Mechanism of Néel Order Switching in Antiferromagnetic Thin Films Revealed by Magnetotransport and Direct Imaging.

We probe the current-induced magnetic switching of insulating antiferromagnet-heavy-metal systems, by electrical spin Hall magnetoresistance measurements and direct imaging, identifying a reversal occurring by domain wall (DW) motion. We observe switching of more than one-third of the antiferromagnetic domains by the application of current pulses. Our data reveal two different magnetic switching mechanisms leading together to an efficient switching, namely, the spin-current induced effective magnetic anisotropy variation and the action of the spin torque on the DWs.

CD40 associates with the MHC class II molecules on human B cells.

The MHC class II and CD40 molecules are two major components of the immune system that are involved in cell-cell interactions and signal transduction. Data obtained in the course of the present investigation show that these two molecules are physically associated on the surface of various human B cell lines and on normal tonsilar B cells. The CD40 / MHC class II complexes were not detected on the germinal center B cell line Ramos. However, stimulation of these cells via CD40 or MHC class II triggered their association, suggesting that the formation of the complex is related to the activation status of the cells. The formation of these complexes did not alter the interaction of MHC class II molecules with one of their natural ligands, the staphylococcal enterotoxin A (SEA), as evidenced by the ability of SEA to bind MHC class II / CD40 complexes. Cross-linking of MHC class II or CD40 molecules leads to the association as well as the co-association of both molecules to the NP-49-insoluble cellular matrix. Such association allowed us to demonstrate that only a fraction of these molecules can be physically associated on the cell surface. Based on previous observations and those presented here, it is highly possible that the CD40 / MHC class II complexes may have an important role in signal(s) induced via both molecules and during T / B cells interactions.

CD40- and HLA-DR-mediated cell death pathways share a lot of similarities but differ in their use of ADP-ribosyltransferase activities.

CD40 and HLA-DR molecules are two major components of the immune system, and their engagement on several cell types leads to various cellular events that modulate cell function. In this study, we demonstrate that signaling via these molecules leads to a rapid B cell death. CD40-mediated cell death was mainly observed in Epstein-Barr virus (EBV)-transformed B cell lines, whereas, HLA-DR-induced response can be triggered in normal activated B cells as well as in EBV-transformed B cell lines. Cell death induced via both molecules does not require de novo protein synthesis, but involves the integrity of the cytoskeleton. The sensitivity of CD40- and HLA-DR-mediated cell death to various inhibitors is very similar to that previously reported for tumor necrosis factor receptor (TNFR)- and Fas-triggered apoptosis; however, caspases leading to poly(ADP-ribose) polymerase cleavage are not implicated in this response. Both B cell death forms do not involve Fas-Fas ligand and TNF-TNFR systems, but require LFA-1-independent cell-cell interactions mediated by still undefined molecules. Although CD40- and HLA-DR-mediated cell death appears to follow a common pathway, inhibitors of poly- and mono-ADP-ribosyltransferase activity differentially affect these responses. Defining the molecules involved in CD40- and HLA-DR-mediated death will provide a possible interrelation between the different B cell death programs that can lead to a better comprehension of regulation of B cell functions.

CD20 is physically and functionally coupled to MHC class II and CD40 on human B cell lines.

Engagement of MHC class II and CD40 on B cell lines triggers intracellular signals that activates cell surface adhesion receptors, resulting in LFA-1-dependent and -independent cell-cell adhesion. In this study, a murine monoclonal antibody (mAb R21) has been produced against a LFA-1-negative human B cell line and proven to completely block MHC class II- and CD40-induced LFA-1-independent homotypic adhesion. However, this mAb failed to prevent MHC class II- or CD40-induced homotypic adhesion in LFA-1-positive Raji B cells, and alone, it triggered LFA-1-dependent cell-cell adhesion. Biochemical characterization indicated that the CD20 molecule, a tetraspan phosphoprotein expressed on B cells that functions as a Ca2+-conductive ion channel, is the target of mAb R21. Interestingly, further biochemical analysis demonstrated that CD20 is physically associated with MHC class II and CD40 molecules on the cell surface of LFA-1-negative and LFA-1-positive B cell lines. Although these three molecules are associated with each other, the complex formation between any two of them is not dependent on the simultaneous expression of the three molecules. Altogether, these results indicate that CD20 is physically and probably functionally coupled to the MHC class II and CD40 molecules; thereby it may have certain modulatory effects on their functions.

Molecular pathogenesis of muscle degeneration in the delta-sarcoglycan-deficient hamster.

The BIO14.6 hamster is an extensively used animal model of autosomal recessive cardiomyopathy and muscular dystrophy. Recently, a large deletion in the 5' end of the delta-sarcoglycan gene was found to be the primary genetic defect in the hamster. In the present investigation, we studied the effects of the delta-sarcoglycan deletion on transcription, expression, and function of the dystrophin-glycoprotein complex in skeletal and cardiac muscle. We demonstrated that in striated muscle the genetic defect leads to the complete deficiency of delta-sarcoglycan and a concomitant loss of alpha-, beta-, and gamma-sarcoglycan. In addition, absence of the sarcoglycan complex reduced the expression of alpha-dystroglycan in striated muscle fibers. These findings indicated that the primary defect in the BIO14.6 hamster leads to the dissociation of the dystroglycan complex from the sarcoglycan complex and disrupted anchorage of alpha-dystroglycan to the cell surface. Using intravenous injection of Evans blue dye as an in vivo tracer assay, we demonstrated that perturbation of the dystrophin-glycoprotein complex caused extensive fiber damage in skeletal and cardiac muscle of the BIO14.6 hamster. Based on our results, we propose that loss of delta-sarcoglycan results in the impairment of sarcolemmal integrity, finally leading to muscular dystrophy and cardiomyopathy.

Molecular characterization and role in T cell activation of staphylococcal enterotoxin A binding to the HLA-DR alpha-chain.

Superantigens bind to MHC class II-positive cells and stimulate T lymphocytes expressing specific V beta regions of the TCR. Two distinct regions of staphylococcal enterotoxin A superantigen (SEA) have been shown to affect the binding to MHC class II molecules. Results presented here demonstrate for the first time that the SEA-DR interaction can be affected by mutations on the class II alpha-chain. Furthermore, we have precisely mapped the interaction of the SEA N-terminal domain with the alpha1 domain of HLA-DR. Scatchard analysis using DAP cells transfected with mutant class II molecules showed a role for residue DR alpha K39 in the binding of SEA. Also, complementation experiments using mutant SEA molecules revealed an interaction between SEA residue F47 and position alphaQ18 on an outer loop of HLA-DR. These interactions between SEAF47 and the DR alpha-chain are critical, as they allow the recognition by an otherwise nonreactive V beta1+ T cell hybridoma and induction of tyrosine phosphorylation through the TCR.

Characterization of delta-sarcoglycan, a novel component of the oligomeric sarcoglycan complex involved in limb-girdle muscular dystrophy.

The sarcoglycan complex is known to be involved in limb-girdle muscular dystrophy (LGMD) and is composed of at least three proteins: alpha-, beta-, and gamma-sarcoglycan. delta-Sarcoglycan has now been identified as a second 35-kDa sarcolemmal transmembrane glycoprotein that shares high homology with gamma-sarcoglycan and is expressed mainly in skeletal and cardiac muscle. Biochemical analysis has demonstrated that gamma- and delta-sarcoglycan are separate entities within the sarcoglycan complex and that all four sarcoglycans exist in the complex on a stoichiometrically equal basis. Immunohistochemical analysis of skeletal muscle biopsies from patients with LGMD2C, LGMD2D, and LGMD2E demonstrated a reduction of the entire sarcoglycan complex in these muscular dystrophies. Furthermore, we have mapped the human delta-sarcoglycan gene to chromosome 5q33-q34 in a region overlapping the recently linked autosomal recessive LGMD2F locus.

A natural mutation of the amino acid residue at position 60 destroys staphylococcal enterotoxin A murine T-cell mitogenicity.

A variety of techniques have been used to identify the amino acid residues of bacterial superantigens involved in their interactions with major histocompatibility complex (MHC) class II and T-cell receptor (TCR). In this study, we isolated a naturally mutated staphylococcal enterotoxin A (SEA) from three different Staphylococcus aureus strains, in which the amino acid at position 60 has been changed from aspartic acid (D) to asparagine (N). We then studied the influence of this change on the immunological activities of SEA. Our results demonstrated that this mutation does not affect the capacity of SEA to bind MHC class II molecules and consequently activates human monocytes and peripheral blood lymphocytes. In contrast, mutated SEA failed to stimulate the proliferation of murine splenic lymphocytes of two different strains, and when presented by human MHC class II molecules, it also failed to activate murine cell line 3DT, which expresses the SEA-specific TCR V beta element (V beta 1). These results indicate that this mutation alters the interaction between SEA and murine TCR. The reactivity patterns of the mutated SEA with two specific anti-SEA monoclonal antibodies suggested that the observed effect of the isolated mutation in the murine system might be due to certain conformational changes in the SEA molecule introduced upon changing the D at position 60 to N. Site-directed mutagenesis of the N residue to D or to glycine reconstituted the ability of SEA to stimulate murine splenic lymphocytes. The different effects of this natural mutation at position 60 on the immunological activities of SEA with murine and human cells highlight the relevance of the affinity and avidity in SEA-TCR interactions in the function of different species or may reflect a difference in epitope specificity.

The expression of P-glycoprotein in canine lymphoma and its association with multidrug resistance.

Canine lymphoma is a spontaneous, naturally occurring disease that is a model for non-Hodgkin's lymphoma in humans. Chemotherapy with antineoplastics results in a high rate of remission; however, relapse and clinical drug resistance are usually seen within 8-10 months. The P-glycoprotein product of the mdr gene is thought to function as an ATP-driven membrane drug efflux pump and appears to play an important role in tumor cell resistance. To assess the role of mdr gene products in drug resistance in canine lymphoma, membrane preparations of lymphoma cells from 31 dogs with high- or intermediate-grade lymphoma were subjected to Western blotting for detection of P-glycoprotein. In this study, one of 30 samples taken from dogs prior to receiving chemotherapy expressed detectable levels of P-glycoprotein. P-glycoprotein was also detected in biopsy samples from 3 of 8 dogs that had become resistant to chemotherapy. This pattern of expression is similar to that in human non-Hodgkin's lymphoma. These studies suggest that canine lymphoma is a useful model for studying multidrug resistance.

Leukotriene synthesis in U937 cells expressing recombinant 5-lipoxygenase.

The U937 human promyelocytic cell line does not express 5-lipoxygenase, but does express 5-lipoxygenase-activating protein (FLAP). U937 cells do not synthesize leukotrienes after stimulation by calcium ionophore A23187. Dimethyl sulfoxide (DMSO) differentiation of U937 cells, towards a more mature monocyte-macrophage lineage, induces the expression of FLAP but not 5-lipoxygenase. These DMSO-differentiated U937 cells also lack the ability to synthesize leukotrienes. We infected viral RNA coding for 5-lipoxygenase into U937 cells using a retroviral vector and measured the synthesis of 5-lipoxygenase, FLAP, leukotrienes and 5-hydroxyeicosatetraenoic acid (5-HETE) by these cells after stimulation with A23187. Undifferentiated U937 cells infected with 5-lipoxygenase RNA expressed 5-lipoxygenase and FLAP but neither leukotrienes nor 5-HETE were detected after these cells were stimulated with A23187. Exposure of the 5-lipoxygenase-infected U937 cells to DMSO increased the expression of 5-lipoxygenase and FLAP, and these cells produced leukotrienes and 5-HETE in response to A23187. The synthesis of these products was inhibited by MK-886, a compound which specifically binds to FLAP.

Pathophysiology and pharmacologic modulation of hepatic fibrosis.

Most chronic liver disorders are accompanied morphologically by the deposition of fibrous tissue within the hepatic parenchyma. This fibrotic tissue compromises hepatic function and contributes significantly to hepatic failure. Fibrosis is a dynamic process associated with the continual deposition and resorption of connective tissue. Therapeutic strategies are emerging whereby this dynamic process can be modulated. Since collagen is the major component of the extracellular matrix deposited in hepatic fibrosis, most anti-fibrotic therapies have been directed toward the control of collagen metabolism. After collagen genes are transcribed and translated into precursor procollagen proteins, a number of post-translational modifications that ensure the deposition of structurally sound collagen within the extracellular matrix occur. A number of drugs can specifically modulate collagen biosynthesis at the transcriptional level or at various post-translational stages. These anti-fibrotic drugs include corticosteroids, azathioprine, penicillamine, colchicine, zinc, prostaglandins, cyclosporine, and interferons. The pharmacologic action of these drugs and the clinical role in veterinary and human fibrotic hepatopathies will be discussed.

Primary structure of dystrophin-associated glycoproteins linking dystrophin to the extracellular matrix.

The primary sequence of two components of the dystrophin-glycoprotein complex has been established by complementary, DNA cloning. The transmembrane 43K and extracellular 156K dystrophin-associated glycoproteins (DAGs) are encoded by a single messenger RNA and the extracellular 156K DAG binds laminin. Thus, the 156K DAG is a new laminin-binding glycoprotein which may provide a linkage between the sarcolemma and extracellular matrix. These results support the hypothesis that the dramatic reduction in the 156K DAG in Duchenne muscular dystrophy leads to a loss of a linkage between the sarcolemma and extracellular matrix and that this may render muscle fibres more susceptible to necrosis.

Dystrophin-related protein is localized to neuromuscular junctions of adult skeletal muscle.

Dystrophin-related protein (DRP) is an autosomal gene product with high homology to dystrophin. We have used highly specific antibodies to the unique C-terminal peptide sequences of DRP and dystrophin to examine the subcellular localization and biochemical properties of DRP in adult skeletal muscle. DRP is enriched in isolated sarcolemma from control and mdx mouse muscle, but is much less abundant than dystrophin. Immunofluorescence microscopy localized DRP almost exclusively to the neuromuscular junction region in rabbit and mouse skeletal muscle, as well as mdx mouse muscle and denervated mouse muscle. DRP is also present in normal size and abundance and localizes to the neuromuscular junction region in muscle from the dystrophic mouse model dy/dy. Thus, DRP is a junction-specific membrane cytoskeletal protein that may play an important role in the organization of the postsynaptic membrane of the neuromuscular junction.

Treatment of posttraumatic carinal stenosis by balloon dilation during thoracotomy in a cat.

A cat with posttraumatic stenosis of the terminal part of the thoracic portion of the trachea and carina was successfully treated by balloon dilation of the stenosis during thoracotomy. Thoracotomy was performed at the right fifth intercostal space, and the stenosis was dilated, using a balloon-tipped catheter passed through an endotracheal tube. A 6-week course of orally administered corticosteroids was instituted after surgery. Balloon dilation during thoracotomy is a technically simple procedure for treatment of stenosis of the thoracic portion of the trachea.

Identification and characterization of proteins in sarcoplasmic reticulum from normal and failing human left ventricles.

Monoclonal and polyclonal antibodies to the major sarcoplasmic reticulum proteins of rabbit skeletal and canine cardiac muscle have been used to identify and characterize the corresponding components of human cardiac sarcoplasmic reticulum. The Ca2(+)-transporting ATPase of human cardiac sarcoplasmic reticulum was identified as a 105,000-Da protein antigenically distinct from its rabbit skeletal muscle counterpart. Human cardiac sarcoplasmic reticulum also contained 53,000- 155,000- and 165,000-Da glycoproteins antigenically related to the low and high molecular weight glycoproteins of canine cardiac and rabbit skeletal muscle sarcoplasmic reticulum. The ryanodine-sensitive Ca2+ channel of human cardiac sarcoplasmic reticulum was identified as a 400,000-Da protein antigenically related to its counterparts in canine cardiac and rabbit skeletal muscle. Human cardiac calsequestrin was identified as a 52,000-Da protein. Human phospholamban was identified as a 29,000-Da substrate for phosphorylation by cAMP-dependent protein kinase. Immunoblots of sarcoplasmic reticulum from the normal left ventricles of four unmatched organ donors and the excised failing left ventricles of nine patients with idiopathic dilated cardiomyopathy were compared in search of qualitative differences in the protein patterns of the failing hearts. No such differences were found with respect to the Ca2+ ATPase, the 53,000-Da glycoprotein, the ryanodine-sensitive Ca2+ channel, calsequestrin or phospholamban. In contrast, the 165,000-Da glycoprotein band, present in all four preparations from nonfailing hearts, was absent from three of nine preparations from failing hearts, and staining of the 155,000-Da glycoprotein in these three preparations appeared to be relatively increased. The absence of the 165,000-Da glycoprotein band may identify or reflect a pathogenetic mechanism in a subset of patients with idiopathic dilated cardiomyopathy.

Correlation between expression of 5-lipoxygenase-activating protein, 5-lipoxygenase, and cellular leukotriene synthesis.

Previous studies involving transfection of cDNAs for 5-lipoxygenase-activating protein (FLAP) and 5-lipoxygenase into osteosarcoma cells have shown that both these proteins are essential for leukotriene synthesis (Dixon, R. A. F., Diehl, R. E., Opas, E., Rands, E., Vickers, P. J., Evans, J. F., Gillard, J. W., and Miller, D. K. (1990) Nature 343, 282-284). In the present study we show that FLAP is present in a variety of cells known to produce leukotrienes, but is absent from a number of cells which do not synthesize leukotrienes. Furthermore, differentiation of the human promyelocytic HL-60 cell line towards granulocytic cells following exposure to dimethylsulfoxide is associated with the concurrent induction of both FLAP and 5-lipoxygenase and an increased capacity to synthesize leukotrienes. Cellular leukotriene synthesis in this system is functionally dependent on FLAP as shown by its inhibition by the leukotriene biosynthesis inhibitor MK-886, a compound which specifically binds to FLAP.

Identification and isolation of a membrane protein necessary for leukotriene production.

Several inflammatory diseases, including asthma, arthritis and psoriasis are associated with the production of leukotrienes by neutrophils, mast cells and macrophages. The initial enzymatic step in the formation of leukotrienes is the oxidation of arachidonic acid by 5-lipoxygenase (5-LO) to leukotriene A4. Osteosarcoma cells transfected with 5-LO express active enzyme in broken cell preparations, but no leukotriene metabolites are produced by these cells when stimulated with the calcium ionophore A23187, indicating that an additional component is necessary for cellular 5-LO activity. A new class of indole leukotriene inhibitor has been described that inhibits the formation of cellular leukotrienes but has no direct inhibitory effect on soluble 5-LO activity. We have now used these potent agents to identify and isolate a novel membrane protein of relative molecular mass 18,000 which is necessary for cellular leukotriene synthesis.

The pharmacology of L-670,596, a potent and selective thromboxane/prostaglandin endoperoxide receptor antagonist.

L-670,596 ((-)6,8-difluoro-9-rho-methylsulfonyl benzyl-1,2,3,4- tetrahydrocarbazol-1-yl-acetic acid) has been shown to be a potent receptor antagonist as evidenced by the inhibition of the binding of 125I-labeled PTA-OH to human platelets (IC50, 5.5 x 10(-9) M), inhibition of U-44069 induced aggregation of human platelet rich plasma (IC50, 1.1 x 10(-7) M), and competitive inhibition of contractions of the guinea pig tracheal chain induced by U-44069 (pA2,9.0). The compound was also active in vivo as shown by inhibition of arachidonic acid and U-44069 induced bronchoconstriction in the guinea pig (ED50 values, 0.04 and 0.03 mg/kg i.v., respectively), U44069 induced renal vasoconstriction in the pig (ED50, 0.02 mg/kg i.v.), and inhibition of ex vivo aggregation of rhesus monkey platelets to U-44069 (active 1-5 mg/kg p.o.). The selectivity of the compound was indicated by the failure to inhibit, first, ADP-induced human or primate platelet aggregation and, second, bronchoconstriction in the guinea pig in vivo and contraction of the guinea pig tracheal chain in vitro to a variety of agonists. It is concluded that L-670,596 is a potent, selective, orally active thromboxane A2/prostaglandin endoperoxide receptor antagonist.