Total chemical synthesis and NMR characterization of the glycopeptide tx5a, a heavily post-translationally modified conotoxin, reveals that the glycan structure is alpha-D-Gal-(1-->3)-alpha-D-GalNAc.

The 13-amino acid glycopeptide tx5a (Gla-Cys-Cys-Gla-Asp-Gly-Trp*-Cys-Cys-Thr*-Ala-Ala-Hyp-OH, where Trp* = 6-bromotryptophan and Thr* = Gal-GalNAc-threonine), isolated from Conus textile, causes hyperactivity and spasticity when injected intracerebral ventricularly into mice. It contains nine post-translationally modified residues: four cysteine residues, two gamma-carboxyglutamic acid residues, and one residue each of 6-bromotryptophan, 4-trans-hydroxyproline and glycosylated threonine. The chemical nature of each of these has been determined with the exception of the glycan linkage pattern on threonine and the stereochemistry of the 6-bromotryptophan residue. Previous investigations have demonstrated that tx5a contains a disaccharide composed of N-acetylgalactosamine (GalNAc) and galactose (Gal), but the interresidue linkage was not characterized. We hypothesized that tx5a contained the T-antigen, beta-D-Gal-(1-->3)-alpha-D-GalNAc, one of the most common O-linked glycan structures, identified previously in another Conus glycopeptide, contalukin-G. We therefore utilized the peracetylated form of this glycan attached to Fmoc-threonine in an attempted synthesis. While the result-ing synthetic peptide (Gla-Cys-Cys-Gla-Asp-Gly-Trp*-Cys-Cys-Thr*-Ala-Ala-Hyp-OH, where Trp* =6-bromotryptophan and Thr* = beta-D-Gal-(1-->3)-alpha-D-GalNAc-threonine) and the native peptide had almost identical mass spectra, a comparison of their RP-HPLC chromatograms suggested that the two forms were not identical. Two-dimensional 1H homonuclear and 13C-1H heteronuclear NMR spectroscopy of native tx5a isolated from Conus textile was then used to determine that the glycan present on tx5a indeed is not the aforementioned T-antigen, but rather alpha-D-Gal-(1-->3)-alpha-D-GalNAc.     

Hybridoma monoclonal antibodies in the analysis fo human cell surface antigens

Summary In the present paper, we will summarize studies we have performed on two distinct human lymphocyte cell surface antigens defined by monoclonal antibodies: Leu-1 and HLA-DR. Presented in the symposium on The Biology of Hybridomas at the 32nd Annual Meeting of the Tissue Culture Association, Washington, D.C., June 7–11, 1981. This work was supported by USPHS-NIH Grants CA-21223, AI-11313, and CA-09302. This symposium was supported in part by the following organizations: Bethesda Research Laboratories, Cetus Corporation, Hybritech Incorporated, MAB-Monoclonal Antibodies, Inc., National Capital Area Branch of the Tissue Culture Association, New England Nuclear Corporation, and Ortho Pharmaceutical Corporation.     

Sphingosine kinase type 1 induces G12/13-mediated stress fiber formation,yet promotes growth and survival independent of G protein-coupled receptors

Sphingosine 1-phosphate (S1P) is the ligand for a family of specific G protein-coupled receptors (GPCRs) that regulate a wide variety of important cellular functions, including growth, survival, cytoskeletal rearrangements, and cell motility. However, whether it also has an intracellular function is still a matter of great debate. Overexpression of sphingosine kinase type 1, which generated S1P, induced extensive stress fibers and impaired formation of the Src-focal adhesion kinase signaling complex, with consequent aberrant focal adhesion turnover, leading to inhibition of cell locomotion. We have dissected biological responses dependent on intracellular S1P from those that are receptor-mediated by specifically blocking signaling of Galphaq, Galphai, Galpha12/13, and Gbetagamma subunits, the G proteins that S1P receptors (S1PRs) couple to and signal through. We found that intracellular S1P signaled "inside out" through its cell-surface receptors linked to G12/13-mediated stress fiber formation, important for cell motility. Remarkably, cell growth stimulation and suppression of apoptosis by endogenous S1P were independent of GPCRs and inside-out signaling. Using fibroblasts from embryonic mice devoid of functional S1PRs, we also demonstrated that, in contrast to exogenous S1P, intracellular S1P formed by overexpression of sphingosine kinase type 1 promoted growth and survival independent of its GPCRs. Hence, exogenous and intracellularly generated S1Ps affect cell growth and survival by divergent pathways. Our results demonstrate a receptor-independent intracellular function of S1P, reminiscent of its action in yeast cells that lack S1PRs.     

Alpha-conotoxins ImI and ImII. Similar alpha 7 nicotinic receptor antagonists act at different sites

A novel conotoxin, alpha-conotoxin ImII (alpha-CTx ImII), identified from Conus imperialis venom ducts, was chemically synthesized. A previously characterized C. imperialis conotoxin, alpha-conotoxin ImI (alpha-CTx ImI), is closely related; 9 of 12 amino acids are identical. Both alpha-CTx ImII and alpha-CTx ImI functionally inhibit heterologously expressed rat alpha7 nAChRs with similar IC(50) values. Furthermore, the biological activities of intracranially applied alpha-CTx ImI and alpha-CTx ImII are similar over the same dosage range, and are consistent with alpha7 nAChR inhibition. However, unlike alpha-CTx ImI, alpha-CTx ImII was not able to block the binding of alpha-bungarotoxin to alpha7 nAChRs. alpha-Conotoxin ImI and alpha-bungarotoxin-binding sites have been well characterized as overlapping and located at the cleft between adjacent nAChR subunits. Because alpha-CTx ImI and alpha-CTx ImII share extensive sequence homology, the inability of alpha-CTx ImII to compete with alpha-BgTx is surprising. Furthermore, functional studies in oocytes indicate that there is no overlap between functional binding sites of alpha-CTx ImI and alpha-CTx ImII. Like alpha-CTx ImI, the block by alpha-CTx ImII is voltage-independent. Thus, alpha-CTx ImII represents a probe for a novel antagonist binding site, or microsite, on the alpha7 nAChR.     

Matrix metalloproteinase secretion by gastric epithelial cells is regulated by E prostaglandins and MAPKs

Because matrix metalloproteinases (MMPs) play roles in inflammatory tissue injury, we asked whether MMP secretion by gastric epithelial cells may contribute to gastric injury in response to signals involved in Helicobacter pylori-induced inflammation and/or cyclooxygenase inhibition. Tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, and epidermal growth factor (EGF) stimulated gastric cell MMP-1 secretion, indicating that MMP-1 secretion occurs in inflammatory as well as non-inflammatory situations. MMP-1 secretion required activation of the MAPK Erk and subsequent protein synthesis but was down-regulated by the alternate MAPK, p38. In contrast, secretion of MMP-13 was stimulated by TNF-alpha/IL-1beta but not EGF and was Erk-independent and mediated by p38. MMP-13 secretion was more rapid (peak, 6 h) than MMP-1 (peak > or =30 h) and only partly depended on protein synthesis, suggesting initial release of a pre-existing MMP-13 pool. Therefore, MMP-1 and MMP-13 secretion are differentially regulated by MAPKs. MMP-1 secretion was regulated by E prostaglandins (PGEs) in an Erk-dependent manner. PGEs enhanced Erk activation and MMP-1 secretion in response to EGF but inhibited Erk and MMP-1 when TNF-alpha and IL-1beta were the stimuli, indicating that the effects of PGEs on gastric cell responses are context-dependent. These data show that secretion of MMPs is differentially regulated by MAPKs and suggest mechanisms through which H. pylori infection and/or cyclooxygenase inhibition may induce epithelial cell signaling to contribute to gastric ulcerogenesis.     

De Novo Loss-of-Function Mutations in SETD5, Encoding a Methyltransferase in a 3p25 Microdeletion Syndrome Critical Region,Cause Intellectual Disability

To identify further Mendelian causes of intellectual disability (ID), we screened a cohort of 996 individuals with ID for variants in 565 known or candidate genes by using a targeted next-generation sequencing approach. Seven loss-of-function (LoF) mutations—four nonsense (c.1195A>T [p.Lys399^∗], c.1333C>T [p.Arg445^∗], c.1866C>G [p.Tyr622^∗], and c.3001C>T [p.Arg1001^∗]) and three frameshift (c.2177_2178del [p.Thr726Asnfs^∗39], c.3771dup [p.Ser1258Glufs^∗65], and c.3856del [p.Ser1286Leufs^∗84])—were identified in SETD5, a gene predicted to encode a methyltransferase. All mutations were compatible with de novo dominant inheritance. The affected individuals had moderate to severe ID with additional variable features of brachycephaly; a prominent high forehead with synophrys or striking full and broad eyebrows; a long, thin, and tubular nose; long, narrow upslanting palpebral fissures; and large, fleshy low-set ears. Skeletal anomalies, including significant leg-length discrepancy, were a frequent finding in two individuals. Congenital heart defects, inguinal hernia, or hypospadias were also reported. Behavioral problems, including obsessive-compulsive disorder, hand flapping with ritualized behavior, and autism, were prominent features. SETD5 lies within the critical interval for 3p25 microdeletion syndrome. The individuals with SETD5 mutations showed phenotypic similarity to those previously reported with a deletion in 3p25, and thus loss of SETD5 might be sufficient to account for many of the clinical features observed in this condition. Our findings add to the growing evidence that mutations in genes encoding methyltransferases regulating histone modification are important causes of ID. This analysis provides sufficient evidence that rare de novo LoF mutations in SETD5 are a relatively frequent (0.7%) cause of ID.     

Affective Priming Using Facial Expressions Modulates Liking for Abstract Art

We examined the influence of affective priming on the appreciation of abstract artworks using an evaluative priming task. Facial primes (showing happiness, disgust or no emotion) were presented under brief (Stimulus Onset Asynchrony, SOA = 20ms) and extended (SOA = 300ms) conditions. Differences in aesthetic liking for abstract paintings depending on the emotion expressed in the preceding primes provided a measure of the priming effect. The results showed that, for the extended SOA, artworks were liked more when preceded by happiness primes and less when preceded by disgust primes. Facial expressions of happiness, though not of disgust, exerted similar effects in the brief SOA condition. Subjective measures and a forced-choice task revealed no evidence of prime awareness in the suboptimal condition. Our results are congruent with findings showing that the affective transfer elicited by priming biases evaluative judgments, extending previous research to the domain of aesthetic appreciation.     

An effector domain mutant of Arf6 implicates phospholipase D in endosomal membrane recycling

In this study, we investigated the role of phospholipase D (PLD) in mediating Arf6 function in cells. Expression of Arf6 mutants that are defective in activating PLD, Arf6N48R and Arf6N48I, inhibited membrane recycling to the plasma membrane (PM), resulting in an accumulation of tubular endosomal membranes. Additionally, unlike wild-type Arf6, neither Arf6 mutant could generate protrusions or recruit the Arf6 GTPase activating protein (GAP) ACAP1 onto the endosome in the presence of aluminum fluoride. Remarkably, all of these phenotypes, including accumulated tubular endosomes, blocked recycling, and failure to make protrusions and recruit ACAP effectively, could be recreated in either untransfected cells or cells expressing wild-type Arf6 by treatment with 1-butanol to inhibit the formation of phosphatidic acid (PA), the product of PLD. Moreover, most of the defects present in cells expressing Arf6N48R or N48I could be reversed by treatment with agents expected to elevate PA levels in cells. Together, these observations provide compelling evidence that Arf6 stimulation of PLD is required for endosomal membrane recycling and GAP recruitment.