Negative regulation of lymphocyte activation by the adaptor protein LAX

The membrane-associated adaptor protein LAX is a linker for activation of T cells (LAT)-like molecule that is expressed in lymphoid tissues. Upon stimulation of T or B cells, it is phosphorylated and interacts with Grb2 and the p85 subunit of PI3K. LAX, however, is not capable of replacing LAT in the TCR signaling pathway. In this study we report that upon T or B cell activation, the LAX protein was up-regulated dramatically. Although disruption of the LAX gene by homologous recombination had no major impact on lymphocyte development, it caused a significant reduction in CD23 expression on mature B cells. Interestingly, naive LAX(-/-) mice had spontaneous germinal center formation. Compared with normal T and B cells, LAX(-/-) T and B cells were hyperresponsive and had enhanced calcium flux, protein tyrosine phosphorylation, MAPK and Akt activation, and cell survival upon engagement of the T or B AgRs. Our data demonstrate that LAX functions as a negative regulator in lymphocyte signaling.     

Molecular cloning and characterization of MFRP, a novel gene encoding a membrane-type Frizzled-related protein

The Frizzled-type cysteine-rich domain (CRD) is a binding motif for soluble-type glycoprotein WNTs, which play key roles in embryogenesis and carcinogenesis. Here, we have cloned and characterized a novel gene MFRP, encoding a type II transmembrane protein with CRD. In addition to CRD, two tandem-repeats containing the Cubilin domain approximately the MFRP domain were present in the extracellular region of MFRP. Although MFRP was homologous to Corin, FZDs, and SFRPs in CRD, amino-acid identities between CRD in MFRP and CRDs in these molecules were less than 40%. The MFRP gene on 11q23 consisted of at least 13 exons. The 4.0-kb MFRP was not detected by Northern blot analysis in normal tissues other than adult and fetal brain. The MFRP mRNA was undetectable in seven gastric cancer cell lines, seven brain tumor cell lines, and other eight tumor cell lines. Regional distribution of the MFRP mRNA in human brain was further investigated, and MFRP was found to be expressed strongly in medulla oblongata, and weakly in hippocampus and corpus callosum. Thus, MFRP with CRD might play key roles in medulla oblongata as a regulator of the WNT signaling pathway.     

Molecular cloning of a light-inducible gene (lipA) encoding a novel pilin from Arthrobacter photogonimos

Development of pili on cells of Arthrobacter photogonimos is induced by photo-oxidative conditions. The nucleotide sequence was determined of a light-inducible gene (lipA) that encodes the precursor of a light-inducible pilin (designated LIP), a polypeptide of 212 amino acids. The N-terminal leader peptide includes a typical signal sequence with a consensus cleavage site for signal peptidase I after residue 28, which should generate N-terminal arginine. However, the next amino acid, alanine, is the N-terminal residue of the mature protein. The abundance of charged amino acids (27% of total), a calculated pI of 9.98, and recovery of mostly monomers when cells were washed with 1 M NaCl suggest that electrostatic interactions play a dominant role in association of LIP, a novel mechanism for assembly of pili.     

Molecular cloning, functional analysis and localization of a novel gene encoding polygalacturonase-inhibiting protein in Chorispora bungeana

Polygalacturonase-inhibiting proteins (PGIPs) are plant defense proteins. To date, no spatial distribution of PGIPs and interaction between PGIPs and nitric oxide (NO) in plant were described. Here, we first reported the full-length cDNA sequence of PGIP of Chorispora bungeana (CbPGIP1). Notably, immunofluorescence localization showed that the CbPGIP was evenly distributed in leaves but it was mainly localized in epidermis and vascular bundle in stems and roots. Further studies indicated that CbPGIP had higher abundance in roots than in stems and leaves. Conversely, the bulk PGIP of C. bungeana showed a higher activity in leaves than in stems and roots. In addition, quantitative real-time polymerase chain reaction demonstrated that CbPGIP1 expression was induced by Stemphylium solani, salicylic acid (SA), 4, ?4°C and NO. This is a first report attempting to predict if NO can induce the PGIP expression. Taken together, these findings showed that the gene was spatially regulated and NO and SA might take part in CbPGIP1 expression induced by biotic and abiotic stresses. This study highlighted the potential importance of CbPGIP1 and NO in plant resistance.     

Negative regulation of Fc epsilonRI-mediated signaling and mast cell function by the adaptor protein LAX

LAX is a transmembrane adaptor protein that is expressed in both T and B cells. Upon stimulation via the antigen receptors, it is tyrosine-phosphorylated and binds Grb2 and the p85 subunit of phosphatidylinositol 3-kinase. Disruption of the Lax gene causes hyperresponsiveness in T and B lymphocytes. Here, we showed that LAX was also expressed in mast cells. Upon engagement of the Fc epsilonRI, LAX was also phosphorylated and interacted with Grb2 and p85. LAX-deficient mast cells were hyperresponsive to stimulation via the Fc epsilonRI, as evidenced by enhanced degranulation, p38 MAPK, Akt, and phosphatidylinositol 3-kinase activation. This hyperresponsiveness was likely a consequence of reduced LAB expression after sensitization of mast cells with anti-dinitrophenyl IgE. In addition, Fc epsilonRI-mediated cytokine production and cell survival were also enhanced. These data suggested that LAX negatively regulates mast cell function.     

Molecular cloning and expression of a cDNA encoding the membrane-associated rat intestinal alkaline phosphatase

Rat intestinal alkaline phosphatase (IAP) has been purified and proteolytic fragments sequenced. A cDNA library was constructed from duodenal poly(A) + RNA and screened for IAP positive clones by a full-length cDNA clone-encoding human IAP. A full length rat IAP clone (2237 bp) was isolated and sequenced, revealing a predicted primary sequence of 519 amino acids (61.974 kDa) with an additional signal peptide of 20 amino acids. 80% of amino acids from residues 1-474 were identical when compared with the human IAP, but there was only 31% identity in the COOH-terminal 45 amino acids. The homology diverges just before the putative binding site for the phosphatidylinositol-glycan (PI-glycan) anchor. The resulting peptide in rat AP contains five hydrophilic amino acids not present in the primary structure of human IAP. Binding of a synthetic 48-mer encoding a portion of this unique and divergent region (residues 476-491) was compared with that of the full-length clone on Northern blots of rat intestinal RNA. Two mRNAs, 3.0 and 2.7 kb, were detected by both probes, confirming earlier results, but the 48-mer bound preferentially to the 3.0 kb mRNA. The protein product of the full-length cDNA in a cell-free system was 62 kDa, corresponding with the smaller of the two IAP proteins produced by rat duodenal RNA. The cDNA transfected into COS-1 cells produced a membrane-bound IAP that was released by phosphatidylinositol-specific phospholipase (PI-PLC). These data provide definitive evidence that IAP is anchored by PI-glycan and conclusively demonstrate that the unique COOH-terminal structure encoded by this rat mRNA supports the addition of a PI-glycan anchor.     

Molecular cloning of a human gene (S31) encoding a novel serotonin receptor mediating inhibition of adenylyl cyclase.

We report the molecular cloning of human gene (S31) containing an open reading frame of 1095 nucleotides, which encodes a protein of 365 amino acids. The encoded protein contains seven hydrophobic putative transmembrane domains considered the hallmark of G protein-coupled receptors. The amino acid sequence shows highest homology to receptors for serotonin (5-hydroxytryptamine). Expression of this receptor in murine Ltk- cells conferred upon these cells the ability to respond to serotonin by inhibition of adenylyl cyclase. No response was observed to isoproterenol, epinephrine, histamine, dopamine or melatonin in the transfected cells. We propose that the human gene S31 encodes a novel serotonin receptor.     

Molecular cloning and sequences of cDNAs encoding alpha (large) and beta (small) isoforms of human pancreatic zymogen granule membrane-associated protein GP2

cDNA clones encoding two different (alpha and beta) forms of human pancreatic zymogen granule membrane-associated protein glycoprotein 2 (GP2) (also referred to as ZAP75), a critical component in regulated membrane trafficking along the apical secretory pathway in pancreatic acinar cells, have been isolated. Structural analysis of the clones revealed that the alpha and beta forms of GP2 consist of 527 and 380 amino acid (aa) residues, respectively. The beta form lacks a 147 aa domain that corresponds to the 25-171 region of the alpha form, suggesting that it is a product of an alternative splicing event. Expression of both forms of GP2 in the human pancreas was confirmed. A unique isoform of GP2 is reported for the first time in humans.     

Modulation of TLR4 signaling by a novel adaptor protein signal-transducing adaptor protein-2 in macrophages

Signal-transducing adaptor protein-2 (STAP-2) is a recently identified adaptor protein that contains pleckstrin and Src homology 2-like domains as well as a YXXQ motif in its C-terminal region. Our previous studies have demonstrated that STAP-2 binds to STAT3 and STAT5, and regulates their signaling pathways. In the present study, STAP-2 was found to positively regulate LPS/TLR4-mediated signals in macrophages. Disruption of STAP-2 resulted in impaired LPS/TLR4-induced cytokine production and NF-kappaB activation. Conversely, overexpression of STAP-2 enhanced these LPS/TLR4-induced biological activities. STAP-2, particularly its Src homology 2-like domain, bound to both MyD88 and IkappaB kinase (IKK)-alphabeta, but not TNFR-associated factor 6 or IL-1R-associated kinase 1, and formed a functional complex composed of MyD88-STAP-2-IKK-alphabeta. These interactions augmented MyD88- and/or IKK-alphabeta-dependent signals, leading to enhancement of the NF-kappaB activity. These results demonstrate that STAP-2 may constitute an alternative LPS/TLR4 pathway for NF-kappaB activation instead of the TNFR-associated factor 6-IL-1R-associated kinase 1 pathway.     

Molecular cloning and characterization of a gene encoding a 13.1 kDa antigenic protein of Naegleria fowleri

An antigen-related gene was cloned from a cDNA expression library of Naegleria fowleri by immunoscreening with sera obtained from mice that were either immunized with an amoebic lysate or infected with trophozoites. The coding nucleotide sequence of the cloned gene consisted of 357 bases that were translated into 119 amino acids. This gene was designated as nfa1. The predicted amino acid sequence of Nfa1 protein has two potential glycosylation and three potential phosphorylation sites, and its predicted secondary structure consists of four helices and three corners. The deduced amino acid sequence of Nfa1 protein shares 43% identity with the myohemerythrin (myoHr) protein from a marine annelid, Nereis diversicolor, including 100% identity in conserved regions and iron-binding residues. A phylogenetic tree constructed from amino acid sequences placed the N. fowleri Nfa1 protein outside of a cluster of myoHr proteins from eight invertebrates. A purified recombinant protein that migrated as a 13.1 kDa species in SDS-PAGE was produced. This recombinant protein exhibited a strong immunoreactivity with infected, immune, and anti-Nfal sera. In addition, an anti-Nfa1 serum reacted with an amoeba lysate in immunoblotting analysis. The present nfal gene encoding the myoHr-like protein is the first myoHr gene cloned from protozoa, and the Nfal antigen may be useful in diagnostic studies     

烟实夜蛾性信息素合成激活肽基因的分子克隆

根据家蚕Bombyx mori和玉米夜蛾Helicoverpa zea的性信息素合成激活肽基因序列,设计若干套引物, 以烟实夜蛾Heliothis assulta基因组DNA为模板进行PCR扩增, 得到0.63 kb的特异性DNA片段。该片段克隆进适当载体,序列测定和同源比较, 查明烟实夜蛾的基因组中存在性信息素合成激活肽基因。烟实夜蛾的性信息素合成激活肽由33个氨基酸组成, C末端是FXPRL结构,是目前发现的第4种昆虫性信息素合成激活肽。在该神经肽第14和第15个氨基酸之间, 插入一个0.42 kb的内含子。 进一步的分析证明了烟实夜蛾的性信息素合成激活肽基因在潜成虫期的食道下神经节中表达。