Molecular cloning and expression pattern of rpr-1, a resiniferatoxin-binding, phosphotriesterase-related protein, expressed in rat kidney tubules

Bacterial phosphotriesterases are enzymes that hydrolyse phosphotriester-containing organophosphate pesticides. Resiniferatoxin is a vanilloid that desensitises nociceptive neurons. By screening a rat cDNA library with labelled resiniferatoxin, we unexpectedly isolated a novel rat phosphotriesterase homologue, here named rpr-1, that encodes a 349 amino acid, 39 kDa protein (confirmed by in vitro translation). Northern blotting and in situ hybridisation show expression primarily in proximal tubules of the kidney, in which rpr-1 distribution correlates with resiniferatoxin-binding activity. These results suggest an unsuspected link between the phosphotriesterase enzyme family and resiniferatoxin toxicity and pharmacology.     

Embigin,a developmentally expressed member of the immunoglobulin super family,is also expressed during regression of prostate and mammary gland

Cell adhesion molecules (CAMs) are intimately involved in a variety of cellular processes, including development, cell growth, apoptosis, and differentiation. Interaction of CAMs with components of the extracellular matrix (ECM), growth factors, and other CAMs provides an intricate regulatory mechanism for a diverse range of cellular responses. Embigin is a developmentally expressed protein that is a member of the immunoglobulin superfamily (IgSF) class of CAMs. We have identified embigin as a gene expressed during tissue regression in rat prostate and lactating mammary gland following hormonal ablation. In the absence of the appropriate hormone, the secretory epithelial cells of these two tissues undergo successive waves of apoptotic cell death co-incident with extensive reorganization of the surviving tissue. Using Northern analysis, in situ hybridization analysis, RT-PCR, and Western analysis, we have characterized the expression of embigin mRNA and protein in both regressing prostate and mammary gland. During development of the prostate gland, increased expression of embigin is correlated with the appearance of highly organized lumenal and ductal structures. Embigin is also expressed in a variety of adult tissues including heart, liver, lung, and brain. Zoo-blot analysis with the rat embigin cDNA indicates that embigin homologs exist in species as diverse as Homo sapiens and Drosophila melanogaster, suggesting that it has been highly conserved during evolution. Embigin protein is expressed at readily detectable levels in a variety of prostate and mammary cancer cell lines, and in some cell lines the expression of embigin appears to be down-regulated in the presence of ECM. Our data have led us to propose a model in which embigin functions as a regulator of cell/ECM interactions during development and in the homeostasis of normal adult tissues. Dev. Genet. 21:268–278, 1997. © 1997 Wiley-Liss, Inc.     

RT1.P, rat class Ib genes related to mouse TL: evidence that CD1 molecules but not authentic TL antigens are expressed by rat thymus

 CD1 and TL were once thought to be genetic homologues because of their thymus-specific expression. We investigated their equivalents in the rat to clarify whether their structure and pattern of expression are conserved in rodents. Two rat class Ib genes, containing 3′ sequences very similar to mouse TL, were identified and designated RT1.P. Neither of them, however, can encode ordinary class I molecules due to the accumulation of harmful mutations in the 5′ regions that are unique to RT1.P, while the 3′TL-like regions still retain protein-coding capacity. Comparison of the structural organization of three types of TL family genes, which include mouse T3/T18-encoding TL antigens, mouse T1/T16, and rat RT1.P1/P2 pseudogenes, revealed the presence of a clear demarcation between the type-specific and TL-specific sequences at intron 3. This finding suggests that recombination plays an important role in creating the TL family genes in rodents. Characteristic features of TL, such as a low level of polymorphism and linkage to the major histocompatibility complex, were also observed in the rat. On the other hand, rat CD1 molecules were expressed at a high level on the surface of thymocytes. Absence of authentic TL antigens and thymic expression of CD1d molecules in the rat suggest the plasticity and conservation of class Ib genes in rodent evolution. Functions of TL may be substituted with CD1 or other class Ib molecules expressed by rat thymus. Received: 16 December 1996 / Revised: 11 March 1997     

The immunoglobulin superfamily: An insight on its tissular, species, and functional diversity

The immunoglobulin superfamily (IgSF) is a heterogenic group of proteins built on a common fold, called the Ig fold, which is a sandwich of two β sheets. Although members of the IgSF share a similar Ig fold, they differ in their tissue distribution, amino acid composition, and biological role. In this paper we report an up-to-date compilation of the IgSF where all known members of the IgSF are classified on the basis of their common functional role (immune system, antibiotic proteins, enzymes, cytokine receptors, etc.) and their distribution in tissue (neural system, extracellular matrix, tumor marker, muscular proteins, etc.), or in species (vertebrates, invertebrates, bacteria, viruses, fungi, and plants). The members of the family can contain one or many Ig domains, comprising two basic types: the constant domain (C), with seven strands, and the variable domain (V), with eight, nine, or ten strands. The different overviews of the IgSF led to the definition of new domain subtypes, mainly concerning the C type, based on the distribution of strands within the two sheets. The wide occurrence of the Ig fold and the much less conserved sequences could have developed from a common ancestral gene and/or from a convergent evolutionary process. Cell adhesion and pattern recognition seem to be the common feature running through the entire family. Received: 4 June 1997 / Accepted: 15 September 1997     

Frequency of family dinner and adolescent body weight status: evidence from the national longitudinal survey of youth, 1997

Objective: To explore associations between overweight status and the frequency of family dinners (FFD) for adolescents and how those associations differ across race and ethnicity. Research Methods and Procedures: A sample of 5014 respondents between 12 and 15 years of age from the 1997 wave of the National Longitudinal Survey of Youth 1997 (NLSY97) was used. BMI was calculated using self‐reported height and weight; 13.3% of respondents qualified as overweight, 16.4% qualified as at‐risk‐of‐overweight, and 1.9% qualified as underweight. The remainder were normal weight. FFD was defined as the number of times respondents had dinner with their families in a typical week in the past year. Multinomial logistic regression models were estimated separately for non‐Hispanic whites vs. blacks and Hispanics for odds of belonging to the other weight categories compared with normal weight. A supplementary longitudinal analysis estimated the odds of change in overweight status between 1997 and 2000. Results: In 1997, the FFD distribution was as follows: 0, 8.3%; 1 or 2, 7.3%; 3 or 4, 13.4%; 5 or 6, 28.1%; 7, 42%. For whites, higher FFD was associated with reduced odds of being overweight in 1997, reduced odds of becoming overweight, and increased odds of ceasing to be overweight by 2000. No such associations were found for blacks and Hispanics. Discussion: Reasons for racial and ethnic differences in the relationship between FFD and overweight may include differences in the types and portions of food consumed at family meals. More research is needed to verify this.     

New member of the Snf1/AMPK kinase family,Melk, is expressed in the mouse egg and preimplantation embryo

The initial phase of mammalian preimplantation development is directed by stored maternal mRNAs and their encoded proteins, yet most of the molecules controlling this process have not been described. We have used differential display analysis of cDNA libraries prepared from unfertilized eggs and preimplantation embryos to isolate three maternal cDNAs that represent novel genes exhibiting different patterns of expression during this developmental period. One of these, Melk, encodes a protein with a kinase catalytic domain and a leucine zipper motif, a new member of the Snf1/AMPK family of kinases. This gene product may play a role in the signal transduction events in the egg and early embryo. Mol. Reprod. Dev. 47:148–156, 1997. © 1997 Wiley-Liss, Inc.     

One-step purification of rat heart-type fatty acid-binding protein expressed in Escherichia coli

Heart-type fatty acid-binding protein (H-FABP) is a member of a family of 14–15 kDa lipid binding proteins which are believed to enhance intracellular transport of lipids by facilitating their cytoplasmic diffusion. To obtain sufficient amounts of protein for in vitro studies, we expressed rat H-FABP in Escherichia coli and compared its biochemical properties with the protein isolated from rat heart. An effective method was developed to purify recombinant rat H-FABP from cell lysates in a single step using anion-exchange chromatography. This method also proved to be applicable for purifying heterologously expressed human H-FABP. Recombinant rat H-FABP, which made up approximately 25% of the soluble proteins in E. coli, was obtained in a yield of 30–40 mg/l culture. Characterization showed that recombinant rat H-FABP was indistinguishable from the protein isolated from rat heart regarding molecular mass and oleic acid binding. Some heterogeneity upon isoelectric focusing was observed, presumably due to differences in N-terminal processing of the proteins. In conclusion, a method is presented for efficient high-yield production of recombinant rat H-FABP.     

A protein associated with the manchette during rat spermiogenesis is encoded by a gene of the TBP-1-like subfamily with highly conserved ATPase and protease domains

We have used a rat pachytene spermatocyte cDNA expression library to clone TBP-1 (for tat-binding protein-1; designated rat testis TBP-1 [rtTBP-1]), a new member of the family of putative ATPases associated with the 26S proteasome complex. The 1.63 kb rtTBP-1 cDNA encodes a 49 kDa protein with 99% amino acid identity to human TBP-1 protein. rtTBP-1 protein contains a heptad repeat of six leucine-type zipper fingers at the amino terminal end and highly conserved ATPase and DNA/RNA helicase motifs towards the carboxyl terminal region. Chromatofocusing fractionation of rat testis sucrose extracts demonstrates that the encoded product, recognized by an antiserum raised to the first 196 amino acids of human TBP-1, consists of a protein triplet with a molecular mass range of 52-48 kDa and acidic pI (5.0–5.9). An identical immunoreactive triplet was detected by immunoblotting in extracts of fractionated pachytene spermatocytes, round spermatids and epididymal sperm. In situ hybridization using digoxigenin-labeled antisense RNA probes shows a predominant distribution of specific mRNA in the seminiferous epithelial region occupied by elongating spermatids and primary spermatocytes. Indirect immunofluorescence and immunogold electron microscopy studies show that rtTBP-1 immunoreactive sites colocalize with α-tubulin-decorated manchettes of elongating spermatids. In addition, rtTBP-1 immunoreactivity was detected in fibrillar and granular cytoplasmic bodies typically observed in spermatocytes and spermatids as well as in association with paraaxonemal mitochondria and outer dense fibers of the developing spermatid tail. Results of this study indicate that rtTBP-1 is a member of the highly evolutionary conserved TBP-1-like subfamily of putative ATPases, sharing regions of identity—including ATP-binding sites—with several subunits of the 26S proteasome, known to be involved in the ATP-dependent degradation of ubiquitin-conjugated proteins. Mol. Reprod. Dev. 48:77–89, 1997. © 1997 Wiley-Liss, Inc.     

Identification and characterization of two related murine genes, Eat2a and Eat2b, encoding single SH2-domain adapters

Human EAT-2 (SH2D1B) and SLAM-associated protein (SAP) (SH2D1A) are single SH2-domain adapters, which bind to specific tyrosine residues in the cytoplasmic tail of six signaling lymphocytic activation molecule (SLAM) (SLAMF1)-related receptors. Here we report that, unlike in humans, the mouse and rat Eat2 genes are duplicated with an identical genomic organization. The coding regions of the mouse Eat2a and Eat2b genes share 91% identity at the nucleotide level and 84% at the protein level; similarly, segments of introns are highly conserved. Whereas expression of mouse Eat2a mRNA was detected in multiple tissues, Eat2b was only detectable in mouse natural killer cells, CD8⁺ T cells, and ovaries, suggesting a very restricted tissue expression of the latter. Both the EAT-2A and EAT-2B coimmunoprecipitated with mouse SLAM in transfected cells and augmented tyrosine phosphorylation of the cytoplasmic tail of SLAM. Both EAT-2A and EAT-2B bind to the Src-like kinases Fyn, Hck, Lyn, Lck, and Fgr, as determined by a yeast two-hybrid assay. However, unlike SAP, the EAT-2 proteins bind to their kinase domains and not to the SH3 domain of these kinases. Taken together, the data suggest that both EAT-2A and EAT-2B are adapters that recruit Src kinases to SLAM family receptors using a mechanism that is distinct from that of SAP. Electronic supplementary material Supplementary material is available for this article at and accessible for authorised users. S. Calpe and E. Erdős contributed equally to this work     

The Drosophila melanogaster homologue of the hsp60 gene is encoded by the essential locus l(1)10Ac and is differentially expressed during fly development

 The hsp60 (heat-shock protein 60) gene family of molecular chaperones has been a subject of study in numerous systems due to its important role in the correct folding of non-native proteins in development as well as after heat-shock treatment. Here we present the characterization of the first Drosophila hsp60 homologue. Drosophila HSP60 is most closely related (72% identity across the entire protein sequence) to the mouse mitochondrial HSP60. Western blot experiments indicate that Drosophila HSP60 is enriched in the mitochondrial fraction. The distribution of HSP60 protein is dynamic during fly embryogenesis, suggesting that various cell types might have different HSP60 requirements. The molecular analysis of a P-element-induced mutation that affects the l(1)10Ac locus shows that the transposon is inserted in a 3-kb intron present in the hsp60 gene. By genetic rescue experiments we prove that Drosophila HSP60 is encoded by the essential locus l(1)10Ac opening the possibility for detailed genetic analysis of HSP60 functions in the fly. Received: 24 March 1997 / Accepted: 16 June 1997     

Characterization of MSH/ACTH-like immunoreactivity in sciatic nerves of Xenopus laevis by immunocytochemistry,Western blotting and radioimmunoassay

Summary Previous studies have indicated that rat neurofilament protein may contain an endogenous MSH-like epitope with neuroregenerative properties. The presence of such an epitope has now been studied in nerve tissue from Xenopus laevis. Western blot analyses of sciatic nerve tissue using an assortment of sequence-specific MSH/ACTH antisera revealed the presence of two major immunoreactive protein bands of 52 and 50 kDa, which contained a mid-region MSH-like epitope. Weaker staining occurred in another protein band at 135 kDa. Immunocytochemistry revealed the immunoreactivity to reside in the axis cylinders of the nerve fibers. Other antisera, recognizing other regions of MSH/ACTH produced strong staining of Xenopus intermediate lobes, but failed to stain sciatic nerves. Thus, the proteins detected have no clear relation to either Xenopus neurofilament proteins or proopiomelanocortin.     

Polymorphisms of the glucocorticoid receptor gene in laboratory and wild rats: steroid binding properties of trinucleotide CAG repeat length variants

The polyglutamine tract, beginning at codon 75 in the N-terminal modulatory domain of rat glucocorticoid receptor (rGR), was analyzed in 61 inbred strains and 155 wild caught Rattus norvegicus. A discontinuous distribution of repeat lengths was found (7, 17–23 repeats). To investigate the possible significance of this distribution, full-length rGR cDNAs with 7, 18, 20, and 21 CAG repeats were expressed in CV-1 cells, and the resulting GR protein analyzed by Western blots and extensive Scatchard analyses. The quantity and steroid binding capacity of GR, together with the binding affinities for dexamethasone and corticosterone, were found to be indistinguishable for the four repeat alleles. From the sequencing of four inbred strains CAG repeat variants were found to be flanked by silent allelic substitutions at nucleotide positions 198, 531, and 711. The four variable sites extended over 471–519 bp of coding sequence, forming six Grl haplotypes. The results are discussed in the light of genetic studies on the Milan hypertensive and normotensive strains of rat. Codon sequence of rat GR required amendment at the following residues: D98, G226, D260, P600, and F602. Received: 30 May 1997 / Accepted: 21 October 1997