BFIT, a unique acyl-CoA thioesterase induced in thermogenic brown adipose tissue: cloning, organization of the human gene and assessment of a potential link to obesity.

We hypothesized that certain proteins encoded by temperature-responsive genes in brown adipose tissue (BAT) contribute to the remarkable metabolic shifts observed in this tissue, thus prompting a differential mRNA expression analysis to identify candidates involved in this process in mouse BAT. An mRNA species corresponding to a novel partial-length gene was found to be induced 2-3-fold above the control following cold exposure (4 degrees C), and repressed approximately 70% by warm acclimation (33 degrees C, 3 weeks) compared with controls (22 degrees C). The gene displayed robust BAT expression (i.e. approximately 7-100-fold higher than other tissues in controls). The full-length murine gene encodes a 594 amino acid ( approximately 67 kDa) open reading frame with significant homology to the human hypothetical acyl-CoA thioesterase KIAA0707. Based on cold-inducibility of the gene and the presence of two acyl-CoA thioesterase domains, we termed the protein brown-fat-inducible thioesterase (BFIT). Subsequent analyses and cloning efforts revealed the presence of a novel splice variant in humans (termed hBFIT2), encoding the orthologue to the murine BAT gene. BFIT was mapped to syntenic regions of chromosomes 1 (human) and 4 (mouse) associated with body fatness and diet-induced obesity, potentially linking a deficit of BFIT activity with exacerbation of these traits. Consistent with this notion, BFIT mRNA was significantly higher ( approximately 1.6-2-fold) in the BAT of obesity-resistant compared with obesity-prone mice fed a high-fat diet, and was 2.5-fold higher in controls compared with ob/ob mice. Its strong, cold-inducible BAT expression in mice suggests that BFIT supports the transition of this tissue towards increased metabolic activity, probably through alteration of intracellular fatty acyl-CoA concentration.

Overexpression of the retinoic acid-responsive gene Stra6 in human cancers and its synergistic induction by Wnt-1 and retinoic acid.

Genetic defects in the Wnt-1 signaling pathway contribute to human tumor progression and are especially prevalent in colorectal cancer. We screened mouse C57MG cells to isolate mRNAs induced by Wnt-1 and identified Stra6, an mRNA known to be up-regulated by retinoic acid. Up-regulation of Stra6 mRNA was also observed in hyperplastic mammary tissue and mammary gland tumors from transgenic mice expressing Wnt-1 and in human tumors that frequently harbor defects in Wnt-1 signaling. Stimulation of C57MG cells with retinoic acid plus Wnt-1 resulted in expression of Stra6 transcript to levels greatly exceeding that observed with either stimulus alone. This synergy could be explained in part by the up-regulation of retinoic acid receptor-gamma that was observed in response to Wnt-1 signaling. Accordingly, treatment of human colorectal cancer cell lines with retinoic acid resulted in the up-regulation of Stra6 mRNA and accumulation of Stra6 protein at the cell membrane. The data support a model in which Wnt-1 signaling synergizes with retinoids to activate retinoic acid receptor-gamma-responsive genes in human cancers.

Independently ligating CD38 and Fc gammaRIIB relays a dominant negative signal to B cells.

CD38 is expressed on a variety of hematopoietic cells and has a unique enzymatic activity that converts nicotinamide adenine dinucleotide (NAD) into cyclic ADP-ribose (cADPR) and then into ADPR. CD38 is expressed at increasingly higher levels on B cells at each stage of B cell differentiation, and is then down-regulated on germinal center B cells and mature plasma cells. Crosslinking of CD38 on the surface of mature, resting B cells induces B-cell proliferation, which is enhanced by co-signals such as IL-4 and LPS. CD38-induced proliferation is abrogated by Fc gammaRIIB ligation and this inhibition can be effected by the addition of anti-Fc gammaRII Ab midway through a 48 h in vitro culture indicating that it delivers a potent negative signal to CD38 activated B cells. The suppressive signal was shown to occur through the Fc gammaRIIB because CD38-induced B-cell activation was not inhibited by the ligation of Fc gammaRIIB in Fc gammaRII-deficient B cells. These results indicate that Fc gammaRIIB can act as a regulatory molecule that modulates CD38 signals in vivo.

Depletion of eosinophils in mice through the use of antibodies specific for C-C chemokine receptor 3 (CCR3).

We have generated rat monoclonal antibodies specific for the mouse eotaxin receptor, C-C chemokine receptor 3 (CCR3). Several anti-CCR3 mAbs proved to be useful for in vivo depletion of CCR3-expressing cells and immunofluorescent staining. In vivo CCR3 mAbs of the IgG2b isotype substantially depleted blood eosinophil levels in Nippostrongyus brasiliensis-infected mice. Repeated anti-CCR3 mAb treatment in these mice significantly reduced tissue eosinophilia in the lung tissue and bronchoalveolar lavage fluid. Flow cytometry revealed that mCCR3 was expressed on eosinophils but not on stem cells, dendritic cells, or cells from the thymus, lymph node, or spleen of normal mice. Unlike human Th2 cells, mouse Th2 cells did not express detectable levels of CCR3 nor did they give a measurable response to eotaxin. None of the mAbs were antagonists or agonists of CCR3 calcium mobilization. To our knowledge, the antibodies described here are the first mAbs reported to be specific for mouse eosinophils and to be readily applicable for the detection, isolation, and in vivo depletion of eosinophils.

Mice deficient for the ecto-nicotinamide adenine dinucleotide glycohydrolase CD38 exhibit altered humoral immune responses.

CD38 is a membrane-associated ecto-nicotinamide adenine dinucleotide (NAD+) glycohydrolase that is expressed on multiple hematopoietic cells. The extracellular domain of CD38 can mediate the catalysis of NAD+ to cyclic adenosine diphosphoribose (cADPR), a Ca2+-mobilizing second messenger, adenosine diphosphoribose (ADPR), and nicotinamide. In addition to its enzymatic properties, murine CD38 has been shown to act as a B-cell coreceptor capable of modulating signals through the B-cell antigen receptor. To investigate the in vivo physiological function(s) of this novel class of ectoenzyme we generated mice carrying a null mutation in the CD38 gene. CD38-/- mice showed a complete loss of tissue-associated NAD+ glycohydrolase activity, showing that the classical NAD+ glycohydrolases and CD38 are likely identical. Although murine CD38 is expressed on hematopoietic stem cells as well as on committed progenitors, we show that CD38 is not required for hematopoiesis or lymphopoiesis. However, CD38-/- mice did exhibit marked deficiencies in antibody responses to T-cell-dependent protein antigens and augmented antibody responses to at least one T-cell-independent type 2 polysaccharide antigen. These data suggest that CD38 may play an important role in vivo in regulating humoral immune responses.

Murine CD38: an immunoregulatory ectoenzyme.

CD38 is an ectoenzyme that utilizes NAD+ and is expressed by many cells of hematopoietic origin. Antibodies to CD38 potentiate many biological activities on lymphocytes, including induction of murine B-cell proliferation. In this article, Frances Lund and colleagues summarize information concerning the expression, enzymatic activity and signal transduction pathway utilized by murine CD38.

A new approach to the study of haematopoietic development in the yolk sac and embryoid bodies.

To understand the mechanisms that control the differentiation of uncommitted mesoderm precursors into haematopoietic stem cells (HSCs) and the activation of haematopoiesis, we conducted a study to identify genes expressed at the earliest stages of both in vivo and in vitro haematopoietic development. Our strategy was to utilize Differential Display by means of the Polymerase Chain Reaction (DD-PCR) to compare patterns of gene expression between mRNA populations representing different levels of haematopoietic activity obtained from the mouse embryo, embryoid bodies (EBs) and mouse cell lines. We report the molecular cloning of two groups of genes expressed in the yolk sac: a group of genes expressed in the day-8.5 yolk sac at higher levels than in the day-8.5 embryo proper and up-regulated during EB development, and another group of day-8.5 yolk sac genes not expressed in the day-8.5 embryo proper or in EBs. Specifically, we describe the molecular cloning of the first nucleobase permease gene to be found in vertebrates, yolk sac permease-like molecule 1 (Ysp11). The Ysp11 gene has the unique property of encoding both intracellular, transmembrane and extracellular protein forms, revealing novel aspects of nucleotide metabolism that may be relevant during mammalian development.

CD38-mediated ribosylation of proteins.

The lymphocyte cell-surface Ag CD38 catabolizes NAD to adenosine 5' diphosphoribose (ADPR) and cyclic ADPR (cADPR). We show here that the soluble extracellular domain of CD38 (sCD38) mediates ADP ribosylation of several proteins. This was demonstrated by mass spectrometric analyses which revealed the addition of mass in units of 541.1 Da to these proteins, presumably corresponding to the covalent attachment of one or more ADPR moieties. Separate experiments showed that the same proteins became specifically radiolabeled following incubation with [32P]NAD plus sCD38. Additionally, it is shown that sCD38 can autoribosylate. Moreover, sCD38-mediated protein ribosylation was found to occur specifically at cysteine residues, since it was effectively blocked by addition of L-cysteine but not by other amino acids, and CD38-mediated protein ribosylation could be reversed by the addition of HgCl2, which specifically cleaves thiol-glycosidic bonds. ADPR purified from the reaction of sCD38 with NAD could itself be covalently transferred to target proteins at rates similar to the sCD38-mediated reaction, indicating that the ribosylation proceeds via the generation of this reactive intermediate. In vitro mutagenesis of a catalytic Glu residue that is conserved in numerous ADP-ribosyl transferases revealed that this amino acid is also important for catalysis in CD38. These data suggest that CD38 has the potential to cause ribosylation of experimental proteins, and raises the possibility that its specific ribosylation of a currently unidentified lymphocyte protein may contribute to its array of immunoregulatory activities.

Signaling through murine CD38 is impaired in antigen receptor-unresponsive B cells.

CD38 is a 42-kDa membrane associated enzyme which converts NAD into cyclic ADP-ribose (cADPR), a Ca(2+)-mobilizing second messenger, and ADP-ribose (ADPR). Agonistic antibodies to murine CD38 deliver a potent growth co-stimulus to mature splenic B lymphocytes. In this report we demonstrate a striking relationship between CD38-mediated mitogenesis and the ability of surface IgM to promote B cell proliferation. Tolerized B lymphocytes obtained from a double-transgenic mouse model of B cell tolerance do not proliferate in response to antigen stimulation through the Ig receptor or to agonistic anti-CD38 antibodies. Similarly, B-1 cells isolated from the peritoneal cavity of normal mice, and splenic B cells isolated from newborn mice were also unresponsive to both anti-IgM and anti-CD38 stimulation. All of these CD38-unresponsive B cells expressed normal levels of cell surface CD38 and responded to numerous other stimuli. CD38 immunoprecipitated from these B cell populations was normal in size and effectively hydrolyzed NAD, suggesting that the defect in CD38 signaling likely occurs downstream of CD38 itself. Signaling through CD38 and IgM does not always have identical effects on B cells since anti-CD38 cannot deliver inhibitory growth or differentiation signals to normal B cells or immature B cell lines. Nevertheless, the correlative data with these multiple B cell models of unresponsiveness suggests that the signaling pathway utilized by CD38 and IgM intersect, possibly sharing at least one of the crucial components of the Ig receptor signaling cascade.

CD38 unresponsiveness of xid B cells implicates Bruton's tyrosine kinase (btk) as a regular of CD38 induced signal transduction.

CD38 is a 42 kDa membrane-associated ectoenzyme expressed by a large proportion of human and mouse lymphocytes. Agonistic antibodies to CD38 induce a strong proliferative response in lymphocytes additionally co-stimulated with other growth co-factors such as IL-4, IL-2 plus accessory cells or sub-mitogenic doses of endotoxin. We show here that B lymphocytes from unstimulated X-linked immunodeficient (xid) mice are unresponsive to CD38 stimulation, both in terms of proliferative response and surface antigen modulation. This CD38 unresponsiveness is evident in the presence of excess quantities of, and normal responses to, the accessory growth co-stimulants required for this response. CD38 molecules expressed on xid B cells are normal in terms of expression levels, size and enzymatic activity, suggesting that CD38 unresponsiveness reflects a down-stream signaling defect. In light of the recent proposal that the xid gene encodes a tyrosine kinase called Bruton's tyrosine kinase (btk), these data suggest that btk is either an integral component or an indirect regulator of the CD38-induced signal transduction pathway.

Formation and hydrolysis of cyclic ADP-ribose catalyzed by lymphocyte antigen CD38.

CD38 is a 42-kilodalton glycoprotein expressed extensively on B and T lymphocytes. CD38 exhibits a structural homology to Aplysia adenosine diphosphate (ADP)-ribosyl cyclase. This enzyme catalyzes the synthesis of cyclic ADP-ribose (cADPR), a metabolite of nicotinamide adenine dinucleotide (NAD+) with calcium-mobilizing activity. A complementary DNA encoding the extracellular domain of murine CD38 was constructed and expressed, and the resultant recombinant soluble CD38 was purified to homogeneity. Soluble CD38 catalyzed the formation and hydrolysis of cADPR when added to NAD+. Purified cADPR augmented the proliferative response of activated murine B cells, potentially implicating the enzymatic activity of CD38 in lymphocyte function.

Expression cloning of a cDNA encoding a novel murine B cell activation marker. Homology to human CD38.

A rat mAb (NIM-R5) has recently been prepared against a novel murine B cell activation marker. We report here isolation of a cDNA (1-19) encoding the B cell-derived protein recognized by NIM-R5 antibody. This cDNA contains an open reading frame that encodes a polypeptide of 304 amino acids with a predicted molecular weight of 34,500. The existence of a 22-amino acid hydrophobic region located 23 amino acids from the amino terminal of the deduced protein, together with four potential N-linked glycosylation sites, characterize the deduced protein encoded by I-19 cDNA as a typical type II transmembrane glycoprotein. Although I-19 cDNA appears to encode a novel murine protein, its nucleotide sequence and deduced amino acid sequence show approximately 70% homology to the previously reported sequence of human CD38, suggesting that I-19 cDNA encodes either the mouse homologue of CD38 or a closely related protein. Northern blot analysis of the expression of this cDNA product in a variety of cell types, together with immunoprecipitation of the recombinant protein expressed in BaF3 cells, indicated that I-19 cDNA encodes not only the epitope recognized by NIM-R5 but also a protein that is indistinguishable biochemically and in terms of distribution from the murine B cell activation marker recognized by NIM-R5 antibody. Chromosomal mapping studies have localized this locus to the proximal region of mouse chromosome 5. We anticipate that the availability of probes for the murine B cell activation marker recognized by NIM-R5, and the recombinant protein itself, will greatly aid efforts to define the role of this molecule in murine B cell development.

Mutation of unique region of Bruton's tyrosine kinase in immunodeficient XID mice.

The cytoplasmic tyrosine kinase, Bruton's tyrosine kinase (Btk, formerly bpk or atk), is crucial for B cell development. Loss of kinase activity results in the human immunodeficiency, X-linked agammaglobulinemia, characterized by a failure to produce B cells. In the murine X-linked immunodeficiency (XID), B cells are present but respond abnormally to activating signals. The Btk gene, btk, was mapped to the xid region of the mouse X chromosome by interspecific backcross analysis. A single conserved residue within the amino terminal unique region of Btk was mutated in XID mice. This change in xid probably interferes with normal B cell signaling mediated by Btk protein interactions.

Genomic structure and chromosomal mapping of the murine CD40 gene.

The B cell-associated surface molecule, CD40, is likely to play a central role in the expansion of Ag-stimulated B cells, and their interaction with activated Th cells. In our study we have isolated genomic clones of murine CD40 from a mouse liver genomic DNA library. Comparison with the murine CD40 cDNA sequence revealed the presence of nine exons that together contain the entire murine CD40 coding region, and span approximately 16.3 kb of genomic DNA. The intron/exon structure of the CD40 gene resembles that of the low affinity nerve growth factor receptor gene, a close homolog of both human and murine CD40. In both cases the functional domains of the receptor molecules are separated onto different exons throughout the genes. Southern blot analysis demonstrated that murine CD40 is a single copy gene that maps in the distal region of mouse chromosome 2.

Evidence for molecular subtypes of HIV-associated lymphoma: division into peripheral monoclonal, polyclonal and central nervous system lymphoma.

The pathogenesis of the HIV-associated lymphomas is not well understood. In order to begin characterizing this class of lymphoma, we initiated a molecular genetic study of DNA extracted from 31 diagnostic biopsy specimens from patients diagnosed with AIDS-associated non-Hodgkin's lymphoma. Analysis of 25 peripheral lymphomas showed that 14 were monoclonal B-cell processes, while 11 appeared to be of polyclonal origin. Five of the 14 monoclonal lymphomas were found to have rearrangements of the c-myc gene. Epstein-Barr virus (EBV) genomes were found in seven out of 14 monoclonal samples, but only two out of nine polyclonal samples. The six primary central nervous system (CNS) lymphoma samples were more homogeneous than the peripheral samples and all were monoclonal, positive for EBV and lacked detectable c-myc gene rearrangements. This study allows us to subdivide the HIV-associated lymphomas into three major molecular subtypes: (1) monoclonal B-cell process frequently associated with c-myc rearrangement or detectable EBV genomes, (2) polyclonal B-cell process typically without evidence of EBV, and (3) monoclonal primary CNS process associated with EBV genomes and lacking detectable c-myc rearrangement.

An additional breakpoint region in the BCL-1 locus associated with the t(11;14)(q13;q32) translocation of B-lymphocytic malignancy.

The t(11;14)(q13;q32) translocation is associated with human B-lymphocytic malignancy. This translocation divides the IgH locus on chromosome 14q32 and may activate a postulated proto-oncogene, bcl-1, located on chromosome 11q13. Two samples of chronic lymphocytic leukemia with the t(11;14)(q32;q13) translocation were studied. The break in one sample was shown to join Jh sequences with the previously described bcl-1 major translocation cluster. DNA blots of the second sample suggested that Jh sequences were joined to a different breakpoint region on chromosome 11. This translocation was cloned and found to link the human Jh3 region and a new breakpoint region 63 kb telomeric of the major translocation cluster. This translocation occurred in part as the result of an aberrant D-J recombination. Recurrent translocations human B-lymphocytic malignancy. The definition of a new breakpoint region may aid the identification of the postulated bcl-1 gene.

The t(5;14) chromosomal translocation in a case of acute lymphocytic leukemia joins the interleukin-3 gene to the immunoglobulin heavy chain gene.

Chromosomal translocations have proven to be important markers of the genetic abnormalities central to the pathogenesis of cancer. By cloning chromosomal breakpoints one can identify activated proto-oncogenes. We have studied a case of B-lineage acute lymphocytic leukemia (ALL) that was associated with peripheral blood eosinophilia. The chromosomal translocation t(5;14) (q31;q32) from this sample was cloned and studied at the molecular level. This translocation joined the immunoglobulin heavy chain joining (Jh) region to the promotor region of the interleukin-3 (IL-3) gene in opposite transcriptional orientations. The data suggest that activation of the IL-3 gene by the enhancer of the immunoglobulin heavy chain gene may play a central role in the pathogenesis of this leukemia and the associated eosinophilia.

Lack of detectable somatic hypermutation in the V region of the Ig H chain gene of a human chronic B lymphocytic leukemia.

Monoclonal human B cell tumors are a model system for the study of somatic hypermutation of the Ig genes of humans. It was previously shown that a number of B cell lymphomas exhibited striking V region point mutation, hypothesized to result from the somatic hypermutation mechanism. In this study we have extended the analysis to chronic lymphocytic leukemia. We have cloned and sequenced the productive Vh representing five different cells from a monoclonal chronic lymphocytic leukemia. All five Vh sequences were identical. Therefore, the Vh region in this leukemia was not the subject of detectable somatic mutation. These data suggest that chronic lymphocytic leukemia might lack the mechanism for somatic hypermutation and represent a stage of normal B lymphocyte differentiation in which the somatic hypermutation mechanism is not active.