SLX4 interacts with RTEL1 to prevent transcription-mediated DNA replication perturbations.

The SLX4 tumor suppressor is a scaffold that plays a pivotal role in several aspects of genome protection, including homologous recombination, interstrand DNA crosslink repair and the maintenance of common fragile sites and telomeres. Here, we unravel an unexpected direct interaction between SLX4 and the DNA helicase RTEL1, which, until now, were viewed as having independent and antagonistic functions. We identify cancer and Hoyeraal-Hreidarsson syndrome-associated mutations in SLX4 and RTEL1, respectively, that abolish SLX4-RTEL1 complex formation. We show that both proteins are recruited to nascent DNA, tightly co-localize with active RNA pol II, and that SLX4, in complex with RTEL1, promotes FANCD2/RNA pol II co-localization. Importantly, disrupting the SLX4-RTEL1 interaction leads to DNA replication defects in unstressed cells, which are rescued by inhibiting transcription. Our data demonstrate that SLX4 and RTEL1 interact to prevent replication-transcription conflicts and provide evidence that this is independent of the nuclease scaffold function of SLX4.

Early prognostic factors in septic shock cancer patients: a prospective study with a proteomic approach.

BACKGROUND: Organ failures are the main prognostic factors in septic shock. The aim was to assess classical clinico-biological parameters evaluating organ dysfunctions at intensive care unit admission, combined with proteomics, on day-30 mortality in critically ill onco-hematology patients admitted to the intensive care unit for septic shock. METHODS: This was a prospective monocenter cohort study. Clinico-biological parameters were collected at admission. Plasma proteomics analyses were performed, including protein profiling using isobaric Tag for Relative and Absolute Quantification (iTRAQ) and subsequent validation by ELISA. RESULTS: Sixty consecutive patients were included. Day-30 mortality was 47%. All required vasopressors, 32% mechanical ventilation, 33% non-invasive ventilation and 13% renal-replacement therapy. iTRAQ-based proteomics identified von Willebrand factor as a protein of interest. Multivariate analysis identified four factors independently associated with day-30 mortality: positive fluid balance in the first 24 h (odds ratio = 1.06, 95% CI = 1.01-1.12, P = 0.02), severe acute respiratory failure (odds ratio = 6.14, 95% CI = 1.04-36.15, P = 0.04), von Willebrand factor plasma level > 439 ng/ml (odds ratio = 9.7, 95% CI = 1.52-61.98, P = 0.02), and bacteremia (odds ratio = 6.98, 95% CI = 1.17-41.6, P = 0.03). CONCLUSION: Endothelial dysfunction, revealed by proteomics, appears as an independent prognostic factor on day-30 mortality, as well as hydric balance, acute respiratory failure and bacteremia, in critically ill cancer patients admitted to the intensive care unit. Endothelial failure is underestimated in clinical practice and represents an innovative therapeutic target.

The formation of an intrachain disulfide bond in the leptin protein is necessary for efficient leptin secretion.

Leptin is a cytokine secreted by the adipose tissue that is involved in the control of body weight. We previously showed that a point mutation (R105W) in leptin results in leptin deficiency, marked obesity and hypogonadism in humans adults. Expression in COS1 cells showed impaired secretion and intracellular accumulation of the mutated protein. However, impaired secretion of the mutant leptin had not been demonstrated in adipose cells. In this work, we demonstrate that secretion of R105W mutant is impaired in rat and human adipocytes. We also show that R105W mutant expressed in COS1 cells and in PAZ6 adipocytes forms large molecular aggregates that cannot cross a filtration membrane with a cut-off of 100 kDa. Moreover, we have engineered, by site directed mutagenesis, the cDNAs coding for leptin in which either Cys 117, Cys 167, or both, were replaced by a serine. When expressed in COS1 cells or PAZ6 adipocytes, cysteine mutants also show impaired secretion and formation of large molecular aggregates. Therefore, our work indicates that the formation of an intramolecular disulfide bridge is necessary for normal processing and secretion of leptin. Moreover, the similarity of the behavior of R105W mutant and cystein mutants suggests that the lack of secretion observed with the naturally occurring mutant could result from impaired disulfide bond formation.

Study of antigen-processing steps reveals preferences explaining differential biological outcomes of two HLA-A2-restricted immunodominant epitopes from human immunodeficiency virus type 1.

Cytotoxic T-lymphocyte (CTL) responses directed to different human immunodeficiency virus (HIV) epitopes vary in their protective efficacy. In particular, HIV-infected cells are much more sensitive to lysis by anti-Gag/p17(77-85)/HLA-A2 than to that by anti-polymerase/RT(476-484)/HLA-A2 CTL, because of a higher density of p17(77-85) complexes. This report describes multiple processing steps favoring the generation of p17(77-85) complexes: (i) the exact COOH-terminal cleavage of epitopes by cellular proteases occurred faster and more frequently for p17(77-85) than for RT(476-484), and (ii) the binding efficiency of the transporter associated with antigen processing was greater for p17(77-85) precursors than for the RT(476-484) epitope. Surprisingly, these peptides, which differed markedly in their antigenicity, displayed qualitatively and quantitatively similar immunogenicity, suggesting differences in the mechanisms governing these phenomena. Here, we discuss the mechanisms responsible for such differences.

[Retrospective study of 55 patients with circulating blood T gama/delta lymphocytosis]. / Etude rétrospective de 55 patients présentant une lymphocytose T gamma/delta dans le sang circulant.

PURPOSE: Gamma/delta T lymphocytes constitute a singular population due to their particular antigenic recognition and their localization inside the epithelium. Their functions are complementary to those of the alpha/beta T-cells and they are involved in the defense and regulation of the immune system. Their role in human diseases is not very well understood and the aim of our study was to analyze a population of patients with a peripheral gamma/delta T-cell lymphocytosis. METHODS: The study included 55 patients, recruited from 1997 to 2000, with a peripheral gamma/delta T lymphocytosis (defined by a proportion of gamma/delta T-cells of over 10% of total peripheral T lymphocytes). Analysis of the lymphocyte population was obtained by cytometry after peripheral blood sampling. RESULTS: Three main groups of diseases were observed: infectious diseases (viral infections and tuberculosis), inflammatory diseases (sarcoidosis and autoimmune diseases) and blood diseases (monoclonal gammopathies and hemopathies). Persistence of gamma/delta T lymphocytosis was dependent on the underlying disease (transitional when associated with an infectious disease and lasting when associated with sarcoidosis). The rest of the immunophenotyping analysis was usually normal. CONCLUSION: Our results confirm the data published in the literature concerning the role of the gamma/delta T lymphocytes in infectious, inflammatory and autoimmune diseases and neoplasias. These data are in agreement with the cytotoxic and regular functions of these lymphocytes.

The iodocyanopindolol and SM-11044 binding protein belongs to the TM9SF multispanning membrane protein superfamily.

SM-11044 is the only beta-adrenergic agonist that inhibits guinea pig eosinophil chemotaxis and induces relaxation of depolarized rat colon tonus. We have previously reported the purification of a 34 kDa photoaffinity-labeled SM-11044 binding protein (SMBP) from rat colon that may mediate the biological effects of the ligand and that differs from all known monoamine receptors (Sugasawa et al., J. Biol. Chem. 272 (1997) 21244). The present report describes partial amino acid sequence of rat SMBP and molecular cloning of corresponding human SMBP (hSMBP) cDNA. This cDNA encodes a 588 amino acid residue polypeptide comprising a signal peptide, a long hydrophilic amino-terminal region, and a highly hydrophobic C-terminal portion organized into nine putative transmembrane domains. The sequence and structure of hSMBP shows homology to members of a new transmembrane protein 9 superfamily (TM9SF). Comparison of hSMBP with related protein sequences from yeast, plant and human revealed two subgroups within TM9SF. The members of these groups differ in length and have characteristic amino acid sequence motifs in their amino-terminal portion. Northern blot analysis revealed two major SMBP mRNAs, at 3.4 and 3.8 kb, that were present in all the human tissues examined. Western blot experiments detected SMBP as a 70 kDa protein that may be further cleaved into an active 34 kDa N-terminal polypeptide. Stable Chinese Hamster Ovary cell transfectants expressing hSMBP cDNA displayed specific binding of [(125)I]iodocyanopindolol that was displaced by SM-11044 in a dose-dependent manner. Thus, SMBP is the first member of TM9SF with functional ligand binding properties, suggesting that some of these integral membrane proteins may function as channels, small molecule transporters or receptors.

Chemokines control fat accumulation and leptin secretion by cultured human adipocytes.

In addition to their role in inflammation, cytokines like TNFalpha have been reported to regulate the adipose tissue function suggesting a role for these soluble mediators in metabolism. However, it is not known whether adipocytes have the capacity to secrete chemokines, a group of low molecular weight inflammatory mediators that control leukocyte migration into tissues. Here we show that primary cultures of human preadipocytes constitutively produce three chemokines, interleukin-8 (IL-8), macrophage inflammatory protein-1alpha (MIP-1alpha) and monocyte chemotactic protein-1 (MCP-1), while their level of expression is low in mature adipocytes. Upon TNFalpha treatment, the expression of all the three chemokines is upregulated in adipocytes differentiated in vitro. In addition, we describe the presence of seven different chemokine receptors, mainly in mature adipocytes, both in vitro and in human fat tissue sections. Prolonged stimulation of cultured human adipocytes with exogenous chemokines leads to a decrease in lipid content in association with the downregulation of PPARgamma mRNA expression. Moreover, chemokines positively control the secretion of leptin, a hormone that regulates appetite, by a post-transcriptional mechanism. These findings reveal a new role for chemokines in the regulation of adipose tissue and suggest a novel therapeutic basis for the treatment of obesity, diabetes and cachexia.

Genetic variation in the stress protein hsp70-2 gene is highly associated with obesity.

BACKGROUND: Tumor necrosis factor-alpha (TNF-alpha) expression is increased in adipose tissue of both rodent models of obesity and obese humans. It has therefore been considered as a candidate gene for obesity. Several studies have indeed shown statistical evidence of linkage between obesity and the chromosomal region encompassing the TNF-alpha gene, suggesting that TNF-alpha and/or a nearby gene (eg hsp70 gene) is involved in the onset and progression of weight gain. We designed a case-controlled study to investigate the potential association of polymorphism of the TNF-alpha and that of a stress protein (hsp70-2) with obesity. METHODS: We used the polymerase chain reaction and restriction enzyme digestion to characterize the variation of the TNF-alpha promoter region and that of the hsp70-2 gene in 343 unrelated Tunisian patients with obesity and 174 healthy control subjects. RESULTS: Analysis of the -308 TNF-alpha polymorphism in patients with obesity and in control subjects did not reveal an association between TNF-alpha alleles and obesity. In contrast, polymorphism analysis of the hsp70-2 gene in patients with obesity demonstrated highly significant differences in genotypic distribution of this bi-allelic locus compared to the control subject group. Homozygosity for one hsp70-2 allele was highly associated with obesity (r2=7.12; P<10(-6)). CONCLUSION: Tunisian persons carrying the P2/P2 genotype of the hsp70-2 gene may have an increased risk of obesity.

Substituting selenocysteine for catalytic cysteine 41 enhances enzymatic activity of plant phospholipid hydroperoxide glutathione peroxidase expressed in Escherichia coli.

The citrus phospholipid hydroperoxide glutathione peroxidase (cit-PHGPx) was the first plant peroxidase demonstrated to exhibit PHGPx-specific enzymatic activity, although it was 500-fold weaker than that of the pig heart analog. This relatively low activity is accounted for the catalytic residue of cit-PHGPx, which was found to be cysteine and not the rare selenocysteine (Sec) present in animal enzymes. Sec incorporation into proteins is encoded by a UGA codon, usually a STOP codon, which, in prokaryotes, is suppressed by an adjacent downstream mRNA stem-loop structure, the Sec insertion sequence (SECIS). By performing appropriate nucleotide substitutions into the gene encoding cit-PHGPx, we introduced bacterial-type SECIS elements that afforded the substitution of the catalytic Cys(41) by Sec, as established by mass spectrometry, while preserving the functional integrity of the peroxidase. The recombinant enzyme, whose synthesis is selenium-dependent, displayed a 4-fold enhanced peroxidase activity as compared with the Cys-containing analog, thus confirming the higher catalytic power of Sec compared with Cys in cit-PHGPx active site. The study led also to refinement of the minimal sequence requirements of the bacterial-type SECIS, and, for the first time, to the heterologous expression in Escherichia coli of a eukaryotic selenoprotein containing a SECIS in its open reading frame.

In vitro generation of endothelial microparticles and possible prothrombotic activity in patients with lupus anticoagulant.

Microparticles (MPs) resulting from vesiculation of platelets and other blood cells have been extensively documented in vitro and have been found in increased numbers in several vascular diseases, but little is known about MPs of endothelial origin. The aim of this study was to analyze morphological, immunological, and functional characteristics of MPs derived from human umbilical vein endothelial cells (HUVECs) stimulated by TNF, and to investigate whether these MPs are detectable in healthy individuals and in patients with a prothrombotic coagulation abnormality. Electron microscopy evidenced bleb formation on the membrane of TNF-stimulated HUVECs, leading to increased numbers of MPs released in the supernatant. These endothelial microparticles (EMPs) expressed the same antigenic determinants as the corresponding cell surface, both in resting and activated conditions. MPs derived from TNF-stimulated cells induced coagulation in vitro, via a tissue factor/factor VII-dependent pathway. The expression of E-selectin, ICAM-1, alphavbeta3, and PECAM-1 suggests that MPs have an adhesion potential in addition to their procoagulant activity. In patients, labeling with alphavbeta3 was selected to discriminate EMPs from those of other origins. We provide evidence that endothelial-derived MPs are detectable in normal human blood and are increased in patients with a coagulation abnormality characterized by the presence of lupus anticoagulant. Thus, MPs can be induced by TNF in vitro, and may participate in vivo in the dissemination of proadhesive and procoagulant activities in thrombotic disorders.

Copurification of selected glycolytic enzymes with retinal S-antigen (arrestin) by hydroxyapatite agarose chromatography of bovine retina.

PURPOSE: A variety of methods have been developed for the purification of S-antigen but a simple and rapid procedure based on hydroxyapatite-agarose (HA) adsorbtion is most widely used. In the present study, we investigated the nature of proteins purified with the aid of HA chromatography. METHODS: After elimination of retinal S-antigen by HA, the soluble extract of retinal tissue was readsorbed on HA. The proteins were thereafter desorbed by 10 to 500 mM phosphate buffer gradient. Two peaks obtained by SDS-PAGE were used for the generation of specific antisera for subsequent analysis by ELISA and Western blotting. RESULTS: Four proteins (two 48 kDa , one 50 kDa and one 46 kDa) were obtained in this manner. Partial amino acid sequencing permitted the identification of these proteins as alpha-enolase (48 kDa), gamma-enolase (48 kDa), Glucose-6-phosphate-Isomerase (50 kDa) and aspartate-amino-transferase (46 kDa). CONCLUSION: The selected glycolytic enzymes co-purified with retinal S-antigen by hydroxyapatite agarose chromatography of bovine retina.

Human preformed IgG combining with membrane-bound porcine serotransferrin lyse porcine endothelial cells through antibody-dependent cellular cytotoxicity.

Preformed antibodies are involved in xenograft rejection. The purpose of this work was to characterize porcine xenoantigens recognized by human preformed IgG (hpIgG), and to investigate the role of hpIgG in xenogeneic rejection. IgG eluted from porcine livers perfused with human plasma, human sera and total human IgG were immunoblotted on porcine aortic endothelial cell extracts. The amino acid sequence of a 76-kDa antigen constantly revealed was 100% homologous with porcine serotransferrin (psTf). hpIgG from human sera, human IgG1 and IgG2 and F(ab')2gamma specifically bound to psTf. Neutralization by psTf abolished that binding. Although alpha1,3-linked galactose residues (Gal(alpha)1,3Gal) is the dominant epitope recognized by preformed antibodies in the swine-to-human combination, the analysis of carbohydrate composition of psTf showed that the molecule was devoid of Gal(alpha)1,3Gal moieties and that preformed anti-psTf IgG bound to epitopes localized on the peptide core of the molecule. Purified human anti-psTf IgG antibodies were able to bind to psTf linked to its receptor on porcine endothelial cells, and to kill those cells through antibody-dependent cellular cytotoxicity.

Amino acid sequence and three-dimensional structure of the Tn-specific isolectin B4 from Vicia villosa.

The partial amino acid sequence of the tetrameric isolectin B4 from Vicia villosa seeds has been determined by peptide analysis, and its three-dimensional structure solved by molecular replacement techniques and refined at 2.9 A resolution to a crystallographic R-factor of 21%. Each subunit displays the thirteen-stranded beta-barrel topology characteristic of legume lectins. The amino acid residues involved in metal- and sugar-binding are similar to those of other GalNAc-specific lectins, indicating that residues outside the carbohydrate-binding pocket modulate the affinity for the Tn glycopeptide. Isolectin B4 displays an unusual quaternary structure, probably due to protein glycosylation.

Purification and characterization of equine testicular cytochrome P-450 aromatase: comparison with the human enzyme.

Cytochrome P-450 aromatase was purified by five chromatographic steps from adult stallion testis. It was first separated from NADPH-cytochrome P-450 reductase (reductase) on omega-aminohexyl-Sepharose 4B then purified to homogeneity on concanavalin A-Sepharose 4B, hydroxyapatite-Sepharose 4B, DEAE-Sepharose CL-6B and on a second hydroxyapatite-Sepharose 4B. On the other hand, purifications of the equine testicular and rat liver reductases, which allowed the reconstitution of aromatase activity in vitro, were achieved for each species in one chromatographic step on an adenosine 2',5'-diphosphate-agarose affinity column. Analysis on SDS/PAGE indicated single bands with apparent molecular masses of 53, 82, and 80 kDa for purified equine testicular cytochrome P-450 aromatase (eAROM), equine testicular reductase and rat liver reductase respectively. eAROM shows a time- and concentration-dependent activity that was stable for at least 2 months when stored at -78 degrees C. It is a highly hydrophobic protein composed from 505 residues and direct sequencing of its N-terminal part showed good homology when compared with human aromatase. When deglycosylated by N-glycosidase-F the apparent molecular mass of eAROM was decreased from 53 to 51 kDa as revealed by electrophoresis, its activity, however, was not impaired. eAROM exhibits much higher affinity for androgens than for 19-norandrogens, Km values were approximately 3, 16 and 170 nM for androstenedione (A), testosterone (T) and 19-nortestosterone (19-NT) respectively. However, it aromatizes 19-norandrostenedione (19-NA) slightly more efficiently than A, the estrone (E1) formed was 4.27 vs 3.54 pmol min-1 micrograms-1 respectively (P < 0.01). After incubation of eAROM with radiolabelled A and separation of steroids on HPLC, E1, 19-hydroxyandrostenedione (19-OHA) and 19-oxoandrostenedione (19-oxoA) were accumulated in the incubation medium in a time-dependent manner. The presence of 4-hydroxyandrostenedione (4-OHA), a suicide inhibitor of aromatase, cause a time-dependent inactivation of the enzyme. Whereas the activity of eAROM was unchanged in the presence of K+ (up to 250 mM), it was increased in the presence of EDTA (up to 50 mM) and decreased in the presence of DTT or Mg2+ (from 25 mM). We conclude that: (a) eAROM is a glycoprotein, however, deglycosylation by N-glycosidase-F does not appear to impair its activity, (b) eAROM aromatizes really both androgens and 19-norandrogens having a higher affinity for androgens, (c) the intermediary compounds of aromatization 19-OHA and 19-oxoA appear to be synthesized by the same active site that synthesizes E1 as the final product, (d) the inhibition of eAROM by increasing concentrations of Mg2+ and the stimulation of its activity by EDTA, taken together, indicate the importance of negatively charged residues in the polypeptide chain of equine aromatase, which play a role in enzymatic activity.

Structural analysis of the principal immunodominant domain of the feline immunodeficiency virus transmembrane glycoprotein.

In the transmembrane envelope glycoprotein (TM) of lentiviruses, including human immunodeficiency virus type 1 (HIV-1) and feline immunodeficiency virus (FIV), two cysteine residues, conserved in most retroviruses, are thought to form a loop containing five to seven amino acids. These elements make up a B-cell epitope recognized by nearly 100% of sera from infected patients or animals, designated the principal immunodominant domain (PID). The PID amino acid sequences are highly conserved between isolates of the same lentivirus but are unrelated, except for the two cysteines, when divergent lentiviruses are compared. The aim of this study was to analyze the relationship between amino acid sequence in the PID and envelope function. We introduced two kinds of mutations in the PID of FIV: mutations which impeded the formation of a loop and mutations which substituted the sequence of FIV with the corresponding sequences from other lentiviruses, HIV-1, visna virus, and equine infectious anemia virus. We analyzed antibody recognition, processing, and fusogenic properties of the modified envelopes, using two methods of Env expression: a cell-free expression system and transfection of a feline fibroblast cell line with gag-pol-deleted FIV proviruses. Most mutations in the PID of FIV severely affected envelope processing and abolished syncytium formation. Only the chimeric envelope containing the HIV-1 PID sequence was correctly processed and maintained the capacity to induce syncytium formation, although less efficiently than the wild-type envelope. We computed three-dimensional structural models of the PID, which were consistent with mutagenesis data and confirmed the similarity of FIV and HIV-1 PID structures, despite their divergence in amino acid sequence. Considering these results, we discussed the respective importance of selection exerted by functional requirements or host antibodies to explain the observed variations of the PIDs in lentiviruses.

Tyr394 and Tyr505 are autophosphorylated in recombinant Lck protein-tyrosine kinase expressed in Escherichia coli.

The activity of the Src family protein-tyrosine kinase p56lck is regulated by phosphorylation and dephosphorylation of two critical tyrosine residues Tyr394 and Tyr505. Tyr394 is autophosphorylated after p56lck activation, whereas phosphorylation of Tyr505 is believed to be due to p50csk which negatively modulates p56lck activity. To determine whether Tyr505 could be autophosphorylated, we used the prokaryotic glutathione S-transferase expression system to express wild-type Lck, the mutants [Y394F]Lck and [Y505F]Lck, a kinase-deficient p56lck with a mutation of the ATP-binding site [K273E]Lck and a double mutant [Y394F, Y505F]Lck. We studied the kinase activities and the patterns of autophosphorylation for tyrosine residues in these mutants and wild-type Lck both in vivo and in vitro. Wild-type Lck, [Y505F]Lck and [Y394F]Lck were phosphorylated on tyrosine. Both the kinase-deficient mutant[K273E]Lck and the double mutant [Y394F, Y505F]Lck did not react with monoclonal anti-phosphotyrosine antibody [anti-Y(P) mAb], thus providing evidence that (a) the bacterial strains used lacked intrinsic protein-tyrosine kinase activities, and therefore tyrosine phosphorylations of wild-type Lck, [Y505F]Lck and [Y394F]Lck are due to autophosphorylation occurring in vivo in bacteria, and (b) that p56lck can only be autophosphorylated on two tyrosine residues, namely Tyr394 and Tyr505. Phosphopeptide mapping analysis confirmed that p56lck can undergo autophosphorylation on these two tyrosine residues. We propose that autophosphorylation at Tyr505 of p56lck may represent an accessory mechanism for the down-regulation of the tyrosine kinase activity of p56lck.

Purification of the plasmin receptor from human carcinoma cells and comparison to alpha-enolase.

We have characterized a receptor for plasmin (Pli-R) from a human tumor cell line, MCF7MF. The Pli-R was purified from a MCF7 0.1% Triton X-100 solubilisate by affinity chromatography. A protein of 55-60 kDa was obtained, which bound plasminogen and plasmin specifically. Chemical cross-linking of M(r) 90 kDa [125I]-Pli to the surface of MCF7 cells with DSP results in the formation of a labelled complex of M(r) 145 kDa, suggesting a M(r) of 55-60 kDa for the receptor. Comparing Pli-R with alpha-enolase (a candidate for plasminogen receptor in U937 cells) we have found a high homology between both proteins, but not an identity.

Anti-CD3 and phorbol ester induce distinct phosphorylated sites in the SH2 domain of p56lck.

P56lck is a protein tyrosine kinase of the Src family specifically expressed in T lymphocytes. Triggering of T cells with anti-CD3 or with phorbol 12-myristate 13-acetate (PMA) results in the appearance of slower migrating forms (shift) of p56lck. To investigate the phosphorylation sites on the shifted forms of p56lck and to assess the role of protein kinase C in this phosphorylation, Jurkat cells were treated with a selective inhibitor of this kinase (GF 109203X). This inhibitor completely reversed the shift induced by PMA but only partially reversed the one induced after triggering with anti-CD3. To analyze the shift further, p56lck was immunoprecipitated from in vivo labeled cells treated either with anti-CD3 or with PMA. Tryptic phosphopeptides were generated and analyzed by using a combination of thin layer chromatography, high reticulation polyacrylamide gel electrophoresis, reverse phase chromatography, and phosphopeptide sequencing. We identified serine 158 as a newly phosphorylated site after PMA treatment and tyrosine 192 and serine 194 in the major tryptic phosphopeptide obtained after anti-CD3 triggering. The three sites identified are located in the SH2 domain of p56lck; this suggests that their phosphorylation may regulate the interaction with other proteins or with other internal domains in p56lck.

Identification, in mouse macrophages and in serum, of a soluble receptor for the Fc portion of IgG (Fc gamma R) encoded by an alternatively spliced transcript of the Fc gamma RII gene.

Low affinity Fc gamma R are a heterogeneous group of glycoproteins which exist in transmembrane (TM) as well as in soluble forms. Two membrane isoforms of the murine type II Fc gamma R, Fc gamma RIIb1 and Fc gamma RIIb2, have been described. They result from the translation of alternatively spliced pre-mRNA, Fc gamma RIIb2 lacking sequences of the first intracytoplasmic domain (IC1). Soluble forms of Fc gamma R (sFc gamma R) have previously been shown to result from proteolysis of membrane receptors. We report here the identification, in macrophages, of a mRNA derived from the Fc gamma RII gene by splicing exons encoding the TM and IC1 domains, i.e. corresponding to a TM-deleted Fc gamma RIIb2 mRNA. A soluble protein possibly encoded by this mRNA was identified in macrophage supernatants. In accordance with Fc gamma R nomenclature, we propose to name this new Fc gamma RII isoform Fc gamma RIIb3. It is the most abundant sFc gamma R present in serum, as compared with sFc gamma R resulting from cleavage of membrane Fc gamma R.

Molecular characterization of salt-stress-associated protein in citrus: protein and cDNA sequence homology to mammalian glutathione peroxidases.

A gene encoding for a citrus salt-stress-associated protein (Cit-SAP) was cloned from Citrus sinensis salt-treated cell suspension. The gene, designated csa, was isolated from a cDNA expression library. The partial amino acid sequence of the protein, as well as that deduced from the nucleotide sequence of csa, revealed a considerable homology to mammalian glutathione peroxidase (GP), and to clone 6P229 from tobacco protoplasts. The increased expression of Cit-SAP in NaCl-treated cultured citrus cells and in citrus plants irrigated with saline water, and its similarity to GP, raise the possibility that one of the effects of salt stress in plants may be the increase of the level of free radicals.