Stromal cell derived factor-2 (Sdf2): A novel protein expressed in mouse

The stromal derived factor (SDFs) family comprises a group of molecules generated by stromal cells. SDF1 and SDF4 are chemokines; SDF2 and SDF5 are not yet functionally and structurally defined. In human and mouse, Sdf2 has a paralogous gene, Sdf2l1, whose protein sequences are 78% similar and 68% identical. Human SDF2L1 is an endoplasmic reticulum-stress inducible-gene. In Arabidopsis thaliana, SDF2-like (39% and 37% amino acid sequence identity with Mus musculus Sdf2 and Sdf2l1) has also been implicated in activating the UPR in ER-stress. Here we have cloned, expressed and purified recombinant Sdf2 and raised an anti-Sdf2 antibody. We demonstrated that the protein is expressed in several tissues and is localized in the endoplasmic reticulum. We suggest that Sdf2, initially predicted as a secretory protein because it lacks the canonical ER retention signals in its C-terminal, could be ER-resident through accessory binding proteins or other amino acid sequence motifs, as suggested for the homolog protein SDF2-like. Furthermore, the crystal structure of SDF2-like from Arabidopsis thaliana is a typical β-trefoil containing three MIR motifs; all hydrophobic residues considered important for maintaining the bottom layer of the β-trefoil barrel seem to be conserved in the Sdf2 family. Multiple alignment using 43 sequences for SDF2 and 38 for SDF2L1 paralogous families also revealed a very similar residue conservation profile. Comparing the amino acid sequence and predicted 3D structure with other Sdf2-like proteins we suggest a role of mouse Sdf2 in the Unfolded Protein Response and ER-stress, similar to that of Sdf2l1 from human and mouse and SDF2-like from Arabidopsis thaliana. Chronic ER stress has been associated with many human diseases including cancer and diabetes. Identification of new factors associated with the ER stress pathway can help to identify and define key targets of this response.     

Identification of genes preferentially expressed in periodontal ligament: specific expression of a novel secreted protein, FDC-SP

Gene expression in human periodontal ligament (PDL) was examined by suppression subtractive hybridization to identify genes that are preferentially expressed in tissue compared to cultured PDL fibroblasts. The most enriched genes in a subtracted cDNA library are primarily genes for extracellular matrix components, types I and III collagen, lumican, periostin, and asporin, among others, whose expression conveys unique mechanical properties to the PDL. Also within this group is the gene for follicular dendritic cell secreted protein (FDC-SP), a small protein like statherin in saliva, not previously found in PDL. FDC-SP's presence in PDL was confirmed by in situ hybridization in mouse which also showed that it was definitely present in the parotid gland, but, surprisingly, not in the other salivary glands: submandibular and sublingual. Since only normal tissue was examined, these findings suggest that FDC-SP plays an important but previously unsuspected role within oral connective tissue.     

T3JAM, a novel protein that specifically interacts with TRAF3 and promotes the activation of JNK(1)

Previous studies suggest that localization of tumor necrosis factor receptor (TNFR)-associated factor (TRAF) family members is important for regulating their signal transduction. During a screen for TRAF3-associated proteins that potentially alter TRAF3 subcellular localization and enable signal transduction, we identified a novel protein, T3JAM (TRAF3-interacting Jun N-terminal kinase (JNK)-activating modulator). This protein associates specifically with TRAF3 but not other TRAF family members. Coexpression of T3JAM with TRAF3 recruits TRAF3 to the detergent-insoluble fraction. More importantly, T3JAM and TRAF3 synergistically activate JNK but not nuclear factor (NF)-kappaB. Our studies indicate that T3JAM may function as an adapter molecule that specifically regulates TRAF3-mediated JNK activation.     

Molecular cloning and characterization of a novel SH3 protein (SLY) preferentially expressed in lymphoid cells

A novel full-length cDNA was isolated from a murine T-cell lymphoma library that has an open reading frame encoding 381 amino acids. The predicted protein (termed SLY) contains a Src homology 3 domain and a sterile alpha motif, suggesting that it functions as a signaling adaptor protein in lymphocytes. Northern blot and in situ hybridization analysis showed a preferential expression in lymphoid tissues. The sly gene is located on the X-chromosome in close proximity to genes involved in various immune disorders. This is consistent with an additional role of SLY in immune pathology.     

Root-specific expression of a Zea mays gene encoding a novel glycine-rich protein,zmGRP3

The isolation and characterization of a cDNA clone from Zea mays coding for a novel glycine-rich protein (GRP) is described. The corresponding 1.4 kb mRNA accumulates exclusively in roots (primary, lateral seminal and crown roots) of young maize seedlings, following developmentally specific patterns. In agreement with previously described GRPs from other plant species the derived protein sequence exhibits a hydrophobic domain at the N-terminal region followed by repeated glycine-rich motifs. Genomic Southern analysis indicates that the zmGRP3 gene is present in the maize genome as one or two copies or at a low copy number.     

Membrane-associated RING-CH 10 (MARCH10 protein) is a microtubule-associated E3 ubiquitin ligase of the spermatid flagella

Spermiogenesis is a complex and dynamic process of the metamorphosis of spermatids into spermatozoa. There is a great deal that is still unknown regarding the regulatory mechanisms for the formation of the sperm flagellum. In this study, we determined that the membrane-associated RING-CH 10 (March10) gene is predominantly expressed in rat testis. We isolated two March10 isoforms encoding MARCH10a and MARCH10b, which are generated by alternative splicing. MARCH10a is a long RING finger protein, and MARCH10b is a short RING finger-less protein. Immunohistochemical staining revealed that the MARCH10 proteins are specifically expressed in elongating and elongated spermatids, and the expression is absent in epididymal spermatozoa. MARCH10 immunoreactivity was observed in the cytoplasmic lobes as well as the principal piece and annulus of the flagella. When overexpressed in COS7 cells, MARCH10a was localized along the microtubules, whereas MARCH10b was distributed throughout the cytoplasm. An in vitro microtubule cosedimentation assay showed that MARCH10a is directly associated with microtubules. An in vitro ubiquitination assay demonstrated that the RING finger domain of MARCH10a exhibits an E3 ubiquitin ligase activity along with the E2 ubiquitin-conjugating enzyme UBE2B. Moreover, MARCH10a undergoes proteasomal degradation by autoubiquitination in transfected COS7 cells, but this activity was abolished upon microtubule disassembly. These results suggest that MARCH10 is involved in spermiogenesis by regulating the formation and maintenance of the flagella in developing spermatids.     

Germ cell nuclear antigen (GCNA1) expression does not require a gonadal environment or steroidogenic factor 1: Examination of GCNA1 in ectopic germ cells and in Ftz-f1 null mice

The germ cell lineage is first recognized as a population of mitotically proliferating primordial germ cells that migrate toward the gonadal ridge. Shortly after arriving at the gonadal ridge, the germ cells begin to initiate a commitment to gamete production in the developing gonad. The mechanisms controlling this transition are poorly understood. We recently reported that a mouse germ cell nuclear antigen 1 (GCNA1) is initially detected in both male and female germ cells as they reach the gonad at 11.5 days postcoitum (dpc). GCNA1 is continually expressed in germ cells through all stages of gametogenesis until the diplotene/dictyate stage of meiosis I. Since GCNA1 expression commences soon after primordial germ cells arrive at the gonadal ridge, we wanted to determine whether the gonadal environment was essential for induction of GCNA1 expression. By examining GCNA1 expression in germ cells that migrate ectopically into the adrenal gland, we determined that both the gonadal and adrenal gland environments allow GCNA1 expression. We also examined GCNA1 expression in Ftz-F1 null mice, which are born lacking gonads and adrenal glands. During embryonic development in the Ftz-F1 null mice, the gonad and most germ cells undergo apoptotic degeneration at about 12.5 dpc. While most of the germ cells undergo apoptosis without expressing GCNA1, a few surviving germs cells, especially outside the involuting gonad clearly express GCNA1. Thus, although the Ftz-F1 gene is essential for gonadal and adrenal development, induction of GCNA1 expression in germ cells does not require Ftz-F1 gene products. The finding that germ cell GCNA1 expression is not restricted to the gonadal environment and is not dependent on the Ftz-F1 gene products suggests that GCNA1 expression may be initiated in the germ cell lineage by autonomous means. Mol. Reprod. Dev. 48:154–158, 1997. © 1997 Wiley-Liss, Inc.     

The Men-10 cDNA encodes a novel form of proline-rich protein expressed in the tapetum of dioecious Silene latifolia

 The Men-10 gene is expressed specifically in the tapetum tissue that surrounds and nourishes the developing microspores in the dioecious plant species, Silene latifolia. Men-10 encodes a proline-rich protein that contains a predicted signal region, indicating that it may be secreted from the tapetal cells and function in the extracellular domain of the tapetum or be translocated to the developing microspores. Here we report the sequence and precise expression pattern of the Men-10 cDNA and demonstrate a high level of restriction fragment length polymorphism associated with the Men-10 locus. The possible classification of Men-10 amongst known groups of proline- and hydroxyproline-rich glycoproteins, such as the arabinogalactan proteins, is discussed. Received: 20 December 1997 / Revision accepted: 2 July 1998     

Molecular and cellular characterization of a water-channel protein,AQP-h3, specifically expressed in the frog ventral skin

The Ca2+ content of pancreatic juice is closely regulated by yet unknown mechanisms. One aim of the present study was to find whether rat pancreatic ducts have a Na+/Ca2+ exchanger, as found in some Ca2+ transporting epithelia. Another aim was to establish whether the exchanger is regulated by hormones/agonists affecting pancreatic secretion. Whole pancreas, pure pancreatic acini and ducts were obtained from rats and used for RT-PCR and Western blot analysis, immunohistochemistry and intracellular Ca2+ measurements using Fura-2. RT-PCR analysis indicated Na+/Ca2+-exchanger isoforms NCX1.3 and NCX1.7 in acini and pancreas. Western blot with NCX1 antibody identified bands of 70, 120 and 150 kDa in isolated ducts, acini and pancreas. Immunofluorescence experiments showed the Na+/Ca2+ exchanger on the basolateral membrane of acini and small intercalated/intralobular ducts, but in larger intralobular/extralobular ducts the exchanger was predominantly on the luminal membrane. Na+/Ca2+ exchange in ducts was monitored by changes in intracellular Ca2+ activity upon reversal of the Na+ gradient. Secretin (1 nM) and carbachol (1 mM) reduced Na+/Ca2+ exchange by 40% and 51%, respectively. Insulin (1 nM) increased Na+/Ca2+ exchange by 230% within 5 min. The present study shows that pancreatic ducts express the Na+/Ca2+ exchanger. Its distinct localization along the ductal tree and regulation by secretin, carbachol and insulin indicate that ducts might be involved in regulation of Ca2+ concentrations in pancreatic juice.     

Structure of the regulatory apparatus of a calcium-dependent protein kinase (CDPK): a novel mode of calmodulin-target recognition

Calcium-dependent protein kinases (CDPKs) are a class of calcium-binding sensory proteins that are found in plants and certain protozoa, including the causative agent of malaria, Plasmodium falciparum. CDPKs have diverse regulatory functions, including involvement in the triggering of the lytic cycle of malarial infection. CDPKs contain an autoinhibitory junction (J) region whose calcium-dependent interaction with the tethered regulatory calmodulin-like domain (CaM-LD) activates the catalytic kinase domain. We report here the X-ray crystal structure of the J-CaM-LD region of CDPK from Arabidopsis thaliana (AtCPK1), determined to 2.0 A resolution using multiple-wavelength anomalous dispersion (MAD). The structure reveals a symmetric dimer of calcium-bound J-CaM-LD with domain-swap interactions, in which the J region of one protomer interacts extensively with the carboxy-terminal EF-hand domain (C-lobe) of the partner protomer. However, as the J-CaM-LD is monomeric in solution, the activated monomer was modelled to account for the intra-molecular recognition of the two domains. While the J-CaM-LD segment mimics certain aspects of target motif recognition by CaM other features are specific to CDPKs, in particular the combination of the strong interaction between the N and C-lobes of the CaM-LD and the exclusive use of only the C-lobe in the recognition of the covalently tethered target region. Combined with our previous observations showing that there is likely to be strong interactions between this tethered J region and the CaM-LD even at basal Ca(2+) concentrations, the new structural data indicate that the response to calcium of CDPKs is clearly unique among the CaM family.     

A novel spermatozoan ultrastructure in the shrimp Hippolyte obliquimanus Dana, 1852 (Decapoda: Caridea: Hippolytidae)

The aim of this study was to describe and illustrate the morphology of the spermatozoon of the Western Atlantic shrimp, Hippolyte obliquimanus. Individuals were sampled from Itaguá Beach (Ubatuba, southern Brazil). The male reproductive system was dissected and morphological analysis was undertaken using a stereomicroscope, a light microscope, and transmission electron and scanning electron microscopes. When viewed from the nuclear or acrosomal poles, each spermatozoon has many translucent radiating arms (about 20) from a denser cell body, while laterally the cell body and arms resemble a “cnidarian medusa”, with all the arms projecting away from the bell-like cell body. This sperm morphology is distinct from the “thumbtack”-shaped spermatozoa observed in the majority of carideans but has similarities to the spermatozoa of Rhynchocinetes spp. The morphology of sperm of several species of the genus Hippolyte resembles the spermatozoon of H. obliquimanus with the presence of posterior nuclear arms, but it is necessary to study other Hippolyte species to place these arms in the context of the genus.