VEGF and angiogenesis in acute and chronic MOG((35-55)) peptide induced EAE.

An increased expression of vascular endothelial growth factor (VEGF) is associated with demyelinated lesions in both multiple sclerosis (MS) and its model (EAE), implicating changes in vasculature as a potential component of CNS plaque formation. The purpose of this study was to investigate the vascular changes in acute and chronic EAE in C57BL/6 mice induced with myelin oligodendrocyte glycoprotein (MOG ((35-55))) peptide. We investigated the functional contribution of VEGF to acute and chronic EAE by treating immunized mice with SU5416 (Semaxinib), a potent and selective inhibitor of VEGF receptor 2 (VEGFR2). Animals received seven daily injections of SU5416 (50 mg/kg) or vehicle beginning on the day after disease onset (acute study) or on day 45 post-immunization (chronic study). Spinal cord sections were collected on the day of sacrifice. Modulation of angiogenic gene expression was determined using RNA isolated from 4 acute and 4 non-immunized controls. MOG peptide induction produced extensive demyelination, immune cell infiltration, tissue laminin deposits, and axonal loss in lesions. VEGF expression was extensively increased in the acute mice, which correlated positively with clinical score. In the acute study, SU5416 treatment produced a significant clinical improvement versus vehicle controls (p<0.001), with less demyelination (-37%) and cellular infiltration (-23%) in the spinal cord (p<0.05). Treated animals also had significantly fewer blood vessels per section than controls (56.1+/-6.1 v. 81.6+/-11.5, p<0.05), and significantly reduced laminin abnormalities (28.9% of lesion area v. 46.8%, p<0.05). There was no improvement in clinical score or tissue pathology, and no difference in vessel number or lesion laminin expression, when SU5416 was administered during the chronic disease (all p>0.05). In the acute study only, VEGF staining correlated with demyelination and the extent of cellular infiltration in both control (r=0.723, r=0.665) and treated (r=0.681, r=0.487) animals (all p<0.05). Laminin staining in lesion areas was strongly correlated with tissue pathology for all animals in both the acute and chronic study (all p<0.001). Vascular alterations in MOG peptide-induced EAE in the mouse are accompanied by increased lesion-specific levels of VEGF, extensive laminin deposits in the tissue and altered transcription of numerous angiogenic factors. In the microarray studies, acute mice showed a significant increase in several angiogenic RNA transcripts, six of which were verified by RT-PCR, alanyl aminopeptidase, caspase 8, Hif1a, MMP-19, plasminogen activator inhibitor, and thrombospondin1.

Experimental allergic encephalomyelitis in connexin 43-heterozygous mice.

Alterations in the expression of gap junction proteins (connexins) have previously been observed in experimental allergic encephalomyelitis (EAE). Demyelinating lesions have significantly decreased Cx43, while recovering lesions have greatly increased Cx43 and increased glial fibrillary acidic protein-expressing astrocytes. This suggests an important role for gap-junctional intercellular communication in astrocytes in the recovery from CNS inflammation. To study the effects of decreased Cx43 expression during acute disease (21 days post-immunization) and in recovering spinal cord tissue (55 days post-immunization) we induced EAE in Cx43 heterozygous and wild-type mice. Mice showed signs of disease by day 10, and signs of recovery by day 25. There were no clinical or pathological differences between heterozygous and wild-type mice in the acute disease stage, except that wild-type male mice had fewer clinical signs of disease. Male mice that were heterozygous for Cx43, and therefore had decreased expression of Cx43, had increased EAE disease severity. All demyelinating lesions had reduced numbers of reactive astrocytes and a significant decrease in Cx43 expression. In the 55-day study, all heterozygous and wild-type mice were clinically improved, showed decreased pathological signs, and showed increased laminin expression, indicative of CNS recovery. Furthermore, all heterozygous mice showed a striking increase in Cx43 expression during recovery, suggesting that the regulatory factors affecting Cx43 expression are still present in mice that have only one wild-type Cx43 allele.

Connexin 43 gap junction proteins are up-regulated in remyelinating spinal cord.

Alterations in the expression of gap junction proteins have previously been observed in several diseases affecting the central nervous system; however, the status of connexin 43 (Cx43) has not yet been reported in spinal cord remyelination. We studied Cx43 expression in demyelination and remyelination by using a chronic guinea pig model of experimental allergic encephalomyelitis (EAE). Hartley guinea pigs were immunized with homogenized whole CNS and complete Freund's adjuvant. Animals became chronically ill by day 40 postimmunization, and animals with paralysis were entered into the study. Animals were treated on days 40-60 postimmunization with either saline or drugs that promote remyelination: an adenosine amine congener (100 mug/kg), an anti-alpha4-integrin blocker (CT301; ELN 69299; 30 mg/kg), or a combination of both drugs. Remyelination was induced in all drug-treated groups. Cx43 expression was virtually absent in demyelinated lesions of saline-treated controls compared with healthy tissue and normal appearing white matter (P < 0.001), whereas Cx43 was considerably increased (300-500%) in remyelinating lesions of all treatment groups (P < 0.001), most notably in CT301-treated animals. These changes in Cx43 expression indicate that Cx43 may beimportant for recovery from neuroinflammation.

Failure of spermatogenesis in mice lacking connexin43.

Connexin43 (Cx43), a gap junction protein encoded by the Gja1 gene, is expressed in several cell types of the testis. Cx43 gap junctions couple Sertoli cells with each other, Leydig cells with each other, and spermatogonia/spermatocytes with Sertoli cells. To investigate the role of this communication pathway in spermatogenesis, we studied postnatal testis development in mice lacking Cx43. Because such mice die shortly after birth, it was necessary to graft testes from null mutant fetuses under the kidney capsules of adult males for up to 3 wk. Grafted wild-type testes were used as controls. In our initial experiments with wild-type testes, histological examination indicated that the development of grafted testes kept pace with that of nongrafted testes in terms of the onset of meiosis, but this development required the presence of the host gonads. When excised grafts were stimulated in vitro with cAMP or LH, there was no significant difference in androgen production between null mutant and wild-type testes, indicating that the absence of Cx43 had not compromised steroidogenesis. Previous research has shown that Cx43 null mutant neonates have a germ cell deficiency that arises during fetal life, and our analysis of grafted testes demonstrated that this deficiency persists postnatally, giving rise to a "Sertoli cell only" phenotype. These results indicate that intercellular communication via Cx43 channels is required for postnatal expansion of the male germ line.

Regulation of Tiam1 nucleotide exchange activity by pleckstrin domain binding ligands.

Rho family GTPases play roles in cytoskeletal organization and cellular transformation. Tiam1 is a member of the Dbl family of guanine nucleotide exchange factors that activate Rho family GTPases. These exchange factors have in common a catalytic Dbl homology and adjacent pleckstrin homology domain. Previous structural studies suggest that the pleckstrin domain, a putative phosphoinositide-binding site, may serve a regulatory function. We identified ascorbyl stearate as a compound that binds to the pleckstrin domain of p120 Ras GTPase-activating protein. Furthermore, ascorbyl stearate appears to be a general pleckstrin domain ligand, perhaps by mimicking an endogenous amphiphilic ligand. Tiam1 nucleotide exchange activity was greatly stimulated by ascorbyl stearate. Certain phosphoinositides also stimulated Tiam1 activity but were less potent than ascorbyl stearate. Tiam1 contains an additional N-terminal pleckstrin domain, but only the C-terminal pleckstrin domain was required for activation. Our results suggest that the pleckstrin domains of Dbl-type proteins may not only be involved in subcellular localization but may also directly regulate the nucleotide exchange activity of an associated Dbl homology domain. In addition, this paper introduces ascorbyl stearate as a pleckstrin domain ligand that can modulate the activity of certain pleckstrin domain-containing proteins.

Direct stimulation of the guanine nucleotide exchange activity of p115 RhoGEF by Galpha13.

Signaling pathways that link extracellular factors to activation of the monomeric guanosine triphosphatase (GTPase) Rho control cytoskeletal rearrangements and cell growth. Heterotrimeric guanine nucleotide-binding proteins (G proteins) participate in several of these pathways, although their mechanisms are unclear. The GTPase activities of two G protein alpha subunits, Galpha12 and Galpha13, are stimulated by the Rho guanine nucleotide exchange factor p115 RhoGEF. Activated Galpha13 bound tightly to p115 RhoGEF and stimulated its capacity to catalyze nucleotide exchange on Rho. In contrast, activated Galpha12 inhibited stimulation by Galpha13. Thus, p115 RhoGEF can directly link heterotrimeric G protein alpha subunits to regulation of Rho.

[125I]EXP985: a highly potent and specific nonpeptide radioligand antagonist for the AT1 angiotensin receptor.

[125I]EXP985 is the first nonpeptide radioligand with high specific activity for the AT1 angiotensin receptor. The biochemical and pharmacological profiles of this ligand were determined using either ligand-receptor binding techniques in rat adrenal cortical microsomes or cellular Ca2+ mobilization in rat smooth muscle cells. Specific binding with 0.1 nM [125I]EXP985 increased slowly with time reaching an equilibrium at 60 min of incubation (22 degrees C). Scatchard analysis of the inhibition/binding data revealed a single class of binding sites having a Kd of 1.49 +/- 0.06 nM and a Bmax of 3.6 +/- 0.1 pmol/mg protein. These sites were saturable and the ligand-receptor complex dissociated with a t1/2 of 58 min. The binding was inhibited by Ang peptides with the following order of potency and IC50 (nM): Ang II (3.7) > Ang III (69) > Ang I (3650), and by the nonpeptide AT1 receptor antagonist, losartan, with an IC50 of 3.2 nM. PD123177, an AT2 selective antagonist, showed minimal inhibitory effect. Specific binding of [125I]EXP985 was found on rat aortic smooth cells. Ang II-induced Ca2+ mobilization in these cells was blocked by EXP985 in a noncompetitive manner. These data show that [125I]EXP985 (or its unlabeled) is a potent and highly specific radioligand or noncompetitive antagonist which represents a novel tool to further our understanding of the biochemistry of AT1 receptors.

AT1 receptors mediate the release of prostaglandins in porcine smooth muscle cells and rat astrocytes.

Angiotensin II (AII) can release arachidonic acid metabolites such as prostacyclin (PGI2) and PGE2 from cells in cultures. It has recently been reported that the AT1 selective nonpeptide AII receptor antagonist losartan had similar effects. The present study was undertaken to further evaluate the effects of AII and losartan on cells which synthesize prostaglandins, including vascular smooth muscle, endothelial, and glial cells. Inhibition of specific [125I]AII binding was demonstrated in porcine smooth muscle cell (PSMC) suspensions with unlabeled AII and losartan. The IC50 values were 1.3 x 10(-9) mol/L and 7.7 x 10(-9) mol/L, respectively. PD123177 (an AT2 selective antagonist) had no effect on binding. AII produced a concentration-related increase in calcium mobilization (fura-2 fluorescence) which was blocked by losartan (IC50 = 8.4 x 10(-8) mol/L) but not by PD123177 (10(-6) mol/L). AII (10(-7) to 10(-5) mol/L) stimulated the basal release of PGI2 by 100%. This response was blocked by losartan (10(-6) to 10(-5) mol/L) but not by PD123177 (10(-6) to 10(-5) mol/L) and neither agent stimulated basal release in PSMC. Similar effects of AII and antagonists were observed upon receptor binding and PGE2 release in primary rat astrocyte (RA) cultures. AII did not release PGI2 from porcine endothelial cells, bovine pulmonary arterial endothelial cells, or rat C6 glioma cells. Losartan had no significant effect at 10(-5) mol/L. By contrast, bradykinin or the calcium ionophore A23187 dramatically increased PGI2 release in each of these cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of biochemical responses of angiotensin II (AT2) binding sites in the rat pheochromocytoma PC12W cells.

Rat pheochromocytoma PC12W cell membranes have previously been shown to exclusively contain the AT2 receptor subtype. The present study extended these binding data and explored the functional expression of these binding sites. Our binding competition studies show a potency series of Ang II = Ang III greater than saralasin greater than Ang I = PD123177 much greater than Ang II(1-7) much much greater than losartan. PD123177 (1 microM) completely eliminated [125I]Ang II binding to PC12W cells. Competitive displacement of [125I]Ang II with Ang II shows a dissociation equilibrium constant (Kd) of 1.79 nM and a binding site maximum (Bmax) of 3.97 fmol/mg protein. Investigating several Ang II signal transduction pathways on these cells, we found that Ang II (10(-8) to 10(-6) M) does not affect basal cAMP, cGMP, arachidonic acid release, prostacyclin release, intracellular Ca2+ mobilization or thymidine incorporation in the PC12W cells. Nerve growth factor, cAMP, 5-fluorouridine deoxyriboside modulation of the number of AT2 receptor sites in PC12W cells failed to unmask any Ang II effects on basal cAMP, cGMP and intracellular Ca2+ mobilization. In conclusion, the present study confirms the exclusive presence of AT2 binding sites in the PC12W cells. However, these binding sites are not functionally coupled to common signal transduction pathways.

History's future: reflections on lesbian and gay history in the community.

From its beginnings in the nineteenth century, the lesbian and gay political movement has been linked to a search for lesbian and gay history. In the post-Stonewall period, community-based historians have been fostering interest in the lesbian and gay past and developing distinctive forms for disseminating their research--in particular, the lesbian/gay archive, the slide-lecture presentation, and the community-based audience. Analyzing the content of these forms reveals how the fascination of the artifact, the image, and the Other fosters the construction of both knowledge and identity. It is these forms of knowledge, rather than their content as such, that are in danger of being forgotten as lesbian and gay studies becomes academically institutionalized.

Inhibition of the thrombin-platelet reactions by DuP 714.

The efficacy and specificity of a novel synthetic thrombin inhibitor, DuP 714, on thrombin-induced elevation of cytoplasmic calcium, fibrinogen binding and aggregation in human platelets were examined. Thrombin (0.5 U/ml) stimulated an increase in [125I]fibrinogen binding in gel-filtered platelets which was blocked by DuP 714 with an IC50 value of 2 nM. Thrombin (1 U/ml)-induced elevation of intracellular [Ca2+]i was also blocked by DuP 714 with an IC50 value of 67 nM. A much higher concentration of thrombin (25 U/ml) was used to stimulate aggregation with heparinized platelet-rich plasma. Under these conditions, micromolar concentrations of DuP 714 were needed to inhibit thrombin. In all of these preparations, DuP 714 at concentrations as high as 10(-5) M had no intrinsic effects and did not affect the responses induced by arachidonate, ADP, collagen, epinephrine, vasopressin and serotonin. These data indicate that DuP 714 is a potent and specific thrombin inhibitor capable of arresting the actions of thrombin on human fibrin formation and platelet aggregation and secretion. It may serve as a potential antithrombotic agent for various forms of thrombotic disorders.

Angiotensin II-1 receptors mediate both vasoconstrictor and hypertrophic responses in rat aortic smooth muscle cells.

The receptor subtypes that mediate the vasoconstrictor and hypertrophic responses of AII were evaluated in cultured aortic smooth muscle cells from rat. The AII receptors were characterized by means of the subtype-specific receptor antagonists DuP 753 (AII-1) and PD123177 (AII-2) using changes of [Ca2+]i (fura-2) as an indirect measurement of vasoconstriction and of [3H]leucine or [3H]thymidine incorporation as indices for hypertrophy and hyperplasia, respectively. AII-induced [Ca2+]i mobilization was blocked by saralasin and DuP 753 with inhibitory potency values of 2 and 1.3 x 10(-8) M, respectively, whereas PD123177 (10(-5) M) was without effect. Addition of AII (10(-7) M) to quiescent cells elicited a 45% increase in protein synthesis and a 56% increase in DNA synthesis after a 24-h exposure. These increases were blocked by both saralasin and DuP 753 (10(-5) M), but not by PD123177 (10(-5) M). Although DNA synthesis was increased by AII, no increase in smooth muscle cell number was detected. These data indicate that the AII receptors on aortic smooth muscle cells of the rat are of the AII-1 receptor subtype, which is responsible for both the vasoconstrictor and hypertrophic responses of AII.

Making history: the challenge of gay and lesbian studies.

This paper addresses a central problem of gay and lesbian studies: how is the subject to be defined? Current essentialist and constructionist positions are ultimately ahistorical and reductionist, reflecting the residual influence of the medical model and its sexual definition. In place of a single-dimensional and a priori sexual category, the author proposes sociosexual specialization as the appropriate focus of gay and lesbian studies and outlines a heuristic, multidimensional model for describing not only contemporary, but historical and cross-cultural evidence. Six dimensions of social and sexual variation are reviewed: sexuality, subjectivity and identity, gender, social roles, economic roles, and spirituality.

Comparison of oxygen tension measurement methodologies: further studies.

The oxygen quantifying techniques of Hill and Rasson and Fatt have been compared by Roscoe and Wilson, who have also derived an "average" equivalent oxygen percentage (EOP) calibration equation. This study provides additional data which support their conclusions. Eight subjects wore three different hydrogel contact lens types of various center thicknesses and oxygen transmissibilities. Open-eyelid oxygen polarographic data were taken for each of these lenses worn by the subjects. The "average" EOP calibration equation was used to calculate the EOP values. The relation between center thickness and the ratio of the oxygen tension values derived by the two methods agrees relatively well with that found previously.

Polarography: an oxygen survey correlating oxygen transmissibility to "EOP" values.

A polarographic oxygen electrode was used to measure the oxygen transmissibility of seven contact lenses of varying water content and center thickness. A similar electrode was used to measure the oxygen uptake following wear of these same contact lenses for both the open- and closed-eyelid conditions on five young healthy subjects. Linear regression revealed a strong correlation between oxygen transmissibility and equivalent oxygen percentage (EOP) values for both the open- and closed-eyelid conditions. This strong correlation between these two oxygen parameters shows both are useful in predicting the oxygen tensions across the tear-epithelial interface during contact lens wear.

Equivalent oxygen percentage (EOP) technique: a standard calibration curve.

Between different oxygen concentrations (mixed with nitrogen) ranging from 0 to 21% were passed over the corneas of six subjects for 5 min. The resulting corneal oxygen responses were measured by polarography. The averages of these responses were used to calculate a "relative oxygen uptake response" (ROUR) value for each oxygen percentage. Analysis of these ordered pairs revealed a curvilinear relation which was best described by a third order equation; marked linearity was demonstrated in the middle of the oxygen range. The use of this third order equation as a "standard" or "average" equivalent oxygen percentage (EOP) calibration equation is suggested.

Oxygen tension measurements: a comparison of methodologies.

The equivalent oxygen percentage (EOP) technique of Hill and the technique of Rasson and Fatt are two commonly used methods to quantify the oxygen tension underneath a contact lens for the open-eyelid case. This study compares these two techniques. Five subjects and six contact lenses of various center thicknesses and oxygen transmissibilities were used. Each subject wore each lens for which the oxygen tension was measured by these two methods. For lenses of approximately 0.23 mm center thickness, the two methods gave similar oxygen values. For contact lenses significantly thicker or thinner, the two methods gave very different oxygen values. However, these oxygen values were related in a specific manner; a strong linear relation was found for contact lens center thickness vs. the ratio of the two oxygen values found by the two methods.

Oxygen measurements: dynamic vs static conditions.

Equivalent oxygen percentage (E.O.P.) responses, using a mammalian corneal model, were measured for open-and closed-eye conditions with the presence of a Permalens contact lens. Under open-eye conditions, the E.O.P. responses agreed with those measured or predicted by other investigators. However, under closed-eye conditions, the E.O.P. responses were significantly different from those measured or predicted by other investigators. This discrepancy is discussed in terms of its clinical significance for extended wear contact lens patients.