Endothelial cell P-selectin mediates a proinflammatory and prothrombogenic phenotype in cerebral venules of sickle cell transgenic mice

Whereas the adhesion of leukocytes and erythrocytes to vascular endothelium has been implicated in the vasooclusive events associated with sickle cell disease, the role of platelet-vessel wall interactions in this process remains undefined. The objectives of this study were to: 1) determine whether the adhesion of platelets and leukocytes in cerebral venules differs between sickle cell transgenic (betaS) mice and their wild-type (WT) counterparts (C57Bl/6) under both resting and posthypoxic conditions, and 2) define the contributions of P-selectin to these adhesion processes. Animals were anesthetized, and platelet and leukocyte interactions with endothelial cells of cerebral postcapillary venules were monitored and quantified using intravital fluorescence microscopy in WT, betaS, and chimeric mice produced by transplanting bone marrow from WT or betaS mice into WT or P-selectin-deficient (P-sel(-/-)) mice. Platelet and leukocyte adhesion to endothelial cells in both unstimulated and posthypoxic betaS mice were significantly elevated over WT levels. Chimeric mice involving bone marrow transfer from betaS mice to P-sel(-/-) mice exhibited a profound attenuation of both platelet and leukocyte adhesion compared with betaS bone marrow transfer to WT mice. These findings indicate that betaS mice assume both an inflammatory and prothrombogenic phenotype, with endothelial cell P-selectin playing a major role in mediating these microvascular responses.     

Efficient recruitment of lymphocytes in inflamed brain venules requires expression of cutaneous lymphocyte antigen and fucosyltransferase-VII

Lymphocyte migration into the brain represents a critical event in the pathogenesis of multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE). However, the mechanisms controlling the recruitment of lymphocytes to the CNS via inflamed brain venules are poorly understood, and therapeutic approaches to inhibit this process are consequently few. In this study, we demonstrate for the first time that human and murine Th1 lymphocytes preferentially adhere to murine inflamed brain venules in an experimental model that mimics early inflammation during EAE. A virtually complete inhibition of rolling and arrest of Th1 cells in inflamed brain venules was observed with a blocking anti-P-selectin glycoprotein ligand 1 Ab and anti-E- and P-selectin Abs. Th1 lymphocytes produced from fucosyltransferase (FucT)-IV(-/-) mice efficiently tethered and rolled, whereas in contrast, primary adhesion of Th1 lymphocytes obtained from FucT-VII(-/-) or Fuc-VII(-/-)FucT-IV(-/-) mice was drastically reduced, indicating that FucT-VII is critical for the recruitment of Th1 cells in inflamed brain microcirculation. Importantly, we show that Abs directed against cutaneous lymphocyte Ag (CLA), a FucT-VII-dependent carbohydrate modification of P-selectin glycoprotein ligand 1, blocked rolling of Th1 cells. By exploiting a system that allowed us to obtain Th1 and Th2 cells with skin- vs gut-homing (CLA(+) vs integrin beta(7)(+)) phenotypes, we observed that induced expression of CLA on Th cells determined a striking increase of rolling efficiency in inflamed brain venules. These observations allow us to conclude that efficient recruitment of activated lymphocytes to the brain in the contexts mimicking EAE is controlled by FucT-VII and its cognate cell surface Ag CLA.     

Diminished nitroprusside-induced relaxation of inflamed colonic smooth muscle in mice.

The dextran sodium sulphate (DSS) induced colitis in mice was used as a experimental model to study the contractility of murine longitudinal colonic smooth muscle during inflammation. Smooth muscle segments of proximal, middle and distal colon were mounted in organ baths. Smooth muscle contraction was induced by carbachol showing an aboral increase in activity, whereas in the inflamed middle colonic segment a marked decrease in activity was observed. The dilatative effect of sodium-nitroprusside (SNP) as a nitric oxide donor was investigated after precontraction by carbachol. Both in normal and DSS segments administration of SNP to isolated mouse colonic smooth muscle preparations caused regional differences in relaxation, the highest relaxation seen in normal proximal colonic tissue. However, this relaxation was markedly reduced in inflamed proximal preparations, associated with a diminished cGMP contents.     

Molecular determinants of sugar transport regulation by ATP

Intracellular ATP inhibits human erythrocyte net sugar transport by binding cooperatively to the glucose transport protein (GluT1). ATP binding produces altered transporter affinity for substrate and promotes substrate occlusion within a post-translocation vestibule formed by GluT1 cytosolic domains. The accompanying paper (Cloherty, E. K., Levine, K. B., Graybill, C., and Carruthers, A. (2002) Biochemistry 41, 12639-12651) demonstrates that reduced intracellular pH promotes high-affinity ATP binding to GluT1 but inhibits ATP-modulation of GluT1-mediated sugar transport. The present study explores the role of GluT1 residues 326-343 (a proposed GluT1 ATP-binding site subdomain) in GluT1 ATP binding by using alanine scanning mutagenesis. Cos-7 and HEK cells were transfected with a cDNA encoding full-length human GluT1 terminating in a carboxyl-terminal hemagglutinin (HA)-His6 epitope. The transporter (GluT1.HA.H6) is expressed at the surface of both cell-types and is catalytically active. In HEK cells, both parental GluT1- and GluT1.HA.H6-mediated sugar transport are acutely sensitive to cellular metabolic inhibition. Isolated, detergent-solubilized GluT1.HA.H6 is photolabeled by [gamma-32P]-azidoATP in an ATP-protectable manner. Alanine substitution of E329 or G332/R333/R334 enhances GluT1.HA.H6 [gamma-32P]azidoATP photoincorporation but blocks acute modulation of net sugar transport by cellular metabolic inhibition. These actions resemble those of reduced pH on ATP binding to and modulation of red cell GluT1. It is proposed that cooperative nucleotide binding to GluT1 and nucleotide modulation of GluT1-mediated sugar transport are regulated by a proton-sensitive saltbridge (Glu329-Arg333/334).     

Molecular determinants of virulence, cell tropism, and pathogenic phenotype of infectious bursal disease virus.

Infectious bursal disease viruses (IBDVs), belonging to the family Birnaviridae, exhibit a wide range of immunosuppressive potential, pathogenicity, and virulence for chickens. The genomic segment A encodes all the structural (VP2, VP4, and VP3) and nonstructural proteins, whereas segment B encodes the viral RNA-dependent RNA polymerase (VP1). To identify the molecular determinants for the virulence, pathogenic phenotype, and cell tropism of IBDV, we prepared full-length cDNA clones of a virulent strain, Irwin Moulthrop (IM), and constructed several chimeric cDNA clones of segments A and B between the attenuated vaccine strain (D78) and the virulent IM or GLS variant strain. Using the cRNA-based reverse-genetics system developed for IBDV, we generated five chimeric viruses after transfection by electroporation procedures in Vero or chicken embryo fibroblast (CEF) cells, one of which was recovered after propagation in embryonated eggs. To evaluate the characteristics of the recovered viruses in vivo, we inoculated 3-week-old chickens with D78, IM, GLS, or chimeric viruses and analyzed their bursae for pathological lesions 3 days postinfection. Viruses in which VP4, VP4-VP3, and VP1 coding sequences of the virulent strain IM were substituted for the corresponding region in the vaccine strain failed to induce hemorrhagic lesions in the bursa. In contrast, viruses in which the VP2 coding region of the vaccine strain was replaced with the variant GLS or virulent IM strain caused rapid bursal atrophy or hemorrhagic lesions in the bursa, as seen with the variant or classical virulent strain, respectively. These results show that the virulence and pathogenic-phenotype markers of IBDV reside in VP2. Moreover, one of the chimeric viruses containing VP2 sequences of the virulent strain could not be recovered in Vero or CEF cells but was recovered in embryonated eggs, suggesting that VP2 contains the determinants for cell tropism. Similarly, one of the chimeric viruses containing the VP1 segment of the virulent strain could not be recovered in Vero cells but was recovered in CEF cells, suggesting that VP1 contains the determinants for cell-specific replication in Vero cells. By comparing the deduced amino acid sequences of the D78 and IM strains and their reactivities with monoclonal antibody 21, which binds specifically to virulent IBDV, the putative amino acids involved in virulence and cell tropism were identified. Our results indicate that residues Gln at position 253 (Gln253), Asp279, and Ala284 of VP2 are involved in the virulence, cell tropism, and pathogenic phenotype of virulent IBDV.     

Molecular determinants for the selective inhibition of cyclooxygenase-2 by lumiracoxib

Lumiracoxib is the first example of a marketed COX-2 inhibitor of the arylacetic acid class, and it is reported to be the most selective COXIB in vivo. However, the molecular basis of its COX-2 inhibition has not been completely defined. Using standard assays, lumiracoxib was found to be a poor inhibitor of purified ovine COX-1 and a relatively weak inhibitor of purified human COX-2. The extent of COX-2 inhibition plateaued at around 50% and suggested that the inhibitor may be reversibly bound to the enzyme. Kinetic studies with lumiracoxib demonstrated that it was a time-dependent and slowly reversible inhibitor of human COX-2 that exhibited at least two binding steps during inhibition. Derivatives of lumiracoxib were synthesized with or without the methyl group on the phenylacetic acid ring and with various substitutions on the lower aniline ring. Inhibition studies demonstrated that the methyl group on the phenylacetic acid ring is required for COX-2 selectivity. The chemical identity and position of the substituents on the lower aniline ring were important in determining the potency and extent of COX inhibition as well as COX-2 selectivity. Mutation of Ser-530 to Ala or Val-349 to Ala or Leu abolished the potent inhibition observed with wild-type human COX-2 and key lumiracoxib analogs. Interestingly, a Val-349 to Ile mutant was inhibited with equal potency to human COX-2 with 2,6-dichloro-, 2,6-dimethyl-, or 2-chloro-6-methyl-substituted inhibitors and, in the case of lumiracoxib, actually showed an increase in potency. Taken together with a recent crystal structure of a lumiracoxib-COX-2 complex, the kinetic analyses presented herein of the inhibition of mutant COX-2s by lumiracoxib allows the definition of the molecular basis of COX-2 inhibition.     

Molecular determinants of KATP channel inhibition by ATP.

ATP-sensitive K+ (KATP) channels are both inhibited and activated by intracellular nucleotides, such as ATP and ADP. The inhibitory effects of nucleotides are mediated via the pore-forming subunit, Kir6.2, whereas the potentiatory effects are conferred by the sulfonylurea receptor subunit, SUR. The stimulatory action of Mg-nucleotides complicates analysis of nucleotide inhibition of Kir6. 2/SUR1 channels. We therefore used a truncated isoform of Kir6.2, that expresses ATP-sensitive channels in the absence of SUR1, to explore the mechanism of nucleotide inhibition. We found that Kir6.2 is highly selective for ATP, and that both the adenine moiety and the beta-phosphate contribute to specificity. We also identified several mutations that significantly reduce ATP inhibition. These are located in two distinct regions of Kir6.2: the N-terminus preceding, and the C-terminus immediately following, the transmembrane domains. Some mutations in the C-terminus also markedly increased the channel open probability, which may account for the decrease in apparent ATP sensitivity. Other mutations did not affect the single-channel kinetics, and may reduce ATP inhibition by interfering with ATP binding and/or the link between ATP binding and pore closure. Our results also implicate the proximal C-terminus in KATP channel gating.     

Molecular determinants of sink strength

The manipulation of sink to source relations has been subject to extensive plant breeding programs aiming to improve harvest index and thereby crop yield. The introduction of molecular and biochemical tools has enabled scientists to investigate the underlying principles. This has opened up the fascinating possibility of identifying molecular determinants of sink strength and to further increase yield on a rational basis. In the past, transgenic plants with alterations in the activity of only one putative molecular determinant have been created and this strategy has not resulted in substantial and reliable increases in yield. Yet, careful molecular and biochemical investigations have provided valuable insight about carbon flux into different metabolic pathways at different stages of sink development and it has become apparent that this metabolic channelling needs to be exploited by using stage- and cell-specific promoters in attempts to increase sink strength.     

Molecular determinants of nef function

The human immunodeficiency virus (HIV), the causative agent of the acquired immune deficiency syndrome (AIDS), in addition to encoding for thegag, pol andenv structural genes common to all retroviruses also encodes six accessory genes:tat, rev, nef, vpr, vpu andvif. These accessory genes are responsible for the regulation of HIV replication. Recent advances in our understanding of the function(s) of these genes have illustrated the complex interplay between HIV, the infected cell and the host. In addition, identification of cellular proteins interacting with accessory gene products have provided new tools to study cellular processes. The topic of this review,nef, has been shown in vitro to induce the cell surface downregulation of CD4, the receptor for HIV, to enhance the infectivity of HIV particles and to associate with at least one cellular serine/threonine kinase. In vivo, Nef is essential for the efficient virus replication responsible for disease progression. In this review, several prominent aspects of Nef function are discussed including its effect on CD4 trafficking, on signaling pathways and on virus infectivity enhancement.     

The effect of essential fatty acid deficiency on eicosanoid production in the inflamed rat colonic mucosa

Eicosanoids are potent mediators of inflammation and are synthesized in increased quantity in active ulcerative colitis. To elucidate the role of prostaglandin E2, thromboxane A2, prostaglandin I2, and leukotriene B2 in acute chemical colitis induced by 4% acetic acid, we utilized an animal model which has a deficiency of arachidonic acid, the precursor of eicosanoids due to an essential fatty acid deficient diet. Forty-eight hours after colitis was induced, mucosal synthesis of the cyclooxygenase products, prostaglandin E2, thromboxane A2, and prostaglandin I2, was significantly decreased in essential fatty acid deficient rats compared to normal controls. However, the 5-lipoxygenase product, leukotriene B4, was not different between groups. The decrease in cyclooxygenase products did not correlate with any change in the severity of colonic inflammation as assessed by gross morphology, histology, or myleoperoxidase activity. Thus inhibition of formation of the cyclooxygenase products of arachidonate metabolism does not appear to improve the degree of inflammation under the experimental conditions employed in this study.     

Molecular determinants of mechanosensation in the muscle spindle

The muscle spindle (MS) provides essential sensory information for motor control and proprioception. The Group Ia and II MS afferents are low threshold slowly-adapting mechanoreceptors and report both static muscle length and dynamic muscle movement information. The exact molecular mechanism by which MS afferents transduce muscle movement into action potentials is incompletely understood. This short review will discuss recent evidence suggesting that PIEZO2 is an essential mechanically sensitive ion channel in MS afferents and that vesicle-released glutamate contributes to maintaining afferent excitability during the static phase of stretch. Other mechanically gated ion channels, voltage-gated sodium channels, other ion channels, regulatory proteins, and interactions with the intrafusal fibers are also important for MS afferent mechanosensation. Future studies are needed to fully understand mechanosensation in the MS and whether different complements of molecular mediators contribute to the different response properties of Group Ia and II afferents.