Human melanoma cells express a novel integrin receptor for laminin

This study sought to determine whether human melanoma cells express integrin-related receptors that mediate their adhesion to laminin. We found that antibodies against the integrin beta 1 chain blocked cell attachment to laminin-coated surfaces. Furthermore, immunofluorescence staining demonstrated beta 1 complexes in vinculin-positive focal adhesion plaques on the basal surface of cells attached to laminin substrates. Chromatography of detergent extracts of 125I-surface-labeled cells on laminin-Sepharose columns recovered two major laminin-binding proteins (100 and 130 kDa, reduced) that bound with high affinity to the columns and were eluted with EDTA. Both proteins were specifically immunoprecipitated from column fractions with monoclonal and polyclonal antibodies to the integrin beta 1 subunit, indicating that they form a noncovalent heterodimer complex. The alpha-like subunit is composed of a 30-kDa light chain that is joined by a disulfide bond to the 100-kDa heavy chain. This complex was not recovered from columns of fibronectin- or collagen type I- or IV-Sepharose. Laminin-binding by the alpha beta 1 complex was independent of Arg-Gly-Asp or Tyr-Ile-Gly-Ser-Arg-like sequences, but required the presence of divalent cations. The 100-kDa alpha-like subunit was electrophoretically and immunochemically distinct from the other known alpha subunits, alpha 1-alpha 6. The results indicate that human melanoma cells express a novel laminin-specific integrin beta 1 complex which may mediate the cells' interactions with this ligand.     

The fibronectin receptor is organized by extracellular matrix fibronectin: implications for oncogenic transformation and for cell recognition of fibronectin matrices

Cells interact with extracellular fibronectin (FN) via adhesive fibronectin receptors (FNRs) that are members of the very late antigens (VLAs) subgroup of the integrin family. In stationary fibroblasts, the FNR is highly organized and distributed identically to extracellular FN fibrils. However, in highly migratory neural crest cells and embryonic somatic fibroblasts, this organization is lost and the FNR appears diffuse. Similarly, oncogenic transformation typically leads to disorganization of the FN receptor and loss of matrix FN. Two models can account for these observations. First, the FN matrix may organize the FN receptor at extracellular matrix contacts on the cell surface. Motile cells not depositing FN matrices thus lack organized receptors. Alternatively, as the FNR is required for optimal FN matrix assembly, (McDonald, J. A., B. J. Quade, T. J. Broekelmann, R. LaChance, K. Forseman, K. Hasegawa, and S. Akiyama. 1987. J. Biol. Chem. 272:2957-2967; Roman, J. R. M. LaChance, T. J. Broekelmann, C. J. R. Kennedy, E. A. Wayner, W. G. Carter, J. A. McDonald. 1989. J. Cell Biol. 108:2529-2543) and has putative cytoskeletal links, it could be organized from within the cell helping to position newly forming FN fibrils. To study this question, we developed peptide antibodies specifically recognizing the alpha 5 subunit of the FNR. Using these antibodies, we examined the organization of FN and of the FNR in normal, matrix assembly inhibited, and SV40-transformed human fibroblasts. On FN-coated substrates, the FNR is found in focal contacts rather than diffusely on the basal cell surface, suggesting FNR interaction with intracellular components. However, when FN fibrils are deposited, the FNR is co-distributed with these fibrils. Preventing FN matrix assembly prevents organization of the FNR. Moreover, when fibroblasts with well established FN matrices and co-distributed FNR are incubated briefly with monoclonal antibodies that block FNR binding to FN, the FNR is no longer co-distributed with the FN matrix. Thus, the FN receptor is organized in fibrils on the cell surface in response to extracellular FN. Because exogenous FN restores a FN matrix and receptor organization to SV40-transformed cells, the diffuse FN receptor phenotype appears to be related to loss of the FN matrix rather than to impaired FNR function. These results explain diffusely distributed FNRs in migratory neural crest and embryonic fibroblasts lacking well organized FN matrices and emphasize the existence of separate but related systems controlling FN deposition and recognition by receptor-armed cells.     

The kinesin-like ncd protein of Drosophila is a minus end-directed microtubule motor

The Drosophila ncd gene is required for chromosome segregation during female meiosis. Previous analyses suggested that the ncd gene encoded a protein with sequence similarity to the kinesin motor domain, which suggested that, like kinesin, the ncd protein might be a plus end-directed microtubule motor. Here we describe the expression of ncd protein in E. coli and the initial characterization of the ncd protein's motor properties. The ncd protein is indeed a microtubule motor, but the polarity of movement is minus end directed. The ncd protein also has microtubule bundling activity. These findings limit possible models for the in vivo functions of the ncd protein and suggest that motor proteins with similar sequence can generate movement in opposite directions along a microtubule.     

Casein kinase II-catalysed phosphorylation of calmodulin is altered by amino acid deletions in the central helix of calmodulin.

Calmodulin is phosphorylated by casein kinase II on Thr-79, Ser-81, Ser-101 and Thr-117. To determine the consensus sequences for casein kinase II in intact calmodulin, we examined casein kinase II-mediated phosphorylation of engineered calmodulins with 1-4 deletions in the central helical region (positions 81-84). Total casein kinase II-catalyzed phosphate incorporation into all deleted calmodulins was similar to control calmodulin. Neither CaM delta 84 (Glu-84 deleted) nor CaM delta 81-84 (Ser-81 to Glu-84 deleted) has phosphate incorporated into Thr-79 or Ser-81, but both exhibit increased phosphorylation of residues Ser-101 and Thr-117. These data suggest that phosphoserine in the +2 position may be a specificity determinant for casein kinase II in intact proteins and/or secondary structures are important in substrate recognition by casein kinase II.     

Amino acid-specific ADP-ribosylation: structural characterization and chemical differentiation of ADP-ribose-cysteine adducts formed nonenzymatically and in a pertussis toxin-catalyzed reaction.

ADP-ribosylation is a posttranslational modification of proteins by amino acid-specific ADP-ribosyltransferases. Both pertussis toxin and eukaryotic enzymes ADP-ribosylate cysteine residues in proteins and also, it has been suggested, free cysteine. Analysis of the reaction mechanisms of cysteine-specific ADP-ribosyltransferases revealed that free ADP-ribose combined nonenzymatically with cysteine. L- and D-cysteine, L-cysteine methyl ester, and cysteamine reacted with ADP-ribose, but alanine, serine, lysine, arginine, N-acetyl-L-cysteine, 2-mercaptoethanol, dithiothreitol, and glutathione did not. The 1H NMR spectrum of the product, along with the requirement for both free sulfhydryl and amino groups of cysteine, suggested that the reaction produced a thiazolidine linkage. ADP-ribosylthiazolidine was labile to hydroxylamine and mercuric ion, unlike the ADP-ribosylcysteine formed by pertussis toxin and NAD in guanine nucleotide-binding (G-) proteins, which is labile to mercuric ion but stable in hydroxylamine. In the absence of G-proteins but in the presence of NAD and cysteine, pertussis toxin generated a hydroxylamine-sensitive product, suggesting that a free ADP-ribose intermediate, expected to be formed by the NADase activity of the toxin, reacted with cysteine. Chemical analysis, or the use of alternative thiol acceptors lacking a free amine, is necessary to distinguish the enzymatic formation of ADP-ribosylcysteine from nonenzymatic formation of ADP-ribosylthiazolidine, thereby differentiating putative NAD:cysteine ADP-ribosyltransferases from NAD glycohydrolases.     

Insulin receptor function is inhibited by guanosine 5''-[gamma-thio]triphosphate (GTP[S]).

The regulatory role of GTP-binding proteins (G-proteins) in insulin receptor function was investigated using isolated insulin receptors and plasma membranes from rat adipocytes. Treatment of isolated insulin receptors with 1 mM-guanosine 5'-[gamma-thio]triphosphate (GTP[S]) inhibited insulin-stimulated phosphorylation of the beta-subunit, histone Hf2b and poly(GluNa4,Tyr1) by 22%, 65% and 65% respectively. Phosphorylation of calmodulin by the insulin receptor kinase was also inhibited by 1 mM-GTP[S] both in the absence (by 88%) and in the presence (by 81%) of insulin. In the absence of insulin, 1 mM-GTP had the same effect on calmodulin phosphorylation as 1 mM-GTP[S]. However, when insulin was present, GTP was less effective than GTP[S] (41% versus 81% inhibition). Concentrations of GTP[S] greater than 250 microM are necessary to inhibit phosphorylation. Although these concentrations are relatively high, the effect of GTP[S] is not due to competition with [32P]ATP for the insulin receptor kinase since (1) other nucleotide triphosphates did not inhibit phosphorylation as much as did GTP[S] (or GTP) and (2) the Vmax of the ATP-dependent kinase reaction was decreased in the presence of GTP[S]. GTP[S] (1 mM) also inhibited insulin binding to isolated receptors and plasma membranes, by 80% and 50% respectively. Finally, an antibody raised to a peptide sequence common to the alpha-subunits of G-proteins Gs, Gi, Go and transducin detected G-proteins in plasma membranes but failed to detect them in the insulin receptor preparation. These results indicate that GTP inhibits insulin receptor function, but does so through a mechanism that does not require a conventional GTP-binding protein.     

Characterisation of Human 5-Hydroxytryptamine2A and 5-Hydroxytryptamine2C Receptors Expressed in the Human Neuroblastoma Cell Line SH-SY5Y: Comparative Stimulation by Hallucinogenic Drugs

Abstract: Stable transfection of the human neuroblastoma cell line SH-SY5Y with the human 5-hydroxytryptamine_2A (5-HT_2A) or 5-HT_2C receptor cDNA produced cell lines demonstrating ligand affinities that correlated closely with those for the corresponding endogenous receptors in human frontal cortex and choroid plexus, respectively. Stimulation of the recombinant receptors by 5-HT induced phosphoinositide hydrolysis with higher potency but lower efficacy at the 5-HT_2C receptor (pEC₅₀ = 7.80 ± 0.06) compared with the 5-HT_2A receptor (pEC₅₀ = 7.30 ± 0.08). Activation of the 5-HT_2A receptor caused a transient fourfold increase in intracellular Ca²⁺ concentration. Whole-cell recordings of cells clamped at ?50 mV demonstrated a small inward current (2 pA) in response to 10 µM 5-HT for both receptors. There were no differences in potency or efficacy of phosphoinositide hydrolysis among four hallucinogenic [d-lysergic acid diethylamide (LSD), 1-(4-iodo-2,5-dimethoxyphenyl)-2-aminopropane (DOI), 5-methoxy-N,N-dimethyltryptamine, and mescaline] and three nonhallucinogenic drugs (m-chlorophenylpiperazine, quipazine, and ergotamine). Comparison of equipotent doses producing 20% of the maximal response induced by 5-HT revealed selective activation of the 5-HT_2A receptor by LSD and to a lesser degree by DOI, mescaline, and ergotamine. Quipazine and 5-methoxy-N,N-dimethyltryptamine were relatively nonselective, whereas m-chlorophenylpiperazine selectively activated the 5-HT_2C receptor. It is unlikely therefore that hallucinosis is mediated primarily by activity at the 5-HT_2C receptor, whereas activity at the 5-HT_2A receptor may represent an important but not unique mechanism associated with hallucinogenic drug action.     

Detecting non-neutral heterogeneity across a region of DNA sequence in the ratio of polymorphism to divergence

Natural selection, in the form of balancing selection or selective sweeps, can result in a decoupling of the amounts of molecular polymorphism and divergence. Thus natural selection can cause some areas of DNA sequence to have greater silent polymorphism, relative to divergence between species, than other areas. It would be useful to have a statistical test for heterogeneity in the polymorphism to divergence ratio across a region of DNA sequence, one that could identify heterogeneity greater than that expected from the neutral processes of mutation, drift, and recombination. The only currently available test requires that a region be arbitrarily divided into sections that are compared with each other, and the subjectivity of this division could be problematic. Here a test is proposed in which runs of polymorphic and fixed sites are counted, where a "run" is a set of one or more sites of one type preceded and followed by the other type. The number of runs is smaller than otherwise expected if polymorphisms are clumped together. By simulating neutral evolution and comparing the observed number of runs to the simulations, a statistical test is possible which does not require any a priori decisions about subdivision.      

Effect of Watering Regimen on Injury to Corn and Grain Sorghum by Pratylenchus Species

The effect of simulated rainfall frequency on the pathogenicity of Pratylenchus zeae and P. brachyurus was studied in four greenhouse experiments. Corn and grain sorghum were watered at different intervals during predetermined cycles to create a gradient of water-stressed plants. Each experiment included nematode and uninoculated treatments. Growth reaction of plants to different frequencies of watering was significant but was not affected by the presence of nematodes. Pratylenchus zeae numbers differed among watering regimens on corn but not on sorghum. Numbers of P. brachyurus did not differ among watering regimens on corn or sorghum. Both lesion nematode species were harmful to corn, but sorghum increased plant growth in response to P. brachyurus. It is concluded that water stress is not the only environmental factor that influences the pathogenicity of these two species on corn and sorghum.