Widespread Tissue Expression Research Articles

Cloning of Human Junctional Adhesion Molecule 3 (JAM3) and Its Identification as the JAM2 Counter-receptor

We have identified a third member of the junctional adhesion molecule (JAM) family. At the protein level JAM3 displays 36 and 32% identity to JAM2 and JAM1, respectively. The coding region is distributed over 9 exons and maps to chromosome 11q25. The gene shows widespread tissue expression with higher levels apparent in the kidney, brain, and placenta. At the cellular level we show expression of JAM3 transcript within endothelial cells. Our major finding is that JAM3 and JAM2 are binding partners. Thus, JAM3 ectodomain binds firmly to JAM2-Fc. This heterotypic interaction is maintained when JAM3-Fc is used to capture Chinese hamster ovary cells expressing full-length JAM2. In static adhesion assays we show that JAM3 is unable to bind to leukocyte cell lines. This is consistent with the lack of JAM2 expression. However, using JAM2-Fc pull-down experiments in combination with polyclonal anti-JAM3 serum, we demonstrate that JAM3 is the previously uncharacterized 43-kDa counter-receptor that mediates JAM2 adhesion to T cells. Most significantly we demonstrate up-regulation of JAM3 protein on peripheral blood lymphocytes following activation. Finally we show the utility of JAM3 ectodomain as an inhibitor of JAM2 adhesion.

The Neural Cell Recognition Molecule Neurofascin Interacts with Syntenin-1 but Not with Syntenin-2, Both of Which Reveal Self-associating Activity

Neurofascin belongs to the L1 subgroup of the immunoglobulin superfamily of cell adhesion molecules and is implicated in axonal growth and fasciculation. We used yeast two-hybrid screening to identify proteins that interact with neurofascin intracellularly and therefore might link it to trafficking, spatial targeting, or signaling pathways. Here, we demonstrate that rat syntenin-1, previously published as syntenin, mda-9, or TACIP18 in human, is a neurofascin-binding protein that exhibits a wide-spread tissue expression pattern with a relative maximum in brain. Syntenin-1 was found not to interact with other vertebrate members of the L1 subgroup such as L1 itself or NrCAM. We confirmed the specificity of the neurofascin-syntenin-1 interaction by ligand-overlay assay, surface plasmon resonance analysis, and colocalization of both proteins in heterologous cells. The COOH terminus of neurofascin was mapped to interact with the second PDZ domain of syntenin-1. Furthermore, we isolated syntenin-2 that may be expressed in two isoforms. Despite their high sequence similarity to syntenin-1, syntenin-2alpha, which interacts with neurexin I, and syntenin-2beta do not bind to neurofascin or several other transmembrane proteins that are binding partners of syntenin-1. Finally, we report that syntenin-1 and -2 both form homodimers and can interact with each other.

Isozyme distribution of ten enzymes and their loci in South American lungfish, Lepidosiren paradoxa (Osteichthyes, Dipnoi)

Scarce bibliographical data exists on the enzymes in Lepidosiren paradoxa and analysis of several enzymes was considered worthy of investigation. Distribution of ADH, ALP, FBALD, GAPDH, G3PDH, G6PDH, GPI, LDH, MDH, and PGM was identified in ten tissues (retina, heart, muscle, liver, kidney, lung, gut, gills, brain, and ovary) of the South American lungfish and compared with patterns previously described in other vertebrates. Compared with earlier results differences in the number of loci expressed were observed for ADH, G3PDH, GPI, and MDH. The number of loci expressed and/or in tissue specificity of several enzymes (ADH, FBALD, GAPDH, G3PDH, G6PDH and PGM) were found to be similar to those of other vertebrates. Differences were detected in ALP due to the absence of an intestinal-specific form typical of fish, amphibians, reptiles and birds; further differences were observed in GPI and MDH due to their tissue expression. The differences in LDH involve the LDH-A 4 isozyme which was most common in tissues. Overall, comparison with other vertebrates reveals that in L. paradoxa the tissue-restricted expressions of some enzymes are similar, while others have retained an ancestral pattern and exhibit a more widespread tissue expression of genes.

Nephrocystin: gene expression and sequence conservation between human, mouse, and Caenorhabditis elegans.

Juvenile nephronophthisis, an autosomal recessive cystic kidney disease, is the primary genetic cause for chronic renal failure in children. The gene (NPHP1) for nephronophthisis type 1 has recently been identified. Its gene product, nephrocystin, is a novel protein of unknown function, which contains a src-homology 3 domain. To study tissue expression and analyze amino acid sequence conservation of nephrocystin, the full-length murine Nphp1 cDNA sequence was obtained and Northern and in situ hybridization analyses were performed for extensive expression studies. The results demonstrate widespread but relatively weak NPHP1 expression in the human adult. In the adult mouse there is strong expression in testis. This expression occurs specifically in cell stages of the first meiotic division and thereafter. In situ hybridization to whole mouse embryos demonstrated widespread and uniform expression at all developmental stages. Amino acid sequence conservation studies in human, mouse, and Caenorhabditis elegans show that in nephrocystin the src-homology 3 domain is embedded in a novel context of other putative domains of protein-protein interaction, such as coiled-coil and E-rich domains. It is concluded that for multiple putative protein-protein interaction domains of nephrocystin, sequence conservation dates back at least to Caenorhabditis elegans. The previously described discrepancy between widespread tissue expression and the restriction of symptoms to the kidney has now been confirmed by an in-depth expression study.

Circadian regulation of prion protein messenger RNA in the rat forebrain: a widespread and synchronous rhythm

Although the expression of the normal prion protein in the host is critical to the development of transmissible spongiform encephalopathies, 16 the physiological role of this protein and the processes regulating its expression remain obscure. We now report that the messenger RNA for the prion protein is regulated in the rat brain in a marked circadian manner not only in the suprachiasmatic nuclei, the principal site for the generation of mammalian circadian rhythms, 14 but also in other forebrain regions. The data show a remarkable consistency in the concurrence of a single peak of prion protein messenger RNA at each of the sites early in the animal's phase of increased locomotor activity; behavioural arousal does not, however, appear to affect this expression. We believe this to be the first study demonstrating that the expression of prion protein messenger RNA can change over a relatively short period in vivo. The results are discussed with reference to the range of recently discovered “clock-related” transcripts which also have widespread tissue expression; these include the messenger RNAs for D-box binding protein and thyroid embryonic factor, transcription factors which bind to the prion protein promoter. 12

Characterization of the cloned human intermediate-conductance Ca2+-activated K+ channel.

The human intermediate-conductance, Ca2+-activated K+ channel (hIK) was identified by searching the expressed sequence tag database. hIK was found to be identical to two recently cloned K+ channels, hSK4 and hIK1. RNA dot blot analysis showed a widespread tissue expression, with the highest levels in salivary gland, placenta, trachea, and lung. With use of fluorescent in situ hybridization and radiation hybrid mapping, hIK mapped to chromosome 19q13.2 in the same region as the disease Diamond-Blackfan anemia. Stable expression of hIK in HEK-293 cells revealed single Ca2+-activated K+ channels exhibiting weak inward rectification (30 and 11 pS at -100 and +100 mV, respectively). Whole cell recordings showed a noninactivating, inwardly rectifying K+ conductance. Ionic selectivity estimated from bi-ionic reversal potentials gave the permeability (PK/PX) sequence K+ = Rb+ (1.0) > Cs+ (10.4) >> Na+, Li+, N-methyl-D-glucamine (>51). NH+4 blocked the channel completely. hIK was blocked by the classical inhibitors of the Gardos channel charybdotoxin (IC50 28 nM) and clotrimazole (IC50 153 nM) as well as by nitrendipine (IC50 27 nM), Stichodactyla toxin (IC50 291 nM), margatoxin (IC50 459 nM), miconazole (IC50 785 nM), econazole (IC50 2.4 microM), and cetiedil (IC50 79 microM). Finally, 1-ethyl-2-benzimidazolinone, an opener of the T84 cell IK channel, activated hIK with an EC50 of 74 microM.

The Novel Receptor TRAIL-R4 Induces NF-κB and Protects against TRAIL-Mediated Apoptosis, yet Retains an Incomplete Death Domain

A fourth member of the emerging TRAIL receptor family, TRAIL-R4, has been cloned and characterized. TRAIL-R4 encodes a 386–amino acid protein with an extracellular domain showing 58%–70% identity to those of TRAIL-R1, TRAIL-R2, and TRAIL-R3. The signaling capacity of TRAIL-R4 is similar to that of TRAIL-R1 and TRAIL-R2 with respect to NF-κB activation, but differs in its inability to induce apoptosis. Yet TRAIL-R4 retains a C-terminal element containing one third of a consensus death domain motif. Transient overexpression of TRAIL-R4 in cells normally sensitive to TRAIL-mediated killing confers complete protection, suggesting that one function of TRAIL-R4 may be inhibition of TRAIL cytotoxicity. Like TRAIL-R1 and TRAIL-R2, this receptor shows widespread tissue expression. The human TRAIL-R4 gene has been mapped to chromosome 8p22-21, clustered with three other TRAIL receptors.

The human α3b is a `full-sized' laminin chain variant with a more widespread tissue expression than the truncated α3a

We report the molecular cloning of the human laminin α3b chain variant and its mRNA expression pattern in adult human tissues when compared to the α3a variant. The mRNA encoding for the α3b variant is about 11 kb and the predicted translation product carries the complete set of domains typical for a `full-sized' laminin α chain. Apart from the similar domain structure of α3b also the sequence of α3 resulted more closely related to the α5 than to the α4 chain. Quantitative analysis of the RNA expression in a broad panel of adult human tissues indicated that the α3b variant is more widely distributed than the α3a shorter variant.

Widespread tissue expression of gastrin-binding-protein mRNA.

Glycine-extended forms of gastrin (gastrin-Gly) are thought to be involved in the autocrine growth control of colorectal carcinomas. The recently described gastrin-binding protein has been suggested to be a gastrin-Gly accepting receptor. Northern blot analysis demonstrated the expression of gastrin-binding-protein mRNA in many tissues of mouse, rat, and man. The gastrin-binding-protein mRNA expression was confirmed by reverse-transcribed PCR analysis. Analysis of the cDNA and the deduced amino acid sequence of the PCR-amplified rat gastrin-binding-protein DNA fragments revealed sequence identity (except in a single position) with the corresponding human and pig gastrin-binding protein and with the alpha-subunit of a rat and human mitochondrial trifunctional enzyme, involved in fatty acid oxidation. The widespread and abundant tissue expression of gastrin-binding-protein mRNA and its sequence identity with a fatty-acid-oxidizing enzyme do not support the view that it represents a genuine gastrin receptor.

Cloning of the Genes Encoding Two Murine and Human Cochlear Unconventional Type I Myosins

Several lines of evidence indicate a crucial role for unconventional myosins in the function of the sensory hair cells of the inner ear. We report here the characterization of the cDNAs encoding two unconventional type I myosins from a mouse cochlear cDNA library. The first cDNA encodes a putative protein named Myo1c, which is likely to be the murine orthologue of the bullfrog myosin Iβ and which may be involved in the gating of the mechanotransduction channel of the sensory hair cells. This myosin belongs to the group of short-tailed myosins I, with its tail ending shortly after a polybasic, TH-1-like domain. The second cDNA encodes a novel type I myosin Myo1f which displays three regions: a head domain with the conserved ATP- and actin-binding sites, a neck domain with a single IQ motif, and a tail domain with the tripartite structure initially described in protozoan myosins I. The tail of Myo1f includes (1) a TH-1 region rich in basic residues, which may interact with anionic membrane phospholipids; (2) a TH-2 proline-rich region, expected to contain an ATP-insensitive actin-binding site; and (3) a SH-3 domain found in a variety of cytoskeletal and signaling proteins. Northern blot analysis indicated that the genes encoding Myo1c and Myo1f display a widespread tissue expression in the adult mouse.Myo1candMyo1fwere mapped byin situhybridization to the chromosomal regions 11D-11E and 17B-17C, respectively. The human orthologuous genesMYO1CandMYO1Fwere also characterized, and mapped to the human chromosomal regions 17p13 and 19p13.2–19p13.3, respectively.

Temporally manipulated rescue of visual and courtship abnormalities caused by a nonA mutation in Drosophila.

The no-on-transient-A gene was identified by independently isolated mutations causing visual-response abnormalities or courtship song defects. The nonA(diss) mutant is abnormal for all of these phenotypes. The pleiotropic effects of this dissonance allele dovetail with the widespread tissue expression of the nonA products. This gene, which encodes a putative RNA-binding protein, is expressed at essentially all stages of the life cycle. To determine whether the behavioral and physiological abnormalities exhibited by nonA mutant adults have a developmental etiology or are the result of an impaired mature nervous (or perhaps neuro-muscular) system, a conditional form of this gene was constructed. Animals from the resulting hsp-nonA (cDNA) transgenic strain were subjected to heat-shock regimes such that the gene's coding sequences were activated during development only, solely in the imaginal stage, or both. Surprisingly, expression during any of these time periods effected rescue of the visual-response and the courtship-song defects.

Differential mRNA regulation of integrin subunits alpha V, beta 1, beta 3, and beta 5 during mouse embryonic organogenesis.

Cell interactions with extracellular matrices play important roles in morphogenetic processes during embryonic development. Extracellular matrix receptors of the integrin family have been implicated in these steps. Recent studies indicate that a variety of integrins can be differentially expressed during development, consistent with diverse roles for integrins in embryogenesis. The present study compares the expression patterns of several major members of the alpha V integrin subfamily, focusing on mRNA expression of alpha V, beta 1, beta 3, and beta 5 subunits during mouse embryonic organogenesis using Northern blot analysis and in situ hybridization. The alpha V and beta 1 subunits showed widespread tissue expression, although most tissues expressed alpha V at relatively low or basal levels. The mRNA for beta 5 was also expressed in a variety of embryonic organs and showed unusual localization patterns in certain organs. Striking, high-level expression of beta 5 transcripts was detected in the ependymal layer of the central nervous system, glomeruli of the kidney, epicardial region of the heart, and in the tooth germs, suggesting specific functions for this molecule during morphogenetic events in these organs. In contrast, few beta 3 transcripts were expressed during mid-gestation mouse embryogenesis except in megakaryocytes within the embryonic liver. These observations of differing expression spectra suggest that members of the alpha V integrin subfamily have distinct roles and also suggests that they have different transcriptional regulation. The beta 5 integrin is unique in its degree of tissue-specific mRNA regulation associated with morphogenesis of embryonic organs.

CTL access to tissue antigen is restricted in vivo.

MHC class I-restricted, hepatitis B surface Ag (HBsAg)-specific, CD8+ polyclonal CTL lines and clones cause a severe necroinflammatory liver disease when they are injected i.v. into transgenic mice that display widespread tissue expression of HBsAg. Surprisingly, the same CTLs fail to cause disease in any other HBsAg-positive tissue in these animals. However, the CTLs are highly cytopathic for HBsAg-positive renal tubules and choroid plexus epithelial cells when they are injected extravascularly, either beneath the kidney capsule or intracerebrally. Analysis of the microvascular anatomy of these tissues reveals that the hepatic sinusoid is characterized by a discontinuous endothelium and the absence of a basement membrane. In contrast, the vasculature of most other tissues displays a continuous endothelium and basement membrane; and the epithelial cells of many of these tissues are also surrounded by a separate basement membrane. These results suggest that viral, tumor, and normal self Ags expressed in the liver are readily accessible to class I-restricted T cells, whereas the vascular endothelium and/or the vascular and cellular basement membranes constitute an extremely effective barrier that precludes CTL access to tissue Ag at many other sites. Because class I-restricted Ag recognition occurs throughout the body in many natural diseases and disease models, the vascular barrier must be breached by other events before CTL access to endogenously synthesized epithelial Ag can occur.

Lipopolysaccharide Treatment in Vivo Induces Widespread Tissue Expression of Inducible Nitric Oxide Synthase mRNA

Nitric oxide (NO) may mediate the hypotension of septic shock, but the effect of endotoxin on inducible NO synthase (iNOS) mRNA expression remains unclear. We studied the effects of lipopolysaccharide (LPS) treatment in vivo on iNOS mRNA expression using reverse transcription and polymerase chain reaction. The iNOS mRNA was absent or negligible in any tissue studied from control rats, but was markedly increased in lung, liver, spleen, skeletal muscle and kidney from LPS-treated rats. The LPS-induced increase in iNOS mRNA was prevented by dexamethasone. Our results indicate that LPS treatment in vivo induces the expression of an iNOS mRNA via a dexamethasone-sensitive mechanism, and thus provide direct molecular evidence for the involvement of NO in septic shock.

Tissue- and treatment-specific usage of multiple preproenkephalin transcriptional start sites.

The significance of the 5' heterogeneity of the transmitter gene ppEnk was evaluated by comparing start site usage (E1-E4) between 12 tissues from untreated rats, using primer extension analysis. In the basal state, we found that E3- and E4-initiated transcripts accounted for 80% of the total striatal RNA present compared with a preferential usage of the E2 start site in all other tissues. To determine whether this selective expression could be modified by biologically relevant pathways, rats were made hypoglycemic. After insulin shock, only E3 + E4-initiated transcripts increased (16-fold at 1 day) in the adrenal medulla but were unaffected in the striatum. As the effects of insulin shock on the adrenal medulla are mediated by cholinergic pathways and the striatum also receives cholinergic inputs, we also compared the effects of cholinergic drug treatments on start site usage in these two tissues. Rats were treated with cholinergic agonists (nicotine + oxotremorine) which induced adrenomedullary E2 and E3 + E4 transcripts (5- and 80-fold, respectively). This effect peaked at 2 days. In contrast, in the same animals, striatal ppEnk RNA (E3 + E4) increased only 10-15-fold after drug treatment. Hence it appears that biologically relevant whole animal stimuli (insulin shock or cholinergic agents) activate biochemical pathways, which affect start site usage in a tissue-specific fashion. Selective RNA start site usage suggests a biological significance, which may be important in the widespread tissue expression of this gene.

1 - Growth factors and the regulation of pre- and postnatal growth

Peptide growth factors represent a largely paracrine level of intercellular communication that is basic to the process of life. Growth factors are present in the ovum and are amongst the first products expressed by the embryonic genome. They function as both signals and progression factors for embryonic tissue growth, induction, differentiation, maturation and function. While a widespread tissue expression is demonstrable during fetal development, and in certain postnatal tissues such as the epiphyseal growth plate, growth factor presence in the adult is restricted to tissues sharing rapid cellular turnover such as ovary. However, a transient re-expression of peptide growth factors occurs during adult tissue repair. In addition to mitogenic peptides such as IGFs or EGF, the family of growth factors also includes physiological growth inhibitors such as TGF beta and certain neuropeptides. Insulin is mitogenic in the early embryo and evidence is presented to support a continuation of this role, under defined nutritional conditions, in late gestation. The importance of insulin to pre- and postnatal growth has prompted an expanding literature dealing with the interactions of nutrients, hormones and growth factors during the growth and functional maturation of the islets of Langerhans. While the expression of growth factors in the early embryo is apparently autonomous, some, such as IGFs, become increasingly dependent on nutrient, insulin and GH availability during fetal development and in childhood growth. This has resulted in circulating IGF I and II determinations becoming useful diagnostic markers of endocrine-based growth disorder and nitrogen balance.

Site-specific mutagenesis of the class I regulatory element the Q10 gene allows expression in non-liver tissues.

Classical transplantation Ag are found on nearly all cells, whereas Ag encoded by the genes of the Qa/Tla region have a restricted tissue distribution. To investigate the cause of these different patterns of expression, we have compared the regulatory regions of Q10, a Qa region gene that is expressed only in liver, with those of H-2Ld. Gel retardation analysis shows that the nuclear factor (rI) that binds to the inverted repeat (TGGGGATTCCCCA) of the class I regulatory element (CRE) present in H-2Ld and other classical class I genes does not bind to the equivalent region of the Q10 gene. However, the Q10 CRE binds another nuclear factor (rII) that binds to the H-2Ld CRE. The sequence of the Q10 CRE differs from the sequence present in classical class I genes by three nucleotides, two of these changes are within the inverted repeat sequence (TGaGGAcTCCCCA) and disrupt the region of dyad symmetry. We have used site-specific in vitro mutagenesis to individually change these two nucleotides and have investigated the ability of this region to promote transcription with and without these substitutions. A change of either base restores transcriptional activity in chloramphenicol acetyl transferase assays and allows for binding of the rI nuclear factor. These results suggest that the failure of the Q10 CRE to bind the rI nuclear factor may play a role in preventing Q10 expression in tissues other than the liver and fetal yolk sac. Thus, the dichotomy between the widespread tissue expression of classical class I genes and the restricted tissue expression of class I genes of the Qa/Tla region may be due in part to differences in the cis acting regulatory sequences that interact with trans-acting nuclear factors to direct transcription.