Expression patterns of class I and class IV alcohol dehydrogenase genes in developing epithelia suggest a role for alcohol dehydrogenase in local retinoic acid synthesis

Vitamin A (retinol) regulates embryonic development and adult epithelial function via metabolism to retinoic acid, a pleiotrophic regulator of gene expression. Retinoic acid is synthesized locally and functions in an autocrine or paracrine fashion, but the enzymes involved remain obscure. Alcohol dehydrogenase (ADH) isozymes capable of metabolizing retinol include class I and class IV ADHs, with class III ADH unable to perform this function. ADHs also metabolize ethanol, and high levels of ethanol inhibit retinol metabolism, suggesting a possible mode of action for some of the medical complications of alcoholism. To explore whether any ADH isozymes are linked to retinoic acid synthesis, herein we have examined the expression patterns of all known classes of ADH in mouse embryonic and adult tissues, and also measured retinoic acid levels. Using in situ hybridization, class I ADH mRNA was localized in the embryo to the epithelia of the genitourinary tract, intestinal tract, adrenal gland, liver, conjunctival sac, epidermis, nasal epithelium, and lung, plus in the adult to epithelia within the testis, epididymis, uterus, kidney, intestine, adrenal cortex, and liver. Class IV ADH mRNA was localized in the embryo to the adrenal gland and nasal epithelium, plus in the adult to the epithelia of the esophagus, stomach, testis, epididymis, epidermis, and adrenal cortex. Class III ADH mRNA, in contrast, was present at low levels and not highly localized in the embryonic and adult tissues examined. We detected significant retinoic acid levels in the fetal kidney, fetal/adult intestine and adrenal gland, as well as the adult liver, lung, testis, epididymis, and uterus--all sites of class I and/or class IV ADH gene expression. These findings indicate that the expression patterns of class I ADH and class IV ADH, but not class III ADH, are consistent with a function in local retinoic acid synthesis needed for the development and maintenance of many specialized epithelial tissues.     

Novel sites of adrenomedullin gene expression in mouse and rat tissues

Adrenomedullin (AM) was originally identified in pheochromocytoma tissue and was characterized as a hypotensive peptide. The tissue distribution and cellular localization of AM messenger RNA (mRNA) were determined in mouse and rat tissues by in situ hybridization. Three probes were used: two nonoverlapping probes to the pro-AM N-terminal 20 peptide (PAMP) and AM peptide regions of mouse pro-AM, and a larger complementary DNA (cDNA) probe spanning both the PAMP- and AM peptide-coding regions. The most intense expression of AM mRNA was in endometrium and epithelial cells lining the uterus and mouse adrenal medulla. Moderate levels of expression were detected in kidney glomerulus and cortical distal tubules, ovarian corpus luteum and follicles, epithelial cells lining the bronchioles, cardiac atrium and ventricle, posterior pituitary (particularly in female rats), stomach, small intestine (microvilli, mucosa and submucosa), spleen, and pancreas. Lower levels were observed in pulmonary alveoli, anterior pituitary, and submandibular gland. No expression was detected in the testis, thymus, skeletal muscle, or liver. The localization of AM mRNA in epithelial cells lining the uterus, bronchioles, and gastrointestinal tract indicates novel roles for AM, possibly as an antimicrobial agent. The strong expression of AM in uterus, ovary, and posterior pituitary suggests that AM plays a role in female reproduction.     

Chicken keratin-19: cloning of cDNA and analysis of expression in the chicken embryonic gut

From many recent studies, it has been argued that keratins (cytokeratins) play important roles in the morphogenesis and differentiation of organ development. To learn the role of keratin in digestive tract development, a cDNA of the chicken homolog of keratin-19 (GK-19) was cloned and its expression pattern was analyzed in the digestive tract of chicken embryos. The GK-19 full-length sequence was approximately 1.6 kb and showed more than 80% similarity to human and mouse keratin-19. The result of in situ hybridization with the proventriculus (glandular stomach) of different developmental stages showed that GK-19 expression disappeared specifically in the glandular epithelium from day 6 to day 9 of incubation. Furthermore, GK-19 was localized in the notochord, floor plate, anterior lobe of the pituitary gland and mesonephros. These results suggest the possibility that GK-19 may have multiple roles in organogenesis during embryogenesis.     

Hyperthyroidism. Etiology, physiopathology, diagnosis, evolution, treatment]

Serum amyloid A (SAA) is an acute-phase reactant whose level in the blood is elevated to 1000-fold as part of the body's responses to various injuries, including trauma, infection, inflammation, and neoplasia. As an acute-phase reactant, the liver has been considered to be the primary site of expression. However, limited extrahepatic SAA expression was described in mouse tissues and in cells of human atherosclerotic lesions. Here we describe nonradioactive in situ hybridization experiments revealing that the SAA mRNA is widely expressed in many histologically normal human tissues. Expression was localized predominantly to the epithelial components of a variety of tissues, including breast, stomach, small and large intestine, prostate, lung, pancreas, kidney, tonsil, thyroid, pituitary, placenta, skin epidermis, and brain neurons. Expression was also observed in lymphocytes, plasma cells, and endothelial cells. RT-PCR analysis of selected tissues revealed expression of the SAA1, SAA2, and SAA4 genes but not of SAA3, consistent with expression of these genes in the liver. Immunohistochemical staining revealed SAA protein expression that co-localized with SAA mRNA expression. These data indicate local production of the SAA proteins in histologically normal human extrahepatic tissues.     

Expression pattern for adrenomedullin during pancreatic development in the rat reveals a common precursor with other endocrine cell types

We cloned a cDNA encoding tissue inhibitor of metalloproteinases-3 (TIMP3) from the frog Xenopus laevis. Similar to TIMP3 from other species, Xenopus TIMP3 has 188 residues including 12 conserved cysteines and Asn184, a putative site for N-linked sugars. Xenopus TIMP3 is 84% identical with human TIMP3. As shown by Northern blotting and RT-PCR, Xenopus TIMP3 mRNA is maternally inherited in eggs and midblastula (stage 8) embryos, downregulated in gastrula and then upregulated in neurula and pretailbud embryos. In select adult tissues, TIMP3 mRNA is present in heart, muscle, liver, skin, intestine and ovaries. These results suggest that TIMP3 is involved in the regulation of expression of matrix metalloproteinases in Xenopus early development and adult tissue remodeling.     

Expression of rat bone sialoprotein promoter in transgenic mice

Bone sialoprotein (BSP) is a major protein of the mineralized bone extracellular matrix that has been implicated in the nucleation of hydroxyapatite crystals. Our previous studies have demonstrated that BSP mRNA is expressed by differentiated osteoblasts, odontoblasts, and cementoblasts involved in de novo mineralized tissue formation in a tissue-specific and developmentally regulated manner. To determine the basis of the selective expression of the BSP gene, we have generated four transgenic mouse lines in which 2.7 kb of the rat BSP promoter ligated to a luciferase reporter gene has been stably integrated into the mouse genome. Assays of luciferase activities in 5-day-old animals has revealed consistently high levels in bone tissues with negligible activities in various other organs including kidney, liver, stomach, intestine, and spleen. In some animals, variable expression was observed in brain and skin. Temporal analyses revealed the highest luciferase expression in neonatal bones, with expression decreasing markedly with subsequent growth and development, as observed previously for the endogenous gene in rats. Immunohistochemical analysis of luciferase activity and in situ hybridization of luciferase mRNA in bone tissues show that differentiated osteoblasts express the highest levels of luciferase, consistent with the induction of endogenous gene expression. These studies demonstrate that the regulation of the BSP gene during osteoblastic differentiation, together with its tissue-specific, developmentally regulated expression, is primarily mediated within the 2.7 kb region of the promoter.     

Aldehyde dehydrogenases of the rat colon: comparison with other tissues of the alimentary tract and the liver

Intracolonic bacteria have previously been shown to produce substantial amounts of acetaldehyde during ethanol oxidation, and it has been suggested that this acetaldehyde might be associated with alcohol-related colonic disorders, as well as other alcohol-induced organ injuries. The capacity of colonic mucosa to remove this bacterial acetaldehyde by aldehyde dehydrogenase (ALDH) is, however, poorly known. We therefore measured ALDH activities and determined ALDH isoenzyme profiles from different subcellular fractions of rat colonic mucosa. For comparison, hepatic, gastric, and small intestinal samples were studied similarly. Alcohol dehydrogenase (ADH) activities were also measured from all of these tissues. Rat colonic mucosa was found to possess detectable amounts of ALDH activity with both micromolar and millimolar acetaldehyde concentrations and in all subcellular fractions. The ALDH activities of colonic mucosa were, however, generally low when compared with the liver and stomach, and they also tended to be lower than in small intestine. Mitochondrial low K(m) ALDH2 and cytosolic ALDH with low K(m) for acetaldehyde were expressed in the colonic mucosa, whereas some cytosolic high K(m) isoenzymes found in the small intestine and stomach were not detectable in colonic samples. Cytosolic ADH activity corresponded well to ALDH activity in different tissues: in colonic mucosa, it was approximately 6 times lower than in the liver and about one-half of gastric ADH activity. ALDH activity of the colonic mucosa should, thus, be sufficient for the removal of acetaldehyde produced by colonic mucosal ADH during ethanol oxidation. It may, however, be insufficient for the removal of the acetaldehyde produced by intracolonic bacteria. This may lead to the accumulation of acetaldehyde in the colon and colonic mucosa after ingestion of ethanol that might, at least after chronic heavy alcohol consumption, contribute to the development of alcohol-related colonic morbidity, diarrhea, and cancer.     

Solution structure of synthetic peptide inhibitor and substrate of cAMP-dependent protein kinase. A study by 2D H NMR and molecular dynamics

Altered hepatic expression of apolipoproteins occurs during the acute phase response. Here we examined whether the acute phase response alters extra hepatic expression of apolipoproteins. Syrian hamsters were injected with endotoxin (LPS), tumor necrosis factor (TNF), interleukin (IL)-1, or the combination of TNF + IL-1 and mRNAs for serum amyloid A (apoSAA), apolipoprotein (apo) J, apo E. apo A-I, and apo D, were analyzed. LPS increased mRNA levels for apoSAA in all tissues examined. LPS and TNF + IL-1 increased mRNA levels for apo J in kidney, heart, stomach, intestine, and muscle. Individually, TNF and IL-1 were less potent than the combination of the two cytokines. LPS decreased mRNA levels for apo E in all tissues, except for mid and distal intestine. TNF and IL-1 were less effective than LPS. LPS, TNF + IL-1 and TNF decreased mRNA levels for apo A-I in duodenum. mRNA for apo D decreased in heart, were unchanged in brain and increased in muscle, following LPS. The widespread extra hepatic regulation of the apolipoproteins during the acute phase response may be important for the alterations in lipid metabolism that occur during infection and inflammation as well as the immune response.     

Molecular cloning and hormonal regulation of PiT-1, a sodium-dependent phosphate cotransporter from rat parathyroid glands

The extracellular concentration of inorganic phosphate (Pi) is an important determinant of parathyroid cell function. The effects of Pi may be mediated through specific molecules in the parathyroid cell membrane, one candidate molecule for which would be a Na+-dependent Pi cotransporter. A complementary DNA encoding a Na+-Pi cotransporter, termed rat PiT-1, has now been isolated from rat parathyroid. The 2890-bp complementary DNA encodes a protein of 681 amino acids that shows sequence identities of 97% and 93% with the type III Na+-Pi cotransporters mouse PiT-1 and human PiT-1, respectively. Expression of rat PiT-1 in Xenopus oocytes revealed that it possesses Na+-dependent Pi cotransport activity. PiT-1 messenger RNA (mRNA) is widely distributed in rat tissues and is most abundant in brain, bone, and small intestine. The amount of PiT-1 mRNA in the parathyroid of vitamin D-deficient rats was reduced compared with that in normal animals and increased markedly after administration of 1,25-dihydroxyvitamin D3. Furthermore, the abundance of PiT-1 mRNA in the parathyroid was much greater in rats fed a low-Pi diet than in those fed a high-Pi diet. Thus, rat PiT-1 may contribute to the effects of Pi and vitamin D on parathyroid function.     

Expression of a soluble form of LFA-1 and demonstration of its binding activity with ICAM-1

The mouse homologues of the breast cancer susceptibility genes, Brca1 and Brca2, are expressed in a cell cycle-dependent fashion in vitro and appear to be regulated by similar or overlapping pathways. Therefore, we compared the non isotopic in situ hybridization expression patterns of Brca1 and Brca2 mRNA in vivo in mitotic and meiotic cells during mouse embryogenesis, mammary gland development, and in adult tissues including testes, ovaries, and hormonally altered ovaries. Brca1 and Brca2 are expressed concordantly in proliferating cells of embryos, and the mammary gland undergoing morphogenesis and in most adult tissues. The expression pattern of Brca1 and Brca2 correlates with the localization of proliferating cell nuclear antigen, an indicator of proliferative activity. In the ovary, Brca1 and Brca2 exhibited a comparable hormone-independent pattern of expression in oocytes, granulosa cells and thecal cells of developing follicles. In the testes, Brca1 and Brca2 were expressed in mitotic spermatogonia and early meiotic prophase spermatocytes. Northern analyses of prepubertal mouse testes revealed that the time course of Brca2 expression was delayed in spermatogonia relative to Brca1. Thus, while Brca1 and Brca2 share concordant cell-specific patterns of expression in most proliferating tissues, these observations suggest that they may have distinct roles during meiosis.     

A new family of heparin-binding growth/differentiation factors: increased midkine expression in Wilms' tumor and other human carcinomas

Midkine (MK) and heparin-binding growth-associated molecule/pleiotrophin form a new family of heparin-binding growth/differentiation factors. We studied MK gene expression in human tumors. In normal human reference tissues, MK was highly expressed in the mucosal tissue of the small intestine, moderately in the thyroid, weakly in the tissues of the lung, colon, stomach, kidney, and spleen, and not at all in the liver. All of 6 surgically removed specimens of Wilms' tumor highly expressed MK. Also, a moderate to intense level of MK expression was noted in the majority of surgically removed hepatocellular carcinomas. The MK mRNA level was analyzed in a number of cultured and nude mice-transplanted lines of human tumors. In stomach, colon, pancreatic, lung, and esophageal carcinomas, a moderate to high level of MK expression was found in the majority of them. These results suggest an important role of MK in the development and/or biological behavior of tumors and raised a possibility to use MK as a diagnostic marker. Heparin-binding growth associated molecule/pleiotrophin mRNA was low or scarcely detectable in samples analyzed thus far except for significant levels of the expression that were observed in PA-1 teratocarcinoma cells and in some surgical specimens of Wilms' tumor.     

Formation of a novel enzymatic metabolite of leukotriene A4 in tissues of Xenopus laevis

Leukotriene-A4, hydrolase catalyzes the final step in the biosynthesis of the potent proinflammatory mediator leukotriene B4. Previously, leukotriene-A4 hydrolase has been characterized from human, mouse and rat sources, i.e. only from mammalian species. In the present investigation, expression of leukotriene-A4, hydrolase was studied in organs of Xenopus laevis. Enzyme activity was found in all nine organs tested with the highest levels in the intestine and the reproductive organs, i.e. oocytes and testes, previously unrecognized rich sources of the enzyme. No immunoreactive leukotriene-A4 hydrolase was detected in Western blots of 10000Xg supernatants of X. laevis organ homogenates, using a polyclonal antiserum raised against human leukotriene-A4 hydrolase. Likewise, Northern blot analysis of liver total RNA did not detect Xenopus leukotriene-A4 hydrolase mRNA using a human CDNA probe. These results indicate significant structural differences between the human and toad enzymes. Incubations of 10000Xg supernatants of organ homogenates with leukotriene A4 revealed the formation of a novel metabolite, denoted compound X. Conversion of leukotriene A4 into compound X was due to an enzymatic activity as judged by its protein dependence, heat sensitivity, and resistance to ultrafiltration, and this activity appeared to be linked, directly or indirectly,, to leukotriene A4 hydrolase. From data obtained by ultraviolet spectrophotometry, gas chromatography coupled to mass spectrometry, ultraviolet-induced isomerization, and comparison with a synthetic standard, compound X was assigned the structure 5S,12R-dihydroxy-6,10-trans-8,14-cis-eicosatetraenoic acid. Finally, compound X was found to exhibit contractile activity in guinea-pig lung parenchyma, apparently elicited via a leukotriene B receptor.     

Mouse hypoxia-inducible factor-1alpha is encoded by two different mRNA isoforms: expression from a tissue-specific and a housekeeping-type promoter

Hypoxic induction of erythropoietin (Epo) and other oxygen-dependent genes is mediated by the hypoxia-inducible factor-1 (HIF-1), a heterodimeric transactivator consisting of an alpha and a beta subunit. We previously found that the mouse gene encoding HIF-1alpha harbors two alternative first exons (I.1 and I.2), giving rise to two different HIF-1alpha mRNA isoforms. Here, we show by RNase protection analysis that the exon I.1-derived mRNA isoform is differentially expressed in mouse tissues, being highest in kidney, tongue, stomach, and testis, but undetectable in liver, whereas the exon I.2 mRNA isoform is ubiquitously expressed. Sequence and methylation analysis showed that, in contrast to exon I.1, exon I.2 resides within a region showing typical features of a CpG island, known to be associated with the 5' end of housekeeping genes. We identified a 232-bp minimal exon I.2 promoter that strongly induced reporter gene expression in mouse L929 fibroblasts and Hepa1 hepatoma cells. In contrast to L929 cells, the exon I.1 promoter was inactive in Hepa1 cells and hypoxic exposure (1% O2) markedly reduced exon I.2 promoter activity in Hepa1 cells. Prolonged exposure of mice to hypoxia (7.5% O2 for up to 72 hours) also caused a decrease in liver HIF-1alpha mRNA, whereas aldolase mRNA levels increased. These findings might be related to the relatively low Epo levels in the adult liver.     

Identification of a gene encoding a hyperpolarization-activated pacemaker channel of brain

The generation of pacemaker activity in heart and brain is mediated by hyperpolarization-activated cation channels that are directly regulated by cyclic nucleotides. We previously cloned a novel member of the voltage-gated K channel family from mouse brain (mBCNG-1) that contained a carboxy-terminal cyclic nucleotide-binding domain (Santoro et al., 1997) and hence proposed it to be a candidate gene for pacemaker channels. Heterologous expression of mBCNG-1 demonstrates that it does indeed code for a channel with properties indistinguishable from pacemaker channels in brain and similar to those in heart. Three additional mouse genes and two human genes closely related to mBCNG-1 display unique patterns of mRNA expression in different tissues, including brain and heart, demonstrating that these channels constitute a widely expressed gene family.     

Evidence for transcriptional modulation but not acid phosphatase expression during programmed cell death in the colonial tunicate Botryllus schlosseri

Botryllus schlosseri is a clonally modular ascidian in which asexually derived adults (zooids) exhibit developmental synchrony. At the conclusion of the blastogenic (asexual) cycle every 5 days at 21 degrees C, all zooids within a colony die simultaneously in 24 hours and are replaced by a new asexual generation of zooids. This cyclical process, called takeover, involves the selective destruction of the zooid's visceral tissues which include the pharynx, esophagus, stomach, intestine, endostyle, neural complex and heart, whereas bud tissues and mesenchymal components (muscle and blood cells) remain unaffected. Ultrastructural analysis indicates that the most prevalent form of cell death occurs by apoptosis, although necrotic changes are also observed in several tissues (i.e., stomach and intestine). Blood-derived macrophages and neighboring cells subsequently engulf visceral tissues, reducing the zooid to the size of a small vesicle. Here, we have tested the possibility that acid phosphatase, a hydrolase whose presence is associated with cell death in several invertebrate systems, could account for some of the regressive changes observed during takeover. Our observations indicate that acid phosphatase (AP) activity was selectively localized in the gut of parent zooids during the growth phase of the cycle, with the stomach exhibiting the most intense histochemical staining on tissue sections. As zooid regression progressed during takeover, stomach AP staining gradually disappeared. Other visceral tissues never became AP-positive. Therefore, this hydrolase appears to play a minimal role in zooid death. In order to characterize genes whose expression pattern was selectively altered during takeover, we have carried out differential mRNA display analysis. We report on two genes, 790.3 and 790.4, that are down- and upregulated, respectively, during this process. Collectively, these findings indicate that the takeover phase of blastogenesis in Botryllus involves modulated gene expression.     

Immunohistochemical localisation of the serotonin 5-HT2B receptor in mouse gut, cardiovascular system, and brain

We recently reported the cloning of a new member of the serotonin 5-HT2 family, the 5-HT2B receptor. We now report the production and characterisation of a specific antiserum directed against the C-terminal portion of the mouse 5-HT2B receptor. After affinity purification, this polyclonal antibody recognises specifically the mouse 5-HT2B receptor. Immunohistochemical analysis of cryosections from various adult mouse tissues reveals a major 5-HT2B receptor expression in stomach, intestine and pulmonary smooth muscles as well as in myocardium. Furthermore, the antiserum recognises specific areas of the mouse brain, including cerebellar Purkinje cells and their projection areas.