OBJECTIVE

Treatment of melanoma patients with selective BRAF inhibitors results in objective clinical responses in the majority of patients with BRAF-mutant tumors. However, resistance to these inhibitors develops within a few months. In this study, we test the hypothesis that BRAF inhibition in combination with adoptive T-cell transfer (ACT) will be more effective at inducing long-term clinical regressions of BRAF-mutant tumors.

METHODS

BRAF-mutated human melanoma tumor cell lines transduced to express gp100 and H-2D(b) to allow recognition by gp100-specific pmel-1 T cells were used as xenograft models to assess melanocyte differentiation antigen-independent enhancement of immune responses by BRAF inhibitor PLX4720. Luciferase-expressing pmel-1 T cells were generated to monitor T-cell migration in vivo. The expression of VEGF was determined by ELISA, protein array, and immunohistochemistry. Importantly, VEGF expression after BRAF inhibition was tested in a set of patient samples.

RESULTS

We found that administration of PLX4720 significantly increased tumor infiltration of adoptively transferred T cells in vivo and enhanced the antitumor activity of ACT. This increased T-cell infiltration was primarily mediated by the ability of PLX4720 to inhibit melanoma tumor cell production of VEGF by reducing the binding of c-myc to the VEGF promoter. Furthermore, analysis of human melanoma patient tumor biopsies before and during BRAF inhibitor treatment showed downregulation of VEGF consistent with the preclinical murine model.

CONCLUSIONS

These findings provide a strong rationale to evaluate the potential clinical application of combining BRAF inhibition with T-cell-based immunotherapy for the treatment of patients with melanoma.

By three criteria, we have demonstrated that B cell stimulatory factor (BSF-1) and B cell differentiation factor (BCDF-gamma) are the same lymphokine. Highly purified preparations of high performance liquid chromatography-purified or affinity-purified BSF-1 had BCDF-gamma activity but not BCDF-mu activity. A monoclonal anti-BSF-1 antibody coupled to Sepharose depleted both BSF-1 and BCDF-gamma activity but not BCDF-mu activity from two different T cell supernatants. Soluble monoclonal anti-BSF-1 blocked the BSF-1 and BCDF-gamma but not the BCDF-mu responses. These results suggest that BSF-1 acts on both resting and activated B cells to induce different effects.

Multiple sclerosis (MS) is a complex trait in which allelic variation in the MHC class II region exerts the single strongest effect on genetic risk. Epidemiological data in MS provide strong evidence that environmental factors act at a population level to influence the unusual geographical distribution of this disease. Growing evidence implicates sunlight or vitamin D as a key environmental factor in aetiology. We hypothesised that this environmental candidate might interact with inherited factors and sought responsive regulatory elements in the MHC class II region. Sequence analysis localised a single MHC vitamin D response element (VDRE) to the promoter region of HLA-DRBBBBBB cells transiently transfected with the HLA-DRBBBBaction with the major locus determining genetic susceptibility. These findings support a connection between the main epidemiological and genetic features of this disease with major practical implications for studies of disease mechanism and prevention.

During early tooth development, multiple signaling molecules are expressed in the dental lamina epithelium and induce the dental mesenchyme. One signal, BMP4, has been shown to induce morphologic changes in dental mesenchyme and mesenchymal gene expression via Msx1, but BMP4 cannot substitute for all the inductive functions of the dental epithelium. To investigate the role of FGFs during early tooth development, we examined the expression of epithelial and mesenchymal Fgfs in wild-type and Msx1 mutant tooth germs and tested the ability of FGFs to induce Fgf3 and Bmp4 expression in wild-type and Msx1 mutant dental mesenchymal explants. Fgf8 expression is preserved in Msx1 mutant epithelium while that of Fgf3 is not detected in Msx1 mutant dental mesenchyme. Moreover, dental epithelium as well as beads soaked in FGF1, FGF2 or FGF8 induce Fgf3 expression in dental mesenchyme in an Msx1-dependent manner. These results indicate that, like BMP4, FGF8 constitutes an epithelial inductive signal capable of inducing the expression of downstream signaling molecules in dental mesenchyme via Msx1. However, the BMP4 and FGF8 signaling pathways are distinct. BMP4 cannot induce Fgf3 nor can FGFs induce Bmp4 expression in dental mesenchyme, even though both signaling molecules can induce Msx1 and Msx1 is necessary for Fgf3 and Bmp4 expression in dental mesenchyme. In addition, we have investigated the effects of FGFs and BMP4 on the distal-less homeobox genes Dlx1 and Dlx2 and we have clarified the relationship between Msx and Dlx gene function in the developing tooth. Dlx1,Dlx2 double mutants exhibit a lamina stage arrest in maxillary molar tooth development (Thomas B. L., Tucker A. S., Qiu M. , Ferguson C. A., Hardcastle Z., Rubenstein J. L. R. and Sharpe P. T. (1997) Development 124, 4811-4818). Although the maintenance of molar mesenchymal Dlx2 expression at the bud stage is Msx1-dependent, both the maintenance of Dlx1 expression and the initial activation of mesenchymal Dlx1 and Dlx2 expression during the lamina stage are not. Moreover, in contrast to the tooth bud stage arrest observed in Msx1 mutants, Msx1,Msx2 double mutants exhibit an earlier phenotype closely resembling the lamina stage arrest observed in Dlx1,Dlx2 double mutants. These results are consistent with functional redundancy between Msx1 and Msx2 in dental mesenchyme and support a model whereby Msx and Dlx genes function in parallel within the dental mesenchyme during tooth initiation. Indeed, as predicted by such a model, BMP4 and FGF8, epithelial signals that induce differential Msx1 and Msx2 expression in dental mesenchyme, also differentially induce Dlx1 and Dlx2 expression, and do so in an Msx1-independent manner. These results integrate Dlx1, Dlx2 and Fgf3 and Fgf8 into the odontogenic regulatory hierarchy along with Msx1, Msx2 and Bmp4, and provide a basis for interpreting tooth induction in terms of transcription factors which, individually, are necessary but not sufficient for the expression of downstream signals and therefore must act in specific combinations.

The Drosophila transforming growth factor beta (TGF-beta) homolog Decapentaplegic (Dpp) acts as a morphogen that forms a long-range concentration gradient to direct the anteroposterior patterning of the wing. Both planar transcytosis initiated by Dynamin-mediated endocytosis and extracellular diffusion have been proposed for Dpp movement across cells. In this work, we found that Dpp is mainly extracellular, and its extracellular gradient coincides with its activity gradient. We demonstrate that a blockage of endocytosis by the dynamin mutant shibire does not block Dpp movement but rather inhibits Dpp signal transduction, suggesting that endocytosis is not essential for Dpp movement but is involved in Dpp signaling. Furthermore, we show that Dpp fails to move across cells mutant for dally and dally-like (dly), two Drosophila glypican members of heparin sulfate proteoglycan (HSPG). Our results support a model in which Dpp moves along the cell surface by restricted extracellular diffusion involving the glypicans Dally and Dly.

The human T-cell lymphotropic virus type III (HTLV-III) is the etiologic agent of the acquired immunodeficiency syndrome (AIDS) and preferentially infects T4 lymphocytes. Other cell types, notably B lymphocytes and other nonlymphoid cells, also have been reported to be infected in vitro by HTLV-III. We now report on the susceptibility of human pulmonary macrophages to infection with HTLV-III in vitro. Alveolar macrophages infected with HTLV-III produced low levels of virus that could be transferred to allogeneic human peripheral blood mononuclear leukocytes as long as 2 weeks after initiation of infection. Unlike HTLV-III infection of T lymphocytes, macrophages appeared more resistant to viral-mediated cytopathic effects. Primary cultures of pulmonary macrophages from two of four patients with AIDS spontaneously produced low levels of virus detected as precipitable reverse transcriptase activity, suggesting that these cells were infected in vivo. Because tissue macrophages are long-lived cells, they may act as a reservoir of HTLV-III, capable of transmitting the virus to other susceptible cells such as T lymphocytes, causing periodic low-level viremia. Macrophage infection with HTLV-III may be one mechanism for the establishment of viral persistence in infected hosts.

We examined pancreas biopsy specimens from 18 newly diagnosed insulin-dependent diabetes mellitus (IDDM) patients to elucidate the mechanism underlying beta cell destruction. Pancreas islets were seen in all patients and insulitis in eight patients. Infiltrating mononuclear cells consisted of CD4+T, CD8+T, B lymphocytes, and macrophages. Among them, CD8+T lymphocytes were predominant and macrophages followed. The expression of MHC class I antigens was increased in islet and endothelial cells in nine patients. MHC class II expression was increased in endothelial cells of the same patients. The expression of intercellular adhesion molecule-1 was increased in endothelial cells in two of the nine patients with MHC hyperexpression; in one of them, lymphocyte function-associated antigen-3 expression was also increased. Out of the eight patients with insulitis, seven showed MHC class I hyper-expression, whereas 2 of the 10 patients without insulitis showed the phenomenon (P < 0.05). The relation between insulitis and the hyperexpression of adhesion molecules was not evident. In conclusion, we revealed the close relation between CD8+T lymphocyte-predominant insulitis and MHC class I hyperexpression in islet cells. This suggests that infiltrating CD8+T lymphocytes recognize islet autoantigens in association with increased MHC class I molecules and act as major effector cells in autoimmune response against islet cells in IDDM pancreases. The role of adhesion molecules in the pathogenesis of IDDM still remains to be elucidated.

By co-injecting fluorescent tubulin and vinculin into fish fibroblasts we have revealed a "cross talk" between microtubules and early sites of substrate contact. This mutuality was first indicated by the targeting of vinculin-rich foci by microtubules during their growth towards the cell periphery. In addition to passing directly over contact sites, the ends of single microtubules could be observed to target several contacts in succession or the same contact repetitively, with intermittent withdrawals. Targeting sometimes involved side-stepping, or the major re-routing of a microtubule, indicative of a guided, rather than a random process. The paths that microtubules followed into contacts were unrelated to the orientation of stress fiber assemblies and targeting occurred also in mouse fibroblasts that lacked a system of intermediate filaments. Further experiments with microtubule inhibitors showed that adhesion foci can: (a) capture microtubules and stabilize them against disassembly by nocodazole; and (b), act as preferred sites of microtubule polymerization, during either early recovery from nocodazole, or brief treatment with taxol. From these and other findings we speculate that microtubules are guided into substrate contact sites and through the motor-dependent delivery of signaling molecules serve to modulate their development. It is further proposed this modulation provides the route whereby microtubules exert their influence on cell shape and polarity.

Hepatitis B virus (HBV) is an enveloped DNA virus with a spherical capsid (or core). The capsid is constructed from 120 copies of the homodimeric capsid protein arranged with T = 4 icosahedral symmetry. We examined in vitro assembly of purified E. coli expressed HBV capsid protein. After equilibration, concentrations of capsid and dimer were evaluated by size exclusion chromatography. The extent of assembly increased as temperature and ionic strength increased. The concentration dependence of capsid assembly conformed to the equilibrium expression: K(capsid) = [capsid]/[dimer](120). Given the known geometry for HBV capsids and dimers, the per capsid assembly energy was partitioned into energy per subunit-subunit contact. We were able to make three major conclusions. (i) Weak interactions (from -2.9 kcal/mol at 21 degrees C in low salt to -4.4 kcal/mol at 37 degrees C in high salt) at each intersubunit contact result in a globally stable capsid; weak intersubunit interactions may be the basis for the phenomenon of capsid breathing. (ii) HBV assembly is characterized by positive enthalpy and entropy. The reaction is entropy-driven, consistent with the largely hydrophobic contacts found in the crystal structure. (iii) Increasing NaCl concentration increases the magnitude of free energy, enthalpy, and entropy, as if ionic strength were increasing the amount of hydrophobic surface buried by assembly. This last point leads us to suggest that salt acts by inducing a conformational change in the dimer from an assembly-inactive form to an assembly-active form. This model of conformational change linked to assembly is consistent with immunological differences between dimer and capsid.

The adenomatous polpyposis coli (APC) protein is mutated in most colorectal tumours. Nearly all APC mutations are truncations, and many of these terminate in the mutation cluster region located halfway through the protein. In cancer cells expressing mutant APC, beta-catenin is stabilized and translocates into the nucleus to act as a transcriptional co-activator of T-cell factor. During normal development, APC also promotes the destabilization of beta-catenin and Drosophila Armadillo. It does so by binding to the Axin complex which earmarks beta-catenin/Armadillo for degradation by the proteasome pathway. APC has a regulatory role in this process, which is poorly understood. Here we show that APC contains highly conserved nuclear export signals 3' adjacent to the mutation cluster region that enable it to exit from the nucleus. This ability is lost in APC mutant cancer cells, and we provide evidence that beta-catenin accumulates in the nucleus as a result. Thus, the ability of APC to exit from the nucleus appears to be critical for its tumour suppressor function.

Suberin and cutin are fatty acid- and glycerol-based plant polymers that act as pathogen barriers and function in the control of water and solute transport. However, despite important physiological roles, their biosynthetic pathways, including the acyl transfer reactions, remain hypothetical. We report the characterization of two suberin mutants (gpat5-1 and gpat5-2) of Arabidopsis thaliana GPAT5, encoding a protein with acyl-CoA:glycerol-3-phosphate acyltransferase activity. RT-PCR and beta-glucuronidase-promoter fusion analyses demonstrated GPAT5 expression in seed coat, root, hypocotyl, and anther. The gpat5 plants showed a 50% decrease in aliphatic suberin in young roots and produced seed coats with a severalfold reduction in very long chain dicarboxylic acid and omega-hydroxy fatty acids typical of suberin but no change in the composition or content of membrane or storage glycerolipids or surface waxes. Consistent with their altered suberin, seed coats of gpat5 mutants had a steep increase in permeability to tetrazolium salts compared with wild-type seed coats. Furthermore, the germination rate of gpat5 seeds under high salt was reduced, and gpat5 seedlings had lower tolerance to salt stress. These results provide evidence for a critical role of GPAT5 in polyester biogenesis in seed coats and roots and for the importance of lipid polymer structures in the normal function of these organs.

Multiple molecular defects trigger cell death in amyotrophic lateral sclerosis (ALS). Among these, altered transcriptional activity may perturb many cellular functions, leading to a cascade of secondary pathological effects. We showed that pharmacological treatment, using the histone deacetylase inhibitor sodium phenylbutyrate, significantly extended survival and improved both the clinical and neuropathological phenotypes in G93A transgenic ALS mice. Phenylbutyrate administration ameliorated histone hypoacetylation observed in G93A mice and induced expression of nuclear factor-kappaB (NF-kappaB) p50, the phosphorylated inhibitory subunit of NF-kappaB (pIkappaB) and beta cell lymphoma 2 (bcl-2), but reduced cytochrome c and caspase expression. Curcumin, an NF-kappaB inhibitor, and mutation of the NF-kappaB responsive element in the bcl-2 promoter, blocked butyrate-induced bcl-2 promoter activity. We provide evidence that the pharmacological induction of NF-kappaB-dependent transcription and bcl-2 gene expression is neuroprotective in ALS mice by inhibiting programmed cell death. Phenylbutyrate acts to phosphorylate IkappaB, translocating NF-kappaB p50 to the nucleus, or to directly acetylate NF-kappaB p50. NF-kappaB p50 transactivates bcl-2 gene expression. Up-regulated bcl-2 blocks cytochrome c release and subsequent caspase activation, slowing motor neuron death. These transcriptional and post-translational pathways ultimately promote motor neuron survival and ameliorate disease progression in ALS mice. Phenylbutyrate may therefore provide a novel therapeutic approach for the treatment of patients with ALS.

The homologous insulin and insulin-like growth factor (IGF) receptors are both expressed in the brain, in overlapping but distinct neuroanatomical patterns. In contrast to insulin, IGF1 is also highly expressed within the brain and is essential for normal brain development. IGF1 promotes projection neuron growth, dendritic arborization and synaptogenesis. IGF1 acts in an autocrine and/or paracrine manner to promote glucose utilization, using phosphatidylinositol 3 kinase (PI3K)/Akt, also known as protein kinase B (PKB)/glycogen synthase kinase 3beta (GSK3beta) pathways similar to insulin signaling in peripheral tissues. IGF1 promotes neuronal survival during normal brain development mainly in hippocampal and olfactory systems that depend on postnatal neurogenesis. IGF1's anabolic and neuroprotective roles may be coordinated by inhibition of GSK3beta. The identification of GSK3beta as a major target of brain IGF1 signaling provides a unifying pathway for IGF1's well-established anabolic and anti-apoptotic functions, with IGF1-induced inhibition of GSK3beta triggering multifaceted anabolic and neuroprotective effects.

To help understand the process of carpel morphogenesis, the roles of three carpel development genes have been partitioned genetically. Mutants of CRABS CLAW cause the gynoecium to develop into a wider but shorter structure, and the two carpels are unfused at the apex. Mutants of a second gene, SPATULA, show reduced growth of the style, stigma, and septum, and the transmitting tract is absent. Double mutants of crabs claw and spatula with homeotic mutants that develop ectopic carpels demonstrate that CRABS CLAW and SPATULA are necessary for, and inseparable from, carpel development, and that their action is negatively regulated by A and B organ identity genes. The third carpel gene studied, AGAMOUS, encodes C function that has been proposed to fully specify carpel identity. When AGAMOUS function is removed together with the A class gene APETALA2, however, the organs retain many carpelloid properties, suggesting that other genes are also involved. We show here that further mutant disruption of both CRABS CLAW and SPATULA function removes remaining carpelloid properties, revealing that the three genes together are necessary to generate the mature gynoecium. In particular, AGAMOUS is required to specify the identity of the carpel wall and to promote the stylar outgrowth at the apex, CRABS CLAW suppresses radial growth of the developing gynoecium but promotes its longitudinal growth, and SPATULA supports development of the carpel margins and tissues derived from them. The three genes mostly act independently, although there is genetic evidence that CRABS CLAW enhances AGAMOUS and SPATULA function.

The ATE1-encoded Arg-transferase mediates conjugation of Arg to N-terminal Asp, Glu, and Cys of certain eukaryotic proteins, yielding N-terminal Arg that can act as a degradation signal for the ubiquitin-dependent N-end rule pathway. We have previously shown that mouse ATE1-/- embryos die with defects in heart development and angiogenesis. Here, we report that the ATE1 Arg-transferase mediates the in vivo degradation of RGS4 and RGS5, which are negative regulators of specific G proteins whose functions include cardiac growth and angiogenesis. The proteolysis of these regulators of G protein signaling (RGS) proteins was perturbed either by hypoxia or in cells lacking ubiquitin ligases UBR1 and/or UBR2. Mutant RGS proteins in which the conserved Cys-2 residue could not become N-terminal were long-lived in vivo. We propose a model in which the sequential modifications of RGS4, RGS5, and RGS16 (N-terminal exposure of their Cys-2, its oxidation, and subsequent arginylation) act as a licensing mechanism in response to extracellular and intracellular signals before the targeting for proteolysis by UBR1 and UBR2. We also show that ATE1-/- embryos are impaired in the activation of extracellular signal-regulated kinase mitogen-activated protein kinases and in the expression of G protein-induced downstream effectors such as Jun, cyclin D1, and beta-myosin heavy chain. These results establish RGS4 and RGS5 as in vivo substrates of the mammalian N-end rule pathway and also suggest that the O2-ATE1-UBR1/UBR2 proteolytic circuit plays a role in RGS-regulated G protein signaling in the cardiovascular system.

Non-prescriptional use of medicinal herbs among cancer patients is common around the world. The alleged anti-cancer effects of most herbal extracts are mainly based on studies derived from in vitro or in vivo animal experiments. The current information suggests that these herbal extracts exert their biological effect either through cytotoxic or immunomodulatory mechanisms. One of the active compounds responsible for the immune effects of herbal products is in the form of complex polysaccharides known as beta-glucans. beta-glucans are ubiquitously found in both bacterial or fungal cell walls and have been implicated in the initiation of anti-microbial immune response. Based on in vitro studies, beta-glucans act on several immune receptors including Dectin-1, complement receptor (CR3) and TLR-2/6 and trigger a group of immune cells including macrophages, neutrophils, monocytes, natural killer cells and dendritic cells. As a consequence, both innate and adaptive response can be modulated by beta-glucans and they can also enhance opsonic and non-opsonic phagocytosis. In animal studies, after oral administration, the specific backbone 1-->3 linear beta-glycosidic chain of beta-glucans cannot be digested. Most beta-glucans enter the proximal small intestine and some are captured by the macrophages. They are internalized and fragmented within the cells, then transported by the macrophages to the marrow and endothelial reticular system. The small beta-glucans fragments are eventually released by the macrophages and taken up by other immune cells leading to various immune responses. However, beta-glucans of different sizes and branching patterns may have significantly variable immune potency. Careful selection of appropriate beta-glucans is essential if we wish to investigate the effects of beta-glucans clinically. So far, no good quality clinical trial data is available on assessing the effectiveness of purified beta-glucans among cancer patients. Future effort should direct at performing well-designed clinical trials to verify the actual clinical efficacy of beta-glucans or beta-glucans containing compounds.

Inappropriate activation of downstream target genes by the oncoprotein beta-catenin is implicated in development of numerous human cancers. beta-catenin and its fruitfly counterpart Armadillo act as a coactivator in the canonical Wnt/Wingless pathway by binding to Tcf/Lef transcription factors. Here we report a conserved nuclear protein, named Chibby, which was identified in a screen for proteins that directly interact with the C-terminal region of beta-catenin. In mammalian cultured cells we demonstrate that Chibby inhibits beta-catenin-mediated transcriptional activation by competing with Lef-1 to bind to beta-catenin. Inhibition of Drosophila Chibby by RNA interference results in segment polarity defects that mimick a wingless gain-of-function phenotype, and overexpression of the wingless target genes engrailed and Ultrabithorax. In addition, epistasis experiments indicate that chibby acts downstream of wingless and upstream of armadillo.

The regulatory signals responsible for the increased biosynthesis of prostaglandins during parturition have not been established. Because interleukin-1 is capable of stimulating prostaglandin production by intrauterine tissues and is an inflammatory mediator, we propose that interleukin-1 may act as a signal for the onset of human labor in the setting of intrauterine infection. The purpose of these studies was to determine interleukin-1 activity in amniotic fluid and to establish its relationship with the onset of term and preterm labor. Amniotic fluid from 182 patients was assayed for interleukin-1 activity. Cell-associated interleukin-1 activity was detected in fluid obtained in the third trimester but not in fluid obtained in the second trimester of pregnancy, suggesting a maturational event in interleukin-1 production. The factor responsible for interleukin-1 activity had biochemical characteristics of interleukin-1 alpha (estimated molecular weight of 14 kilodaltons, isoelectric point = 4.9), and its activity was blocked with an anti-interleukin-1 alpha antisera. Women in spontaneous labor at term were likely to have fluid phase interleukin-1 activity in amniotic fluid than women who were not in labor at term. Preterm labor in the setting of intraamniotic infections was associated with significant interleukin-1 activity in amniotic fluid. This bioactivity was predominantly attributable to interleukin-1 beta. A strong correlation between interleukin-1 and amniotic fluid concentrations of prostaglandin E2 and prostaglandin F2 alpha was found in women in preterm labor. These findings support the hypothesis that interleukin-1 may play a role in the initiation of preterm labor associated with intraamniotic infection.

The mammalian heart rate is regulated by the vagus nerve, which acts via muscarinic acetylcholine receptors to cause hyperpolarization of atrial pacemaker cells. The hyperpolarization is produced by the opening of potassium channels and involves an intermediary guanosine triphosphate-binding regulatory (G) protein. Potassium channels in isolated, inside-out patches of membranes from atrial cells now are shown to be activated by a purified pertussis toxin-sensitive G protein of subunit composition alpha beta gamma, with an alpha subunit of 40,000 daltons. Thus, mammalian atrial muscarinic potassium channels are activated directly by a G protein, not indirectly through a cascade of intermediary events. The G protein regulating these channels is identified as a potent Gk; it is active at 0.2 to 1 pM. Thus, proteins other than enzymes can be under control of receptor coupling G proteins.

Insulators are DNA sequence elements that can serve in some cases as barriers to protect a gene against the encroachment of adjacent inactive condensed chromatin. Some insulators also can act as blocking elements to protect against the activating influence of distal enhancers associated with other genes. Although most of the insulators identified so far derive from Drosophila, they also are found in vertebrates. An insulator at the 5' end of the chicken beta-globin locus marks a boundary between an open chromatin domain and a region of constitutively condensed chromatin. Detailed analysis of this element shows that it possesses both enhancer blocking activity and the ability to screen reporter genes against position effects. Enhancer blocking is associated with binding of the protein CTCF; sites that bind CTCF are found at other critical points in the genome. Protection against position effects involves other properties that appear to be associated with control of histone acetylation and methylation. Insulators thus are complex elements that can help to preserve the independent function of genes embedded in a genome in which they are surrounded by regulatory signals they must ignore.

A common structural motif consisting of a cystine knot and a small triple-stranded beta-sheet has been defined from comparison of the 3-dimensional structures of the polypeptides omega-conotoxin GVIA (Conus geographus), kalata BI (Oldenlandia affinis DC), and CMTI-I (Curcurbita maxima). These 3 polypeptides have diverse biological activities and negligible amino acid sequence identity, but each contains 3 disulfide bonds that give rise to a cystine knot. This knot consists of a ring formed by the first 2 bonds (1-4 and 2-5) and the intervening polypeptide backbone, through which the third disulfide (3-6) passes. The other component of this motif is a triple-stranded, anti-parallel beta-sheet containing a minimum of 10 residues, XXC2, XC5X, XXC6X (where the numbers on the half-cysteine residues refer to their positions in the disulfide pattern). The presence in these polypeptides of both the cysteine knot and antiparallel beta-sheet suggests that both structural features are required for the stability of the motif. This structural motif is also present in other protease inhibitors and a spider toxin. It appears to be one of the smallest stable globular domains found in proteins and is commonly used in toxins and inhibitors that act by blocking the function of larger protein receptors such as ion channels or proteases.

OBJECTIVE

The goal of this study was to investigate the mechanism of reduced vasoconstrictor sensitivity to norepinephrine in women compared with men.

BACKGROUND

<em>beta</em>2-adrenergic agonists such as albuterol dilate forearm resistance vessels, partly by activating the L-arginine/nitric oxide pathway. Norepinephrine (which <em>acts</em> as <em>beta</em>- as well as alpha-adrenergic receptors) causes less forearm vasoconstriction in women than it does in men. This could be explained by a greater sensitivity to <em>beta</em>2-receptor stimulation in women than in men.

METHODS

Forearm blood flow was measured by venous occlusion plethysmography in healthy women (days 10 to 14 of the menstrual cycle) and in men. Drugs were administered via the brachial artery in three separate protocols: albuterol +/- NG-monomethyl-L-arginine (an inhibitor of nitric oxide synthase); substance P, nitroprusside and verapamil (control vasodilators); norepinephrine (+/- propranolol, a beta-adrenergic receptor antagonist).

RESULTS

Vasodilator responses to albuterol were greater in women than they were in men (p = 0.02 by analysis of variance). NG-monomethyl-L-arginine reduced these similarly in men and women. Responses to control vasodilators were less in women than they were in men (each p < 0.05). Norepinephrine caused less vasoconstriction in women than it did in men (p = 0.02). Propranolol did not influence basal flow in either gender nor responses of men to norepinephrine but increased vasoconstriction to each dose of norepinephrine in women (p < 0.0001 for interaction between gender and propranolol). Responses to norepinephrine coinfused with propranolol were similar in men and women.

CONCLUSIONS

Stimulation of <em>beta</em>2-adrenergic receptors causes greater forearm vasodilation in premenopausal women, at midmenstrual cycle, than it does in men. This is sufficient to explain why vasoconstriction to brachial artery norepinephrine is attenuated in such women.

Translocation of conventional protein kinases C (PKCs) to the plasma membrane leads to their specific association with transmembrane-4 superfamily (TM4SF; tetraspanin) proteins (CD9, CD53, CD81, CD82, and CD151), as demonstrated by reciprocal co-immunoprecipitation and covalent cross-linking experiments. Although formation and maintenance of TM4SF-PKC complexes are not dependent on integrins, TM4SF proteins can act as linker molecules, recruiting PKC into proximity with specific integrins. Previous studies showed that the extracellular large loop of TM4SF proteins determines integrin associations. In contrast, specificity for PKC association probably resides within cytoplasmic tails or the first two transmembrane domains of TM4SF proteins, as seen from studies with chimeric CD9 molecules. Consistent with a TM4SF linker function, only those integrins (alpha(3)beta(1), alpha(6)beta(1), and a chimeric "X3TC5" alpha(3) mutant) that associated strongly with tetraspanins were found in association with PKC. We propose that PKC-TM4SF-integrin structures represent a novel type of signaling complex. The simultaneous binding of TM4SF proteins to the extracellular domains of the integrin alpha(3) subunit and to intracellular PKC helps to explain why the integrin alpha3 extracellular domain is needed for both intracellular PKC recruitment and PKC-dependent phosphorylation of the alpha(3) integrin cytoplasmic tail.

The lasR gene of Pseudomonas aeruginosa is required for transcription of the genes for elastase (lasB) and LasA protease (lasA), two proteases associated with virulence. We report here that the alkaline protease gene (apr) also requires the lasR gene for transcription. Alkaline protease mRNA was absent in the lasR mutant PAO-R1 and present when an intact lasR gene was supplied in trans as determined by Northern (RNA) analysis. The lasR gene also enhances exotoxin A production. Exotoxin A activity in supernatants of PAO-R1 were 30% less than in supernatants of the parental strain, PAO-SR. Multiple copies of lasR in trans in PAO-R1 in increased toxin A activity to twice the parental levels. Analysis of PAO-R1 containing the toxA promoter fused to beta-galactosidase suggests that LasR acts at the toxA promoter or at upstream toxA mRNA sequences. beta-Galactosidase activity was approximately 40% lower in PAO-R1 than in the parental strain, PAO-SR. Furthermore, the effect of LasR on the toxA promoter is not due to the stimulation of transcription of regA, a transcriptional activator of toxA. No difference in chloramphenicol acetyltransferase (CAT) activity was noted between PAO-SR and PAO-R1 containing transcriptional regA promoter-CAT gene fusions. These results broaden the regulatory dominion of lasR and suggest that the lasR gene plays a global role in P. aeruginosa pathogenesis.