and 19% of HTLV-II (OR = 0.56, 95% CI 0.22-1.44). HTLV-I- and -II-infected persons appear to have intact delayed hypersensitivity skin test responses to mumps and Candida albicans antigens.
Adult↗
Developmentally regulated expression of GABA receptor rho1 and rho2 subunits, L7 and cone-rod homeobox (CRX) genes in mouse retina.
In this study, we compared the temporal expression pattern of four retinal genes; rho1 and rho2 that encode subunits of GABA(c) receptors, L7 that encodes Purkinje cell protein and CRX that encodes the cone-rod homeobox transcription factor. A reverse-transcription-polymerase chain reaction (RT-PCR) strategy that generated a linear correlation between the amount of retinal RNA and the amount of amplified product was used to quantify transcripts from each gene. Results with this method showed that the rho1 and L7 have similar developmental patterns. Both exhibit basal level expression before P7. From P7 to P20, the RNA levels for both genes were increased about 12-fold. After P20, the RNA levels remained unchanged. Compared to rho1 and L7, expression of rho2 began later, since the rho2 RNA could not be detected until P10. At P10, the rho2 RNA level was about 10% of its level at P35. Expression of rho2 reached its peak at a later developmental stage compared to that of rho1 and L7. The different temporal patterns were confirmed by co-amplification of rho1, rho2, and L7 in a single PCR tube. CRX RNA was detected at embryonic day 15 (E15) and increased progressively, in agreement with a prior study using in situ hybridization. These data, combined with evidence that the tissue distribution of rho1 and L7 RNA in the CNS are similar, indicates that rho1 and L7 may share common transcriptional regulatory elements. Furthermore, the difference in the timing of rho subunit expression suggests that the subunit composition of GABA(c) receptors vary during retinal development.
Aging↗
Selective transcription and modulation of resting T cell activity by preintegrated HIV DNA.
The quiescent nature of most peripheral T cells poses an effective limitation to human immunodeficiency virus (HIV) replication and, in particular, to viral integration into the host chromatin. Two HIV proteins, Nef and Tat, increase T cell activity, but a requirement of integration for viral gene expression would preclude a role for these proteins in resting cells. Here, we report that HIV infection leads to selective transcription of the nef and tat genes before integration. This preintegration transcription in quiescent cells leads to increased T cell activation and viral replication.
Anti-HIV Agents↗
Loss of presenilin 1 is associated with enhanced beta-catenin signaling and skin tumorigenesis.
Presenilin 1 (PS1) is required for the proteolytic processing of Notch and the beta-amyloid precursor protein (APP), molecules that play pivotal roles in cell-fate determination during development and Alzheimer's disease pathogenesis, respectively. In addition, PS1 interacts with beta-catenin and promotes its turnover through independent mechanisms. Consistent with this activity, we report here that PS1 is important in controlling epidermal cell proliferation in vivo. PS1 knockout mice that are rescued through neuronal expression of human PS1 transgene develop spontaneous skin cancers. PS1-null keratinocytes exhibit higher cytosolic beta-catenin and beta-catenin/lymphoid enhancer factor-1/T cell factor (beta-catenin/LEF)-mediated signaling. This effect can be reversed by reintroducing wild-type PS1, but not a PS1 mutant active in Notch processing but defective in beta-catenin binding. Nuclear beta-catenin protein can be detected in tumors. Elevated beta-catenin/LEF signaling is correlated with activation of its downstream target cyclin D1 and accelerated entry from G(1) into S phase of the cell cycle. This report demonstrates a function of PS1 in adult tissues, and our analysis suggests that deregulation of beta-catenin pathway contributes to the skin tumor phenotype.
Animals↗
Vezf1/DB1 is an endothelial cell-specific transcription factor that regulates expression of the endothelin-1 promoter.
Coordinated gene regulation within the vascular endothelium is required for normal cardiovascular patterning during development and for vascular homeostasis during adulthood, yet little is known about the mechanisms that regulate endothelial transcriptional events. Vascular endothelial zinc finger 1 (Vezf1)/DB1 is a recently identified zinc finger-containing protein that is expressed specifically within endothelial cells during development. In this report, we demonstrate that Vezf1/DB1 is a nuclear localizing protein that potently and specifically activates transcription mediated by the human endothelin-1 promoter, in a Tax-independent manner, in transient transfection assays. Using a combination of deletion mutagenesis and electrophoretic mobility shift assays, a novel Vezf1/DB1-responsive element was localized to a 6-base pair (bp) motif, ACCCCC, located 47 bp upstream of the endothelin-1 transcription start site. Recombinant Vezf1/DB1 also bound to this sequence, and a 2-bp mutation in this element abolished Vezf1/DB1 responsiveness by the endothelin-1 promoter. Vezf1/DB1 could be identified with a specific antibody in nuclear complexes from endothelial cells that bound to this element. Regulation of endothelin-1 promoter activity by Vezf1/DB1 provides a mechanism for endothelin-1 expression in the vascular endothelium during development and to maintain vascular tone; Vezf1/DB1 itself is a candidate transcription factor for modifying endothelial cell phenotypes in order to appropriately assemble and maintain the cardiovascular system.
3T3 Cells↗
Efforts toward expansion of the genetic alphabet: replication of DNA with three base pairs.
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Base Pair Mismatch↗
Cross-linking of EphB6 resulting in signal transduction and apoptosis in Jurkat cells.
Eph kinases are the largest family of receptor tyrosine kinases (RTK), and their ligands are cell surface molecules. The known functions of Eph kinases are mainly pattern formation in the CNS. Although several Eph kinases are expressed at high levels in hemopoietic cells and in the thymus, we have no knowledge of the functions of any Eph kinase in the immune system. In this study, we have demonstrated that an Eph kinase, EphB6, was expressed at high levels in Jurkat leukemic T cells. Co-cross-linking of EphB6 and CD3 led to an altered profile of lymphokine secretion along with proliferation inhibition of Jurkat cells. The cells subsequently underwent Fas-mediated apoptosis. Although EphB6 has no intrinsic kinase activity, its cross-linking triggered general protein tyrosine phosphorylation in Jurkat cells. EphB6 was found to associate with a number of molecules in the signaling pathways, notably Cbl. EphB6 cross-linking resulted in Cbl dephosphorylation and dissociation from Src homology 2 domain-containing tyrosine phosphatase-1 (SHP-1). Our results show that EphB6 has important functions in T cells, and it can transduce signals into the cells via proteins it associates with.
Animals↗
Increased entry into the IFN-gamma effector pathway by CD4+ T cells selected by I-Ag7 on a nonobese diabetic versus C57BL/6 genetic background.
IFN-gamma-mediated Th1 effects play a major role in the pathogenesis of autoimmune diabetes in nonobese diabetic (NOD) mice. We analyzed functional responses of CD4(+) T cells from NOD and B6.G7 MHC congenic mice, which share the H2(g7) MHC region but differ in their non-MHC genetic background. T cells from each strain proliferated equally to panstimulation with T cell lectins as well as to stimulation with glutamic acid decarboxylase 524-543 (self) and hen egg lysozyme 11-23 (foreign) I-A(g7)-binding peptide epitopes. Despite comparable proliferative responses, NOD CD4(+) T cells had significantly increased IFN-gamma intracellular/extracellular protein and mRNA responses compared with B6.G7 T cells as measured by intracellular cytokine analysis, time resolved fluorometry, and RNase protection assays. The increased IFN-gamma production was not due to an increase in the amount of IFN-gamma produced per cell but to an increase in the number of NOD CD4(+) T cells entering the IFN-gamma-producing pathway. The increased IFN-gamma response in NOD mice was not due to increased numbers of activated precursors as measured by activation/memory markers. B6.G7 lymphoid cells demonstrated an absolute decrease in IFN-gamma mRNA, an increase in IL-4 mRNA production, and a significantly decreased IFN-gamma:IL-4 mRNA transcript ratio compared with NOD cells. CD4(+) T cells from C57BL6 mice also showed significantly decreased IFN-gamma production compared with CD4(+) T cells from NOD.H2(b) MHC-congenic mice (which have an H2(b) MHC region introgressed onto an NOD non-MHC background). Therefore, the NOD non-MHC background predisposes to a quantitatively increased IFN-gamma response, independent of MHC class II-mediated T cell repertoire selection, even when compared with a prototypical Th1 strain.
Animals↗