A mouse zinc finger gene which is transiently expressed during spermatogenesis

Zinc finger proteins are polypeptides with sequence-specific, nucleic acid-binding properties. Substantial evidence has established them as a class of trans-acting molecules with regulatory roles in cellular growth and differentiation. We have screened an 11.5 day post coitum urogenital ridge cDNA library with an oligonucleotide encoding a sequence conserved between a variety of zinc finger proteins. By cDNA cloning and sequencing we show that a novel mouse gene, Zfp-35, encodes a protein with a block of 18 zinc finger domains and an N-terminal region rich in acidic residues. The 2.4 kb mRNA encoding this polypeptide is selectively expressed in adult testis, by comparison with other organs. We have analysed Zfp-35 expression in whole testes of sex-reversed mice, whole testes of prepuberal XY animals, germ cell fractions from XY adult testes and by in situ hybridization to sections from adult XY testes. Our studies show that a considerable increase in expression is restricted to spermatocytes at the pachytene stage of meiotic prophase. These experiments suggest that Zfp-35 may act to control gene activity during this particular stage of spermatogenesis.     

The IFN-inducible GTPase LRG47 (Irgm1) negatively regulates TLR4-triggered proinflammatory cytokine production and prevents endotoxemia

LRG47/Irgm1, a 47-kDa IFN-inducible GTPase, plays a major role in regulating host resistance as well as the hemopoietic response to intracellular pathogens. LRG47 expression in macrophages has been shown previously to be stimulated in vitro by bacterial LPS, a TLR4 ligand. In this study, we demonstrate that induction of LRG47 by LPS is not dependent on MyD88 signaling, but rather, requires STAT-1 and IFN-beta. In addition, LRG47-deficient mice are highly susceptible to LPS, but not TLR2 ligand-induced shock, an outcome that correlates with enhanced proinflammatory cytokine production in vitro and in vivo. Further analysis revealed that LPS-stimulated LRG47-deficient macrophages display enhanced phosphorylation of p38, a downstream response associated with TLR4/MyD88 rather than IFN-beta/STAT-1 signaling. In contrast, LPS-induced phosphorylation of IFN regulatory factor-3 and expression of IFN-beta or the type I IFN-regulated genes, CCL5 and CCL10, were unaltered in LRG47(-/-) cells. Together, these observations indicate that in LPS-stimulated murine macrophages LRG47 is induced by IFN-beta and negatively regulates TLR4 signaling to prevent excess proinflammatory cytokine production and shock. Thus, our findings reveal a new host-protective function for this GTPase in the response to pathogenic encounter.     

Novel membrane protein containing glycerophosphodiester phosphodiesterase motif is transiently expressed during osteoblast differentiation

Osteoblast maturation is a multistep series of events characterized by an integrated cascade of gene expression that are accompanied by specific phenotypic alterations. To find new osteoblast-related genes we cloned differentially expressed cDNAs characteristic of specific differentiation stages in the mouse osteoblast-like MC3T3-E1 cells by a differential display method. We identified a novel cDNA encoding a putative glycerophosphodiester phosphodiesterase, GDE3, which specifically was expressed at the stage of matrix maturation. Interestingly, the deduced amino acid sequence contains 539 amino acids including seven putative transmembrane domains and a glycerophosphodiester phosphodiesterase region in one of the extracellular loops. Northern blot analysis revealed that GDE3 was also expressed in spleen as well as primary calvarial osteoblasts and femur. We next transfected HEK293T cells with GDE3 with green fluorescent protein fused to the C terminus. The green fluorescent protein-fused protein accumulated at the cell periphery, and the transfected cells overexpressing the protein changed from a spread form to rounded form with disappearance of actin filaments. Immunofluorescence staining with GDE3 antibody and phalloidin in MC3T3-E1 cells indicated that endogenous GDE3 might be co-localized with the actin cytoskeleton. To identify a role for GDE3 in osteoblast differentiation, MC3T3-E1 cells stably expressing the full-length protein were constructed. Expression of GDE3 showed morphological changes, resulting in dramatic increases in alkaline phosphatase activity and calcium deposit. These results suggest that GDE3 might be a novel seven-transmembrane protein with a GP-PDE-like extracellular motif expressed during the osteoblast differentiation that dramatically accelerates the program of osteoblast differentiation and is involved in the morphological change of cells.     

Interferon RNA of embryonic origin is expressed transiently during early pregnancy in the ewe

Ovine trophoblast protein-1 (oTP-1), an interferon of embryonic origin, is produced during the peri-implantation period of early pregnancy. Secretion of oTP-1 is detectable between days 13 and 21, but not beyond. In this study, the levels of oTP-1 mRNA in embryos were analyzed to determine if they reflected the transient nature of oTP-1 production. Total cellular RNA (tcRNA) was isolated from embryos representing day 12 (n = 5), 14 (n = 7), 16 (n = 5), 18 (n = 6), 20 (n = 4), and 22 (n = 5) of pregnancy and spotted on nylon membranes. Complementary RNA was transcribed from a specific oTP-1 cDNA (550 base pairs) template and applied (16-1000 pg) to nylon membranes to develop a standard curve. The fixed RNA samples were then allowed to hybridize with the 32P-labeled oTP-1 cDNA. oTP-1 mRNA was not detectable on day 12, increased to high levels (3.6 +/- 1.6 ng/ug of embryo DNA) on day 14, decreased about 5-fold by day 16, 15-fold by day 18, 170-fold by day 20, and 200-fold by day 22 of pregnancy. At day 14 oTP-1 mRNA comprised 0.060 +/- 0.019% of the tcRNA and was more abundant than actin mRNA. Northern analyses of pooled tcRNA representing each day of pregnancy showed that the oTP-1 probe hybridized to a single class of mRNA (approximately 1.1 kilobases) and confirmed the results obtained with dot blots.     

A macrophage protein, Ym1, transiently expressed during inflammation is a novel mammalian lectin

Oral infections of mice with Trichinella spiralis induce activation of peritoneal exudate cells to transiently express and secrete a crystallizable protein Ym1. Purification of Ym1 to homogeneity was achieved. It is a single chain polypeptide (45 kDa) with a strong tendency to crystallize at its isoelectric point (pI 5.7). Co-expression of Ym1 with Mac-1 and scavenger receptor pinpoints macrophages as its main producer. Protein microsequencing data provide information required for full-length cDNA cloning from libraries constructed from activated peritoneal exudate cells. A single open reading frame of 398 amino acids with a leader peptide (21 residues) typical of secretory protein was deduced and later deposited in GenBank (accession number M94584) in 1992. By means of surface plasmon resonance analyses, Ym1 has been shown to exhibit binding specificity to saccharides with a free amine group, such as GlcN, GalN, or GlcN polymers, but it failed to bind to other saccharides. The interaction is pH-dependent but Ca2+ and Mg2+ ion-independent. The binding avidity of Ym1 to GlcN oligosaccharides was enhanced by more than 1000-fold due to the clustering effect. Specific binding of Ym1 to heparin suggests that heparin/heparan sulfate may be its physiological ligand in vivo during inflammation and/or tissue remodeling. Although it shares approximately 30% homology with microbial chitinases, no chitinase activity was found associated with Ym1. Genomic Southern blot analyses suggest that Ym1 may represent a member of a novel lectin gene family.     

Hu-K4 is a ubiquitously expressed type 2 transmembrane protein associated with the endoplasmic reticulum

Hu-K4 is a human protein homologous to the K4L protein of vaccinia virus. Due to the presence of two HKD motifs, Hu-K4 was assigned to the family of Phospholipase D proteins although so far no catalytic activity has been shown. The Hu-K4 mRNA is found in many human organs with highest expression levels in the central nervous system. We extended the ORF of Hu-K4 to the 5' direction. As a consequence the protein is 53 amino acids larger than originally predicted, now harbouring a putative transmembrane domain. The exon/intron structure of the Hu-K4 gene reveals extensive alternative splicing in the 5' untranslated region. Due to the absence of G/C-rich regions and upstream ATG codons, the mRNA isoform in brain may be translated with higher efficacy leading to a high Hu-K4 protein concentration in this tissue. Using a specific antiserum produced against Hu-K4 we found that Hu-K4 is a membrane-bound protein colocalizing with protein disulfide isomerase, a marker of the endoplasmic reticulum. Glycosylation of Hu-K4 as shown by treatment with peptide N-glycosidase F or tunicamycin indicates that Hu-K4 has a type 2 transmembrane topology.     

The small GTPase AtRAC2/ROP7 is specifically expressed during late stages of xylem differentiation in Arabidopsis

The RAC/ROP family of small GTPases are central regulators of important cellular processes in plants. AtRAC2/ROP7 is an ancient member of the RAC/ROP gene family in Arabidopsis thaliana whose functions are generally unknown. In order to study the spatial expression pattern of the AtRAC2/ROP7 gene, transgenic plants expressing GUS or GFP under the control of the AtRAC2/ROP7 promoter were analysed. Functional analysis of AtRAC2/ROP7 was done using transgenic plants overexpressing wild-type and constitutively activated AtRAC2/ROP7 (Val15Gly), and an AtRAC2/ROP7T-DNA insertion mutant. The AtRAC2/ROP7 promoter directs a highly specific xylem-specific expression in the root, hypocotyl, stem, and leaves. The expression is developmentally limited to the late stages of xylem differentiation, and coincides with the formation of secondary cell walls. Leaf epidermal cells of transgenic plants overexpressing constitutively active AtRAC2/ROP7 exhibited highly impaired lobe formation, suggesting that AtRAC2/ROP7 is able to regulate polar cell expansion. Finally, GFP-AtRAC2/ROP7 fusion proteins were localized to the plasma membrane. The results indicate a role for AtRAC2/ROP7 in the development of secondary cell walls of xylem vessels.     

Phosphorylation of neutrophil 47-kDa cytosolic oxidase factor. Translocation to membrane is associated with distinct phosphorylation events

Activation of the phagocytic cell superoxide-generating NADPH oxidase requires interaction of cytosolic and membrane-associated components. With most stimuli activation of the oxidase is accompanied by multisite phosphorylation of the 47-kDa cytosolic oxidase factor (p47) which translocates from cytosol to membranes. Native p47 is a highly basic protein that undergoes stepwise charge shifts with successive phosphorylation events. Phosphorylation of p47 was studied by immunoprecipitation from neutrophil cytosol and membrane fractions followed by two-dimensional gel electrophoresis and autoradiography. In the resting cell p47 was not phosphorylated. In the cytosol of phorbol myristate acetate-activated neutrophils eight distinct p47 phosphoproteins were present. The membrane fraction from these activated cells contained a family of p47 phosphoproteins of electrophoretic mobilities identical to those seen in cytosol plus an additional, more acidic p47 phosphoprotein not present in cytosol. Very early after activation (30 s) only the four most acidic p47 phosphoproteins were present in the membrane fraction. Only at later times (5-15 min) was the full spectrum of p47 phosphoproteins present in the membrane fraction. In contrast, the full spectrum of p47 phosphoproteins was present in the cytosol over the entire time course we studied. In neutrophils from patients with cytochrome b558-deficient chronic granulomatous disease p47 phosphorylation was incomplete and p47 translocation to membrane did not occur. These studies demonstrated that the cytochrome was essential for formation of the three most acidic p47 phosphoproteins and greatly augmented formation of the fourth most acidic p47 phosphoprotein found in normal neutrophils. The temporal correlation between specific p47 phosphorylation events and p47 translocation to membrane is consistent with a model of oxidase activation in which a series of p47 phosphorylation events which occurs in cytosol precedes and may be required for p47 interaction with membrane.     

A precursor of the reserve-protein, phaseolin, is transiently associated with the endoplasmic reticulum of developing Phaseolus vulgaris cotyledons

Developing cotyledons of Phaseolus vulgaris L. were labeled for 30 min with [³H] amino acids, homogenized, and the proteins fractionated on sodium dodecylsulfate (SDS) polyacrylamide gels. Fluorographs of these gels showed that the polypeptides of phaseolin, the major reserve protein of P. vulgaris, were synthesized as precursors which could be distinguished from the polypeptides of mature phaseolin by their slightly lower mobility. When extracts of cotyledons labeled for 45 min with [³H] amino acids were fractionated on isopynic sucrose gradients, radioactive phaseolin banded at the same density (1.14 g cm^-3) as the endoplasmic reticulum (ER)-marker enzyme NADH-cytochrome c reductase. Fractionation in the presence of 3 mM MgCl₂ indicated that the newly-synthesized phaseolin was associated with the rough ER. Pulse-chase experiments showed that phaseolin was transiently associated with the ER, and later accumulated in the protein bodies. Treatment of isolated ER with proteinase K showed that phaseolin polypeptides were degraded only if Triton X-100 was present, indicating that phaseolin was membrane-protected, probably enclosed within the vesicles. ER-associated phaseolin associated to an 18S form at pH 4.5 in the presence of 0.3 M NaCl and 100 mM sodium acetate. The polypeptides of ER-associated phaseolin had a slightly lower mobility on SDS-gels than polypeptides of protein body phaseolin. ER-associated phaseolin had a carbohydrate content of 6.8%, while protein body-derived phaseolin had a carbohydrate content of 6.2%. When cotyledons were labeled simultaneously with [¹⁴C] amino acids and [³H] glucosamine or with [¹⁴C] amino acids and [³H] mannose, the [³H]/[¹⁴C] ratio of ER-derived phaseolin was similar to that of protein body derived phaseolin, indicating that the faster mobility on SDS-gels was not due to the detachment of carbohydrate. Experiments in which the carbohydrate side chains were removed with endoglycosidase H, and the resulting polypeptides subjected to electrophoresis in SDS-gels showed that the differential mobility of the glycopolypeptides of phaseolin resided in their polypeptide chains.     

The Vibrio harveyi GTPase CgtAV is essential and is associated with the 50S ribosomal subunit

It was previously reported that unlike the other obg/cgtA GTPases, the Vibrio harveyi cgtAV is not essential. Here we show that cgtAV was not disrupted in these studies and is, in fact, essential for viability. Depletion of CgtAV did not result in cell elongation. CgtAV is associated with the large ribosomal particle. In light of our results, we predict that the V. harveyi CgtAV protein plays a similar essential role to that seen for Obg/CgtA proteins in other bacteria.     

Endogenous meltrin alpha is ubiquitously expressed and associated with the plasma membrane but exogenous meltrin alpha is retained in the endoplasmic reticulum

Meltrin alpha (ADAM12) is a member of the ADAM (MDC) protein family characterized by the presence of metalloprotease and disintegrin domains. ADAM proteins contain single transmembrane domains, and the processed mature proteins are postulated to span the plasma membrane. It has been reported that transfection of a truncated meltrin alpha cDNA lacking the prodomain and metalloprotease domain promotes skeletal muscle cell fusion. We show here that meltrin alpha was constitutively expressed in both undifferentiated and differentiated C2 skeletal muscle cells and also in fibroblasts. Both its precursor and processed mature forms were present in these cells. Thus, meltrin alpha may play general roles in addition to its roles in myogenesis. Since endogenous meltrin alpha cannot be detected by immunofluorescence microscopy, we examined the location of the exogenously expressed protein by transfection. Unexpectedly, the exogenously expressed meltrin alpha was located to a network structure of the endoplasmic reticulum (ER) but not to the plasma membrane. Cell fractionation revealed that the intrinsic mature protein was associated with the plasma membrane. However, the exogenously expressed protein remained unprocessed. These results seem to imply that the exogenously expressed meltrin alpha is not translocated from the ER to the trans-Golgi network, where a processing enzyme resides, and that it is consequently not converted to the mature form. Thus, the transfected meltrin alpha is unlikely to exert its physiological functions. Conversely, the ER may serve as a reservoir of the latent form of intrinsic meltrin alpha.     

Heart C-protein is transiently expressed during skeletal muscle development in the embryo, but persists in cultured myogenic cells

The expression of cardiac and white skeletal C-protein isoforms was analyzed in developing chicken embryos and in primary skeletal muscle cell cultures by immunoblot and immunofluorescence staining using polyclonal antibodies specific for both of the two different proteins. In the embryo, cardiac C-protein was detected in the developing heart from very early stages through adulthood. In skeletal muscle, cardiac C-protein is shown to be transiently expressed between Days 3 and 15 during development. In contrast, the expression of white skeletal C-protein is gradual and progressive starting approximately from Day 15 on in development. In primary cell cultures of skeletal muscle, however, cardiac C-protein remained expressed throughout prolonged culture time, this in conjunction with white skeletal C-protein. Thus the down regulation of cardiac C-protein and the transition from cardiac C-protein to adult skeletal (white) C-protein which was observed during skeletal muscle development in vivo, does not seem to go to completion in the in vitro system.     

The murine chemokine CXCL11 (IFN-inducible T cell alpha chemoattractant) is an IFN-gamma- and lipopolysaccharide-inducible glucocorticoid-attenuated response gene expressed in lung and other tissues during endotoxemia

A new murine chemokine was identified in a search for glucocorticoid-attenuated response genes induced in the lung during endotoxemia. The first 73 residues of the predicted mature peptide are 71% identical and 93% similar to human CXCL11/IFN-inducible T cell alpha chemoattractant (I-TAC) (alias beta-R1, H174, IFN-inducible protein 9 (IP-9), and SCYB9B). The murine chemokine has six additional residues at the carboxyl terminus not present in human I-TAC. Identification of this cDNA as murine CXCL11/I-TAC is supported by phylogenetic analysis and by radiation hybrid mapping of murine I-TAC (gene symbol Scyb11) to mouse chromosome 5 close to the genes for monokine induced by IFN-gamma (MIG) and IP10. Murine I-TAC mRNA is induced in RAW 264.7 macrophages by IFN-gamma or LPS and is weakly induced by IFN-alphabeta. IFN-gamma induction of murine I-TAC is markedly enhanced by costimulation with LPS or IL-1beta in RAW cells and by TNF-alpha in both RAW cells and Swiss 3T3 fibroblasts. Murine I-TAC is induced in multiple tissues during endoxemia, with strongest expression in lung, heart, small intestine, and kidney, a pattern of tissue expression different from those of MIG and IP10. Peak expression of I-TAC message is delayed compared with IP10, both in lung after i.v. LPS and in RAW 264.7 cells treated with LPS or with IFN-gamma. Pretreatment with dexamethasone strongly attenuates both IFN-gamma-induced I-TAC expression in RAW cells and endotoxemia-induced I-TAC expression in lung and small intestine. The structural and regulatory similarities of murine and human I-TAC suggest that mouse models will be useful for investigating the role of this chemokine in human biology and disease.     

The p47 GTPase Lrg-47 (Irgm1) links host defense and hematopoietic stem cell proliferation

Hematopoietic stem cells (HSCs) are self-renewing bone marrow cells that give rise to all blood lineages and retain a remarkable capacity to proliferate in response to insult. Although some controls on HSC activation are known, little is understood about how this process is linked to natural signals. We report that the interferon-inducible GTPase Lrg-47 (Irgm1), previously shown to play a critical role in host defense, inhibits baseline HSC proliferation and is required for a normal HSC response to chemical and infectious stimuli. Overproliferating Lrg-47(-/-) HSCs are severely impaired in functional repopulation assays, and when challenged with hematopoietic ablation by 5-fluorouracil or infection with Mycobacterium avium, Lrg-47(-/-) mice fail to achieve the expected expansion response in stem and progenitor cell populations. Our results establish a link between the response to infection and HSC activation and demonstrate a novel function for a member of the p47 GTPase family.