Identification and characterization of galectin-9, a novel beta-galactoside-binding mammalian lectin

A 36-kDa beta-galactoside mammalian lectin protein, designated as galectin-9, was isolated from mouse embryonic kidney by using a degenerate primer polymerase chain reaction and cloning strategy. Its deduced amino acid sequence had the characteristic conserved sequence motif of galectins. Endogenous galectin-9, extracted from liver and thymus, as well as recombinant galectin-9 exhibited specific binding activity for the lactosyl group. It had two distinct N- and C-terminal carbohydrate-binding domains connected by a link peptide, with no homology to any other protein. Galectin-9 had an alternate splicing isoform, exclusively expressed in the small intestine with a 31-amino acid insertion between the N-terminal domain and link peptide. Sequence homology analysis revealed that the C-terminal carbohydrate-binding domain of mouse galectin-9 had extensive similarity to that of monomeric rat galectin-5. The presence of galectin-5 in the mouse could not be demonstrated by polymerase chain reaction or by Northern or Southern blot genomic DNA analyses. Sequence comparison of rat galectin-5 and rat galectin-9 cDNA did not reveal identical nucleotide sequences in the overlapping C-terminal carbohydrate-binding domain, indicating that galectin-9 is not an alternative splicing isoform of galectin-5. However, galectin-9 had a sequence identical with that of its intestinal isoform in the overlapping regions in both species. Southern blot genomic DNA analyses, using the galectin-9 specific probe derived from the N-terminal carbohydrate-binding domain, indicated the presence of a novel gene encoding galectin-9 in both mice and rats. In contrast to galectin-5, which is mainly expressed in erythrocytes, galectin-9 was found to be widely distributed, i.e. in liver, small intestine, thymus > kidney, spleen, lung, cardiac and skeletal muscle > reticulocyte, brain. Collectively, these data indicate that galectin-9 is a new member of the galectin gene family and has a unique intestinal isoform.     

B cells in the bursa of Fabricius express a novel C-type lectin gene

The avian bursa of Fabricius provides an essential microenvironment for B cell development and Ab repertoire expansion by a gene conversion mechanism. To explore regulatory interactions between B lineage cells and the bursal microenvironment, we sought to identify genes encoding cell surface molecules selectively expressed on bursal B cells. We report in this work the identification of the chB1 gene that encodes a C-type lectin molecule that is a distant relative of the mammalian B cell differentiation Ag, CD72. The chB1 gene is expressed by intrabursal B cells and a B cell line, DT40, that also diversifies its Ig V region genes by gene conversion. Two forms of this type II membrane protein, differing in their cytoplasmic domains, are generated by the differential usage of two translational initiation sites. The longer chB1 isoform, which is the most abundant, contains a consensus immunoreceptor tyrosine-based inhibitory motif in its cytoplasmic domain. Cross-linkage of the chB1 molecules inhibits proliferation of bursal B cells and the DT40 cell line. The chB1 lectin-like molecule may thus modulate intrabursal B cell development.     

Functional characterization of a novel type of 1 alpha,25-dihydroxyvitamin D3 response element identified in the mouse c-fos promoter

The measurement characteristics of two asthma symptom diary scales developed for use as health outcome measures in clinical trials of asthma therapy were investigated. A daytime diary scale was designed to capture the frequency and inconvenience of daytime asthma symptoms and their effects on activities, and a nocturnal asthma symptom diary scale was designed to capture awakenings with asthma symptoms. The internal consistency, reliability, validity and responsiveness of both asthma diary scales were assessed in 346 adult asthma patients in two placebo-controlled clinical trials of an investigational asthma therapy, a leukotriene biosynthesis inhibitor. The daytime symptom scale showed sufficient internal consistency (0.90-0.92), and the daytime and nocturnal symptom scales showed sufficient test retest reliability (0.69-0.87). Construct validity was demonstrated by generally moderate-to-strong correlations for changes in the diary scales with changes in other measures of asthma status, such as forced expiratory volume in one second (FEV1), peak expiratory flow (PEF), and puffs of beta-agonist inhaler. Both scales demonstrated significant responsiveness to change in asthma due to therapy in one of the clinical trials. Based on these results, the daytime and nocturnal asthma symptom diary scales show measurement characteristics appropriate for use as asthma outcome measures in clinical trials of asthma therapy.     

Identification of a novel polymorphism in the promoter region of the bovine growth hormone gene

Polyorchidism is defined as the presence of more than two testes. We report the case of a 3-year-old boy and review the embryology and surgical management of the condition.     

Rhodocytin, a functional novel platelet agonist belonging to the heterodimeric C-type lectin family, induces platelet aggregation independently of glycoprotein Ib

We isolated and characterized a functionally novel platelet agonist, designated as rhodocytin, from the Calloselasma rhodostoma venom. Rhodocytin was a disulfide-linked heterodimer consisting of 18- and 15-kDa subunits. The respective N-terminal amino acid sequences of both subunits were homologous to each other and to those of the carbohydrate-recognition domains (CRD) of C-type lectins. Rhodocytin alone induced platelet aggregation. Platelet agonists and antagonists constructed with CRD-like subunits from snake venoms bind to glycoprotein Ib directly or indirectly. However, rhodocytin induced platelet aggregation not by binding to glycoprotein Ib, because rhodocytin-induced platelet aggregation was not influenced by echicetin, a glycoprotein Ib-binding protein, that completely inhibits platelet agglutination by bovine von Willebrand factor. These findings indicate that rhodocytin is a novel protein structurally related to heterodimers of CRD-like subunits, but functionally distinct from venom proteins that induce platelet aggregation via glycoprotein Ib.     

Molecular cloning and characterization of CDEP, a novel human protein containing the ezrin-like domain of the band 4.1 superfamily and the Dbl homology domain of Rho guanine nucleotide exchange factors

BACKGROUND/AIMS: Liver cirrhosis and portal hypertension are associated with hyperdynamic circulation. Portacaval shunts are widely used to prevent recurrent hemorrhage, but the hemodynamic effects caused by these procedures have not been well characterized in cirrhosis. We therefore compared the hemodynamic effects of both end-to-side and side-to-side portacaval shunts in normal and cirrhotic rats. METHODS: Sprague-Dawley rats were divided into six groups according to the operations they underwent. End-to-side or side-to-side portacaval shunts were performed in both rats with cirrhosis induced by bile duct ligation and sham-operated rats. Systemic and regional blood flows were measured by the radioactive microsphere method. RESULTS: Portal pressures in the shunted rats decreased significantly. Cardiac index in cirrhotic rats (557 +/- 27 ml.min-1.kg-1) was significantly higher than controls (455 +/- 21 ml.min-1.kg-1), but the two types of shunts did not further increase cardiac index in either the cirrhotic or the sham-operated rats. After shunting, hepatic arterial flows approximately doubled. Portal tributary blood flows in the end-to-side shunted sham (108 +/- 13 ml.min-1.kg-1) and cirrhotic (139 +/- 19 ml.min-1.kg-1 groups were significantly higher than their respective controls (62 +/- 8 and 76 +/- 5 ml.min-1.kg-1). Portosystemic shunting indices were > 99% in both the end-to-side and side-to-side shunted groups in cirrhotic and sham-operated rats. CONCLUSIONS: The hyperdynamic circulation in cirrhotic rats was not augmented by portacaval shunting operations (either end-to-side or side-to-side), despite essentially total portosystemic blood diversion. Compensatory increase in the hepatic arterial blood flow to the liver remained intact even in cirrhotic rats. A selective redistribution of cardiac output to the mesenteric vascular bed was observed after the shunting procedure. However, there were no significant differences in hemodynamics between the end-to-side and side-to-side shunted groups.     

Regulation of Na+/H+ exchanger gene expression. Role of a novel poly(dA.dT) element in regulation of the NHE1 promoter

In this study we examine regulation of expression of the Na+/H+ exchanger promoter in L6 and NIH 3T3 cells. We have identified a highly conserved poly(dA dT)-rich region that appears to be important in regulation of expression of the NHE1 gene. Deletion or mutation of this region results in dramatic decreases in promoter activity in both L6 and NIH 3T3 cells. In addition, DNase I footprinting experiments demonstrated that this region is protected by nuclear extracts from both cell types, and gel mobility shift assays showed that a protein or proteins specifically binds to the poly(dA dT)-rich element. Using Southwestern blotting, we determined that a 33-kDa protein binds to the poly(dA dT)-containing region. Mutations that abolished protein binding to this element diminished activity of the promoter. Insertion of the poly(dA dT)-rich element into a plasmid containing the SV40 promoter demonstrated that this element can also enhance the activity of a foreign promoter. Together, the results we have presented here show that the poly(dA dT)-rich region is important in regulation of NHE1 expression in different cell types.     

A novel cis-acting element is essential for cytokine-mediated transcriptional induction of the serum amyloid A gene in nonhepatic cells

Serum amyloid A (SAA) is a plasma protein which has been associated with several diseases, including amyloidosis, arthritis, and atherosclerosis, and its abnormal expression, particularly in nonhepatic cells, is implicated in the pathogenesis of these diseases. Transfection and DNA-binding studies were performed to investigate the mechanism controlling cytokine-induced, nonhepatic expression of the SAA gene. We have identified a novel promoter, located between positions -280 and 224, that confers interleukin-6 (IL-6) inducibility to an SAA-chloramphenicol acetyltransferase reporter gene in both nonhepatic and hepatic cells. DNase I protection assays revealed, within this region, three homologous highly pyrimidine rich octanucleotide sequence motifs, termed SAA-activating sequences (SAS). Specific mutations within these three SAS motifs severely reduced IL-6-mediated induction of the reporter gene in transfected nonhepatic cells but not in liver cells. A nuclear factor activated by IL-6 in both hepatic and nonhepatic cells efficiently interacts with the SAS. The induction kinetics and cycloheximide sensitivity of this SAS-binding factor (SAF) suggested that de novo synthesis of this factor itself or an activator protein is essential. Loss of DNA-binding ability as a result of in vitro dephosphorylation, induction of SAA-chloramphenicol acetyltransferase reporter gene activity in the presence of genistein, a protein kinase inhibitor, further indicate that a phosphorylation step is necessary for the activation of SAF. Our results suggest that SAF is a key regulator of cytokine-mediated SAA gene expression in some nonhepatic cells.     

The lipopolysaccharide-binding protein participating in hemocyte nodule formation in the silkworm Bombyx mori is a novel member of the C-type lectin superfamily with two different tandem carbohydrate-recognition domains

We recently isolated and characterized the lipopolysaccharide (LPS)-binding protein, BmLBP, from the larval hemolymph of the silkworm Bombyx mori. BmLBP is a pattern recognition molecule that recognizes the lipid A portion of LPS and participates in a cellular defense reaction. This paper describes the cDNA cloning of BmLBP. The deduced amino acid sequence of BmLBP revealed that BmLBP is a novel member of the C-type lectin superfamily with a unique structural feature that consists of two different carbohydrate-recognition domains in tandem, a short and a long form.     

Isolation and characterization of a 2.3-kilobase-pair cDNA fragment encoding the binding domain of the bovine leukemia virus cell receptor

An immunoscreening strategy was used to isolate a cDNA clone encoding the binding domain for the external glycoprotein gp51 of the bovine leukemia virus (BLV). Three recombinant phages demonstrating BLV binding activity and containing 2.3-kbp cDNA inserts with identical nucleotide sequences were isolated from a lambda gt11 cDNA library of bovine kidney cells (MDBK). One clone, BLVRcp1, hybridized with a 4.8-kb mRNA from cells of bovine origin and was also found to be conserved as a single-copy gene in murine, bovine, ovine, primate, canine, feline, and porcine DNAs. The same gene is amplified in caprine DNA isolated from a BLV-induced tumor. The longest open reading frame of BLVRcp1 encodes a protein fragment of 729 amino acids with a putative receptor structure. BLVRcp1 cDNA was cloned in the eucaryotic expression vector pXT-1 and transfected into murine NIH 3T3 and human HEp-2 cells. Cells expressing BLVRcp1 mRNA became susceptible to BLV infection. BLVRcp1 has no known physiological function and has no significant homology with sequences registered in the GenBank and EMBL data libraries (31 July 1992). Expression of deleted constructs of BLVRcp1 indicates that the BLV binding region is encoded at the 5' side of the receptor clone.     

Deletional and mutational analyses of the human CD4 gene promoter: characterization of a minimal tissue-specific promoter

Chloroanilines (CA) are widely used chemical intermediates which induce numerous toxicities including hematotoxicity, splenotoxicity, hepatotoxicity and nephrotoxicity. Although chloroaniline-induced hematotoxicity has been studied in detail, little information is available on the organ-directed toxicity seen following exposure to these agents. The purpose of this study was to examine and compare the excretion and distribution of two nephrotoxicant and hepatotoxicant chloroanilines (2- and 4-chloroaniline) to liver, kidney, spleen, plasma and erythrocytes. Subcellular distribution and covalent binding in kidney and liver were also determined. Male Fischer 344 rats (four per group) were administered [14C]-2-chloroaniline or [14C]-4-chloroaniline (0.5 or 1.0 mmol/kg; approximately 50 microCi/rat) intraperitoneally (i.p.). Urine, feces, blood and tissues were collected at 3 and 24 h. Both 2- and 4-chloroaniline-derived radioactivity were primarily renally excreted with < 1% excretion in the feces by 24 h post-treatment. Both chloroanilines accumulated mainly in liver (percentage of administered dose/total tissue), but kidney generally had similar or higher equivalent concentrations (micromol/g tissue) compared to liver. Subcellular distribution revealed that for both chloroanilines, the cytosolic fraction generally had the highest level of radioactivity independent of time or dose. Covalent binding was detected in both liver and kidney, with the highest concentration (pmol/mg protein) of binding observed in the hepatic microsomal fraction regardless of compound, dose or time studied. In general, 2-chloroaniline derived radioactivity was excreted faster, reached peak tissue concentrations earlier, disappeared from tissues faster and had less covalent binding in target tissue at 24 h than 4-chloroaniline-derived radioactivity. These results suggest that the increased toxic potential of 4-chloroaniline as compared to 2-chloroaniline may be due in part to a more prolonged and persistent accumulation of 4-chloroaniline and/or its metabolites in target tissue.