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Objective : In neuropsychological evaluations, assessing cognitive functioning is often achieved using objective neuropsychological measures, whereas subjective informant reports are typically obtained to determine manifest daily functioning. Informant reports of participant functioning and their associations with objective participant performance on neuropsychological testing have been shown to vary based on informant characteristics. However, associations among informant characteristics, reported functioning, and neuropsychological performance have not been adequately examined with Mexican American or other Hispanic/Latino samples, despite these populations' disproportionately higher rates of dementia due to Alzheimer's disease and related disorders. Method: We examined associations of informant characteristics with informant reports of participant functioning (assessed via the Functional Activities Questionnaire [FAQ]), and potential moderating effects of these characteristics on associations between reported functioning and participant performance on neuropsychological testing, for Mexican American adult participants in the National Alzheimer's Coordinating Center cohort ( n  = 294). Results: Female informants reported significantly worse participant functioning compared to male informants ( p = .035, r = .126). Moreover, significant associations between reported functioning and memory performance were observed for participants with female informants, but not for those with male informants ( p = .024, r = .138). Higher levels of informant education were associated with significantly worse participant functioning ( p = .011, r = .151). However, informant education did not moderate associations between reported functioning and neuropsychological performance ( p s > .05). Conclusions: Compared to male informants, female informants may provide subjective reports of Mexican American participant functioning that more closely corroborate objective participant performance in memory.

Long QT syndrome, either inherited or acquired from drug treatments, can result in ventricular arrhythmia (torsade de pointes) and sudden death. Human ether-a-go-go-related gene (hERG) channel inhibition by drugs is now recognized as a common reason for the acquired form of long QT syndrome. It has been reported that more than 100 known drugs inhibit the activity of the hERG channel. Since 1997, several drugs have been withdrawn from the market due to the long QT syndrome caused by hERG inhibition. Food and Drug Administration regulations now require safety data on hERG channels for investigative new drug (IND) applications. The assessment of compound activity on the hERG channel has now become an important part of the safety evaluation in the process of drug discovery. During the past decade, several in vitro assay methods have been developed and significant resources have been used to characterize hERG channel activities. However, evaluation of compound activities on hERG have not been performed for large compound collections due to technical difficulty, lack of throughput, and/or lack of biological relevance to function. Here we report a modified form of the FluxOR thallium flux assay, capable of measuring hERG activity in a homogeneous 1536-well plate format. To validate the assay, we screened a 7-point dilution series of the LOPAC 1280 library collection and reported rank order potencies of ten common hERG inhibitors. A correlation was also observed for the hERG channel activities of 10 known hERG inhibitors determined in this thallium flux assay and in the patch clamp experiment. Our findings indicate that this thallium flux assay can be used as an alternative method to profile large-volume compound libraries for compound activity on the hERG channel.

The voltage-gated sodium channel Na(v)1.6 plays unique roles in the nervous system, but its functional properties and neuromodulation are not as well established as for Na(V)1.2 channels. We found no significant differences in voltage-dependent activation or fast inactivation between Na(V)1.6 and Na(V)1.2 channels expressed in non-excitable cells. In contrast, the voltage dependence of slow inactivation was more positive for Na(v)1.6 channels, they conducted substantially larger persistent sodium currents than Na(v)1.2 channels, and they were much less sensitive to inhibition by phosphorylation by cAMP-dependent protein kinase and protein kinase C. Resurgent sodium current, a hallmark of Na(v)1.6 channels in neurons, was not observed for Na(V)1.6 expressed alone or with the auxiliary beta(4) subunit. The unique properties of Na(V)1.6 channels, together with the resurgent currents that they conduct in neurons, make these channels well-suited to provide the driving force for sustained repetitive firing, a crucial property of neurons.

Voltage-gated sodium channels are responsible for action potential initiation and propagation in neurons, and modulation of their function has an important impact on neuronal excitability. Sodium channels are regulated by a Src-family tyrosine kinase pathway, and this modulation can be reversed by specifically bound receptor phosphoprotein tyrosine phosphatase-beta. However, the specific tyrosine kinase and signaling pathway are unknown. We found that the sodium channels in rat brain interact with Fyn, one of four Src-family tyrosine kinases expressed in the brain. Na(V)1.2 channels and Fyn are localized together in the axons of cultured hippocampal neurons, the mossy fibers of the hippocampus, and cell bodies, dendrites, and axons of neurons in many other brain areas, and they coimmunoprecipitate with Fyn from cotransfected tsA-201 cells. Coexpression of Fyn with Na(V)1.2 channels decreases sodium currents by increasing the rate of inactivation and causing a negative shift in the voltage dependence of inactivation. Reconstitution of a signaling pathway from brain-derived neurotrophic factor (BDNF) to sodium channels via the tyrosine receptor kinase B (TrkB)/p75 neurotrophin receptor and Fyn kinase in transfected cells resulted in an increased rate of inactivation of sodium channels and a negative shift in the voltage dependence of inactivation after treatment with BDNF. These results indicate that Fyn kinase is associated with sodium channels in brain neurons and can modulate Na(V)1.2 channels by tyrosine phosphorylation after activation of TrkB/p75 signaling by BDNF.

Opioid requirements in neonatal patients are reported to be lower than older infants and this may be a reflection of the developmental regulation of opioid receptors. In this study we have investigated the postnatal regulation of Mu opioid receptor (MOR) function in both rat lumbar dorsal root ganglion (DRG) cultures and behavioural mechanical and thermal reflex tests in rat pups. Immunostaining with MOR and selective neurofilament (NF200) antibodies was combined with calcium imaging of MOR function in cultured neonatal and adult rat dorsal root ganglion cells. Calcium imaging showed that a significantly greater number of neonatal DRG neurons expressed functional MOR compared to adult (56.5+/-3.4 versus 39.9+/-1.5%, n=8, mean+/-SEM, P<0.001). This expression is confined to the large, neurofilament positive sensory neurons, while expression in small, nociceptive, neurofilament negative neurons remains unchanged. Sensory threshold testing in rat pups showed that the analgesic potency of systemic morphine to mechanical stimulation is significantly greater in the neonate and declines with postnatal age. Morphine analgesic potency in thermal nociceptive tests did not change with postnatal age. These experiments show that the MOR expressed on large DRG neurons in neonates are functional and are subject to postnatal developmental regulation. This changing functional receptor profile is consistent with greater morphine potency in mechanical, but not thermal, sensory tests in young animals. These results have important clinical implications for the use of morphine in neonates and provide a possible explanation for the differences in morphine requirements observed in the youngest patients.

Several mechanisms have been implicated in underlying the perception of cold, most notably the activation of TRPM8 and TRPA1. We have used ratiometric calcium imaging to reveal a population of neurons in the superior cervical ganglion (SCG) of the mouse that respond to cooling but are insensitive to menthol. Furthermore we show that the expression of the mRNA transcripts encoding the recently identified noxious cold-sensitive channel TRPA1 but not TRPM8 are expressed in the SCG. These data provide evidence for a population of cold-responsive neurons in the SCG whose cold-responsiveness could be mediated by the activation of TRPA1 and suggest that the sympathetic nervous system may play a direct role in mediating sympathetic responses to cold temperatures.

The GP Ib complex can participate in endothelial cell (EC) migration on von Willebrand factor (vWF) or the mixed matrix of vWF and type I collagen (vWF/collagen). In this study, viper venom proteins alboaggregin (albo) A or B blocked GP Ibalpha, and echistatin inhibited alphavbeta3 binding. Albo A, B and echistatin inhibited EC migration on vWF and vWF/collagen. Albo B or the anti-GP Ibalpha monoclonal antibody (mAb) 1b1 did not affect the migration of smooth muscle cells or fibroblasts, which lack GP Ib. EC also migrate on albo A- or albo B-coated dishes. PD98059, which blocks ERK activation, abolished EC migration on vWF, vWF/collagen, collagen or albo B. Soluble albo A or 1b1 dramatically inhibited ERK activation during EC migration on vWF or albo B. Echistatin inhibited ERK activation on vWF and vitronectin (VN), but not albo B. Thus, in addition to alphavbeta3, EC GP Ibalpha initiates ERK activation, and regulates ERK-induced EC migration on vWF.

We have isolated and characterized EMS16, a potent and selective inhibitor of the alpha2beta1 integrin, from Echis multisquamatus venom. It belongs to the family of C-lectin type of proteins (CLPs), and its amino acid sequence is homologous with other members of this protein family occurring in snake venoms. EMS16 (M(r) approximately 33K) is a heterodimer composed of two distinct subunits linked by S-S bonds. K562 cells transfected with alpha2 integrin selectively adhere to immobilized EMS16, but not to two other snake venom-derived CLPs, echicetin and alboaggregin B. EMS16 inhibits adhesion of alpha2beta1-expressing cells to immobilized collagen I at picomolar concentrations, and the platelet/collagen I interaction in solution at nanomolar concentrations. EMS16 inhibits binding of isolated, recombinant I domain of alpha2 integrin to collagen in an ELISA assay, but not the interaction of isolated I domain of alpha1 integrin with collagen IV. Studies with monoclonal antibodies suggested that EMS16 binds to the alpha2 subunit of the integrin. EMS16 inhibits collagen-induced platelet aggregation, but has no effect on aggregation induced by other agonists such as ADP, thromboxane analogue (U46619), TRAP, or convulxin. EMS16 also inhibits collagen-induced, but not convulxin-induced, platelet cytosolic Ca(2+) mobilization. In addition, EMS16 inhibits HUVEC migration in collagen I gel. In conclusion, we report a new, potent viper venom-derived inhibitor of alpha2beta1 integrin, which does not belong to the disintegrin family.

In this study we introduce a novel in vitro 'oil-drop' assay system for the measurement of endothelial cell (EC) migration, based on the original concept of the Teflon fence assay (Pratt et al., 1984; Am. J. Pathol. 117: 349-354). An aliquot of 15-20,000 human umbilical vein EC (HUVEC) is pipetted through a layer of mineral oil. The cells readily attach, spread and migrate on the surface of a matrix-coated tissue culture dish as a confluent circular monolayer. Migration is measured as the net increase in the total area covered at 24 hours. We have used this system to quantify EC migration on matrices composed of a mixture of type I collagen and either von Willebrand factor (vWF) or fibronectin (FN) in the presence or absence of tumor necrosis factor alpha (TNFalpha). Plating efficiency on both vWF/collagen and FN/collagen, measured by counting cells after attachment and spreading, is about 80%. With this method, migration on vWF/collagen was about 6.4 mm(2) and 5.3 mm(2) for TNFalpha-treated and untreated HUVEC, respectively. HUVEC migration on FN/collagen was slightly greater - 6.4 mm(2) and 6.5 mm(2) with and without TNFalpha treatment, respectively. During the 24 hour time period, HUVEC numbers increased 30-40% on vWF/collagen, and 60-80% on FN/collagen, with increased proliferation observed with TNF-alpha treatment. EC proliferation could be completely inhibited by 2 mM hydroxyurea. This assay system has proven useful in our studies to quantify cell migration and proliferation.

We have previously shown that, in addition to the vitronectin receptor (VNR, alpha(v)beta(3)), the GP Ib complex can participate in endothelial cell (EC) attachment to von Willebrand Factor (vWF) (D. A. Beacham, M. S. Cruz, and R. I. Handin, 1995, Thromb. Haemostas. 73, 309-317; D. A. Beacham, L.-P. Tran, and S. S. Shapiro, 1997, Blood 89, 4071-4077). In this study we have investigated the functional roles of these vWF receptors in the migration of untreated and TNFalpha-treated EC on vWF, a mixture of vWF and type I collagen, and on vitronectin (VN). In agreement with previous studies (D. I. Leavesley, M. A. Schwartz, M. Rosenfeld, and D. A. Cheresh, 1993, J. Cell Biol. 121, 163-170), the migration of untreated and TNFalpha-treated EC on VN was dependent entirely on the VNR. Migration of untreated EC on vWF was inhibited 10-15% by recombinant vWF-A1, the GP Ibalpha-binding domain on vWF which abrogates the platelet GP Ibalpha-vWF interaction. In contrast, migration of TNFalpha-treated EC on vWF was inhibited 50-60% by vWF-A1 or the anti-GP Ibalpha mAb AS-7 but only 20% by the anti-VNR mAb LM609. On a mixed vWF-collagen substratum, vWF-A1 inhibited untreated EC migration by 45%, and TNFalpha-treated EC migration by 75%. The possible role of EC proliferation was eliminated, since hydroxyurea completely inhibited EC proliferation without reducing migration significantly. The anti-GP Ibalpha mAb Ib1 inhibited EC migration by 50%, but reduced proliferation by only 15%. Taken together, our data demonstrate that EC migration on vWF-containing substrata involves the GP Ib complex as well as the VNR and raises the possibility that the VNR and GP Ib act cooperatively in supporting EC migration., (Copyright 1999 Academic Press.)

Exposure to shear stress has been shown to alter the expression of a number of surface components of cultured endothelial cells (EC). However, relatively few studies have examined the status of human EC surface proteins after prolonged flow, more closely corresponding to the steady state in vivo. Since the promoter region of glycoprotein (Gp) Ib alpha contains several copies of a putative shear stress response element, 5'-GAGACC-3', we investigated the response of cultured human umbilical vein EC (HUVEC) GpIb alpha to shear stress over a 72 h time period. In response to 30 dynes/cm2 of shear stress, total cell content of GpIb alpha protein was markedly increased above static levels at 7 and 24 h, as determined immunohistochemically. Western blot analysis of whole cell lysates after 24, 48, and 72 h of shear treatment demonstrated a 2.4-, 4.1-, and 3.2-fold increase in total GpIb alpha protein, respectively. Cell surface protein expression of GpIb alpha increased 2.5-fold at 7 h, as measured by quantitative immunofluorescence, and remained at that level at 24 h. After 48 h of shear stress, cell surface GpIb alpha, GpIX, and GpV, analyzed by flow cytometric analysis, were further increased over the levels observed at 24 h. The increase in cell surface membrane expression of GPIb alpha at 24, 48, and 72 h was confirmed by immunoprecipitation of biotinylated surface proteins. No upregulation of GpIb alpha was noted after exposure to shear stress of 1-3 dynes/cm2. These observations imply that under steady-state arterial shear conditions endothelial expression of the GpIb complex is significantly greater than observed in static EC cultures, and raise the possibility of a more important role for this complex under flow, rather than static conditions.

Whole-cell patch-clamp recordings were used to study Ba2+ currents through voltage-dependent Ca2+ channels in dorsal root ganglion x mouse neuroblastoma hybrid (F-11) cells. Opioid agonists selective for either mu (Tyr-D-Ala-Gly-Mephe-Gly-ol; DAMGO) or delta (Tyr-D-Pen-Gly-Phe-D-Pen-OH; DPDPE) receptors inhibited high-threshold Ba2+ currents. The inhibition was reversible, naloxone-sensitive, and dose-dependent. The inhibitory effects of both DAMGO and DPDPE were blocked by pretreatment of the cells with pertussis toxin (PTX) as well as by brief exposure to the sulfhydryl alkylating agent, N-ethylmaleimide (NEM). The N-type Ca2+ channel antagonist omega-conotoxin GVIA (omega-CTX GVIA) irreversibly inhibited high threshold Ba2+ currents by 66% and blocked the inhibitory effect of DAMGO or DPDPE. In contrast, the L-type Ca2+ channel blocker nifedipine inhibited high threshold Ba2+ currents by 15% and failed to block the inhibitory effect of DAMGO or DPDPE. These results demonstrate that mu and delta opioid receptors are negatively coupled to N-type Ca2+ channels via PTX- and NEM-sensitive GTP-binding proteins in F-11 cells.

Endothelial cells (EC) possess at least two membrane receptors for von Willebrand factor (vWF), the vitronectin receptor (VNR, alpha(v)beta3), which recognizes an Arg-Gly-Asp (RGD) sequence in the C-terminus of vWF, and glycoprotein Ib alpha (GP Ib alpha), which interacts with a region in the N-terminal A1 domain of vWF. In the absence of added cytokines, EC attachment to a vWF substratum is mediated largely through the alpha(v)beta3, with a smaller contribution by GP Ib alpha. In the present study, we have examined the effect of cytokines on the receptor specificity of EC attachment to wild-type vWF (WT-vWF) and to vWF, which had been mutated in the C-terminal RGDS sequence (RADS-vWF). Exposure of human umbilical vein EC (HUVEC) to tumor necrosis factor-alpha (TNF-alpha) or to TNF-alpha in combination with interferon-gamma (IFN-gamma), but not to interleukin-1beta (IL-1), increased attachment to RADS-vWF by about twofold. The TNF-alpha-induced increase in EC attachment was accompanied by an increase in cell surface GP Ib alpha expression; GP Ib alpha surface expression was not increased by IL-1. Attachment of untreated HUVEC to WT-vWF could be inhibited 60% to 70% by a monoclonal antibody (MoAb) (LM609) to the VNR and 30% to 40% by the A1 fragment of vWF (containing the GP Ib alpha binding domain). The pattern of inhibition of attachment to WT-vWF was largely unchanged after TNF-alpha treatment of HUVEC. In contrast, the attachment to WT-vWF of HUVEC, treated with TNF-alpha +IFN-gamma was completely inhibited by vWF-A1 and inhibited only 35% by the anti-VNR antibody LM609. Two MoAbs to GP Ib alpha produced similar, but incomplete, inhibition. Pretreatment of HUVEC with the combination of TNF-alpha + IFN-gamma produced a dramatic decrease in VNR expression, confirming previous findings of Defilippi et al. These results suggest that in the presence of the inflammatory cytokines TNF-alpha + IFN-gamma, the endothelial GP Ib complex is a major determinant of HUVEC adhesion to surface-bound vWF.

Introduction of single amino acid substitutions into the C-terminal Arg-Gly-Asp-Ser (RGDS) site of von Willebrand Factor, referred to as RGD mutant vWF, selectively abrogated vWF binding to platelet glycoprotein IIb/IIIa (GpIIb/IIIa alpha IIb beta 3) and abolished human umbilical vein endothelial cell (HUVEC) spreading, but not attachment, to RGD mutant vWF (Beacham, D.A., Wise, R.J., Turci, S.M. and Handin, R. I. 1992. J. Biol. Chem. 167, 3409-3415). These results suggested that in addition to the vitronectin receptor (VNR, alpha V beta 3), a second endothelial membrane glycoprotein can mediate HUVEC adhesion to vWF. HUVEC attachment to wild-type (WT) and RGD-mutant vWF was reduced by two proteins known to block the vWF-platelet glycoprotein Ib/IX (GpIb/IX) interaction, the monoclonal antibody AS-7 and the recombinant polypeptide, vWF-A1. The addition of cytochalasin B or DNase I to disrupt potential GPIb alpha-cytoskeletal interactions enhanced the immunoprecipitation of endothelial GPIB alpha, caused HUVEC to round up, and increased HUVEC adhesion to RGD mutant vWF. These results indicate that while the VNR is the primary adhesion receptor for vWF, endothelial GPIb alpha can mediate HUVEC attachment to vWF. GpIb-dependent attachment could contribute to HUVEC adhesion under conditions when cell surface expression of the VNR is downregulated, and VNR-dependent adhesion is reduced.

In order to assess the requirement for the Arg-Gly-Asp-Ser (RGDS) consensus adhesion sequence in von Willebrand factor (vWF) for vWF binding to platelets and endothelial cells, point mutations were introduced into this sequence by site-directed mutagenesis. A glycine to alanine substitution yielded RADS-vWF, while an aspartate to glutamate substitution resulted in RGES-vWF. Recombinant RADS-vWF and RGES-vWF, purified from transformed Chinese hamster ovary cells, were compared with recombinant wild type vWF (WT-vWF) in functional assays with platelets and human umbilical vein endothelial cells (HU-VECs). High molecular weight RADS-vWF and RGES-vWF multimers inhibited binding of 125I-vWF to a mixture of insolubilized native type I and III collagen and competed effectively with 125I-vWF for binding to formalin-fixed platelets in the presence of ristocetin, indicating functional collagen and platelet glycoprotein Ib binding. However, RADS-vWF and RGES-vWF were unable to displace the binding of 125I-vWF to thrombin or ADP-activated platelets. The attachment of HUVECs to either RADS-vWF or RGES-vWF coated surfaces was reduced and spreading was almost completely inhibited, compared with WT-vWF. We conclude that point mutations of the RGDS sequence in vWF selectively impair binding to platelet glycoprotein IIb/IIIa and the HUVEC vitronectin receptor.

Three HeLa cell surface collagen receptors of apparent molecular mass 102/58, 87, and 38/33 kDa were eluted from gelatin-Sepharose with salt gradients or Arg-Gly-Asp-containing peptides. To understand how the collagen receptors are involved in HeLa cell spreading on collagen we investigated the effects of divalent cations and Arg-Gly-Asp-containing peptides on adhesion to gelatin, since HeLa cells behave similarly on both native type I collagen and gelatin substrata and also whether Arg-Gly-Asp-containing substrata would substitute for gelatin in facilitating cell spreading. Gly-Arg-Gly-Asp-Ser-containing peptides in solution inhibited HeLa cell spreading onto gelatin and promoted only partial HeLa cell spreading when bound to tissue culture plastic. Both partial spreading of HeLa cells on the Gly-Arg-Gly-Asp-Ser substratum and full spreading on gelatin was dependent on Mg2+, but not on Ca2+. Binding of the 102/58-, 87-, and 38/33-kDa collagen receptors to gelatin-Sepharose was increased fourfold in the presence of Mg2+, and subsequent elution of the collagen receptors and a 45-kDa collagen-binding protein not thought to be involved in HeLa cell spreading was achieved with EDTA. In contrast, affinity chromatography on Gly-Arg-Gly-Asp-Ser-Sepharose eluted predominantly the 45-kDa collagen-binding protein and the 38/33-kDa collagen receptor. In summary, the Mg2(+)-dependent interaction of the collagen receptors with the Arg-Gly-Asp sequence in collagen appears to be essential for the initial events in HeLa cell spreading but is not sufficient for full cell spreading.