1,721,120 research outputs found
Evolution of scales and their hard proteins in reptiles in relation to cornification of skin appendages in the amniote integument
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Isolation of a new class of cysteine-glycine-proline-rich beta-proteins (beta-keratins) and their expression in snake epidermis.
No full textScales of snakes contain hard proteins (beta-keratins), now referred to as keratin-associated beta-proteins. In the present study we report the isolation, sequencing, and expression of a new group of these proteins from snake epidermis, designated cysteine-glycine-proline-rich proteins. One deduced protein from expressed mRNAs contains 128 amino acids (12.5 kDa) with a theoretical pI at 7.95, containing 10.2% cysteine and 15.6% glycine. The sequences of two more snake cysteine-proline-rich proteins have been identified from genomic DNA. In situ hybridization shows that the messengers for these proteins are present in the suprabasal and early differentiating beta-cells of the renewing scale epidermis. The present study shows that snake scales, as previously seen in scales of lizards, contain cysteine-rich beta-proteins in addition to glycine-rich beta-proteins. These keratin-associated beta-proteins mix with intermediate filament keratins (alpha-keratins) to produce the resistant corneous layer of snake scales. The specific proportion of these two subfamilies of proteins in different scales can determine various degrees of hardness in scales
Tissue-specific transcriptional initiation of the CYP19 genes in rainbow trout, with analysis of splicing patterns and promoter sequences
No full textThe rainbow trout (Oncorhynchus mykiss Walbaum) genome contains three separate CYP19 genes for distinct isoforms of cytochrome P450arom: CYP19A encoding the prevalently ovarian isoform P450aromA, and CYP19B-I and II, encoding forms I and II of the mainly cerebral variant P450aromB. RNA Ligase-Mediated 5'-Rapid Amplification of cDNA Ends analysis was used to determine the 5'-untranslated terminal regions (5'-UTRs) of the corresponding mRNAs, which are actually all expressed in the ovary, brain and gills. CYP19A is transcribed at different transcription start sites (TSSs) in each tissue, the most distal TSS being found in the brain, the intermediate one in the gills, and the proximal one in the ovary. CYP19B-I also displays tissue-specific TSSs, but transcripts undergo three distinct splicing patterns: the same pattern as previously reported for the brain and occurring also in the gills, and two novel patterns, established in the ovary and brain, which include two cryptic 3'-splice sites in intron 1, leading to the inclusion of intronic sequences of 92/94 and 66 b in the 5'-UTRs. Lastly, the CYP19B-II transcript in the ovary shows the same splicing pattern previously described for the brain. A PCR-based gene walking strategy was used to explore the promoter regions of the rainbow trout CYP19 genes, which were found to contain potential binding sites for a variety of transcription factors
Ultrastructural Localization of Hair Keratin Homologs in the Claw of the Lizard Anolis carolinensis
No full texthe claw of lizards is largely composed of beta-keratins, also referred to as keratin-associated beta-proteins. Recently, we have reported that the genome of the lizard Anolis carolinensis contains alpha keratin genes homologous to hair keratins typical of hairs and claws of mammals. Molecular and immunohistochemical studies demonstrated that two hair keratin homologs named hard acid keratin 1 (HA1) and hard basic keratin 1 (HB1) are expressed in keratinocytes forming the claws of A. carolinensis. Here, we extended the immunocytochemical localization of the novel reptilian keratins to the ultrastructural level. After sectioning, claws were subjected to immunogold labeling using antibodies against HA1, HB1, and, for comparison, beta-keratins. Electron microscopy showed that the randomly organized network of tonofilaments in basal and suprabasal keratinocytes becomes organized in long and parallel bundles of keratin in precorneous layers, resembling cortical cells of hairs. Entering the cornified part of the claw, the elongated corneous cells fuse and accumulate corneous material. HA1 and HB1 are absent in the basal layer and lower spinosus layers of the claw and are expressed in the upper and precorneous layers, including the elongating corneocytes. The labeling for alpha-keratin was loosely associated with filament structures forming the fibrous framework of the claws. The ultrastructural distribution pattern of hard alpha-keratins resembled that of beta-keratins, which is compatible with the hypothesis of an interaction during claw morphogenesis. The data on the ultrastructural localization of hair keratin homologs facilitate a comparison of lizard claws and mammalian hard epidermal appendages containing hair keratins
Rat cytochrome P450c17 gene transcription is initiated at different start sites in extraglandular and glandular tissues
No full textIn the male rat, the mRNA of the steroidogenic cytochrome P450c17 is expressed extraglandularly in the stomach, duodenum, kidney and liver, throughout the animal's lifespan, as demonstrated by reverse transcriptase and polymerase chain reaction (RT-PCR) analysis with specific primers. Northern analysis indicated that all tissues, except the kidney, contain high levels of such mRNA, but the relative mobility of liver mRNA is slightly less than that of the testis and other tissues. Thus, we analysed their 5'- and 3'-untranslated terminal regions (UTRs) by means of 5'- and 3'-rapid amplification of cDNA ends (RACE) techniques. All tissues utilised the same polyadenylation site as the testis. In the 5'-UTR of liver mRNA, however, we found a distal transcription start site (TSS) located 252b upstream of that used in testicular P450c17 mRNA, which is placed 41b upstream of the first ATG. The 5'-UTR sequence of liver P450c17 cDNA exactly matched the contiguous upstream untranslated region of the gene, suggesting that alternative splicing was not involved in the synthesis of liver P450c17 transcript. The other tissues used the same TSS present in the testis. Nevertheless, a second TSS located 125b upstream of the first ATG was found in the stomach and duodenum. These results show that the transcriptional regulation of the CYP17 gene in the rat is complex and differs between tissues in the use of TSSs
Characterisation of three variants of estrogen receptor beta mRNA in the common sole, Solea solea L. (Teleostei).
No full textIn all vertebrates, estrogen action is mediated by cognate nuclear receptors. In this study, we cloned the different transcripts of the estrogen receptor beta (ERbeta) gene of the common sole, Solea solea. 5'-RLM-RACE (RNA Ligase-Mediated 5'-Rapid Amplification of cDNA Ends) and 3'-RACE analyses revealed three isoforms of different length, called Long, Intermediate and Short isoforms, consisting of 2212, 1531 and 1207 b, respectively. The Long isoform is characterised by an open reading frame (ORF) encoding 589aa, with an estimated molecular weight of 65kDa. Phylogenetic analysis established that it belongs to the teleost ERbeta1 or ERbetaa cluster. The Intermediate isoform encodes a 490-aa protein, which lacks the first 99aa of the Long isoform, but still retains a complete DNA-binding domain (DBD). In the Short variant (363aa-long), all the N-terminal region, down to the two zinc fingers included, is missing, thus crippling DBD. ERbeta transcription was analysed by semiquantitative RT-PCR with specific primers, common to the Long and Intermediate isoforms, in various sole tissues, such as brain, gills, muscle, stomach, intestine, spleen, head kidney, kidney, liver and gonads. This analysis revealed that ERbeta displays a widespread or ubiquitous pattern of transcription, with the highest levels being found in the gonads and liver
Use of random amplification to develop a PCR detection method for the causative agent of fish pasteurellosis, Photobacterium damselae subsp piscicida (Vibrionaceae)
No full textRandom amplification of polymorphic DNA (RAPD) was used to generate species-specific markers, which were exploited in the construction of a fast and convenient PCR-based diagnostic assay for the causative agent of fish pasteurellosis or pseudotuberculosis, the bacterium Photobacterium damselae subsp. piscicida, formerly known as Pasteurella piscicida. In RAPD genomic fingerprints of this bacterium, two distinctive fragments were observed. These fragments were cloned, sequenced and found to bear no significant homology with known DNA sequences. Two sets of primers were then synthesized to amplify, by PCR, fragments within the sequences between the terminal RAPD primers of the fragments. Another primer pair was designed to perform nested PCR internally to one of the amplified fragments. The species specificity of the PCR products was analyzed, by dot blotting and Southern hybridization, with DNA from 13 other bacterial species. Cross-reactivity was obtained only with isolates of P. damselae subsp. damselae (formerly Vibrio damselae) a bacterial species with a high degree of overall genomic homology with P. d. piscicida. The detection limit of the assay was 20 fg of template DNA by PCR and 2 fg by nested PCR. The assay was effective in revealing P. d. piscicida when applied to an aliquot of DNA extracted from 50-mg samples of kidney, spleen and liver tissues from naturally infected fish. In conclusion, these results demonstrate the utility of the RAPD plus PCR approach in the development of genetic diagnostic assays for fish pathogens
Extraglandular hormonal steroidogenesis in aged rats.
No full textWe have examined the metabolisM in Vitro of [4-C-14]pregnenolone by the following organs of 2.4-year-old rats: submandibular gland, stomach, duodenum, liver, lung, heart, spleen, kidney, skin, prostate, testis and adrenal. All tissues converted pregnenolone to progesterone, the highest yields being observed with adrenal, testis and skin. Androgen formation was intense in the testis and absent in the adrenal. Moreover, 17alpha-hydroxylation of pregnenolone occurred moderately in kidney, skin and submandibular gland and markedly in duodenum and stomach, which also produced high amounts of dehydroepiandrosterone and/or 5-androstene-3beta,17beta-diol. Extratesticular synthesis of androstenedione and testosterone was very low. A significant formation of 20alpha-dihydropregnenolone was observed in all tissues but stomach, duodenum and steroidogenic endocrines. Corticosteroids were not synthesized extraadrenally, except a small amount of 11-deoxycorticosterone in the testis. These results indicate that key steroid-biosynthetic enzymes, such as 3beta-hydroxysteroid dehydrogenase/DELTA5-DELTA4 isomerase, 17beta- and 20alpha-hydroxysteroid dehydrogenases and steroid 17alpha-monooxygenase/17,20-lyase are also expressed extraglandularly in the rat
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