Expression analysis of steroid hormone receptor mRNAs during zebrafish embryogenesis.

We have analyzed by qRT-PCR and/or RT-PCR the abundance and degradation rate of maternal mRNAs for nine steroid hormone receptors and their possible replacement by corresponding embryonic transcripts in both ovulated oocytes and embryos of zebrafish collected at 0, 1, 2, 4, 8, 12, 24 and 48 h post-fertilization (hpf). The mRNAs encoded the nuclear receptors for progesterone (pr), androgen (ar), estrogen (er alpha, er beta 1 and er beta 2), glucocorticoids (gr), mineralocorticoids (mr) and the membrane progestin receptor-alpha and beta (mpr alpha and beta). gr mRNA was the most abundant maternal transcript in oocytes and early embryos followed by er beta 2 and ar mRNAs. They declined during the first 8 hpf, being replaced, thereafter, by the embryonic messengers. er beta 1 and mr transcript levels were low until 8 hpf, but increased steadily during embryonic transcription from 24 to 48 hpf. pr transcripts were detectable only in ovulated oocytes and at 24 and 48 hpf. At these stages, there was a slight increase of er alpha mRNA that initially was very low. mPr alpha and beta mRNAs were expressed in ovulated oocytes and faintly persisted during the first 4 hpf. There was no subsequent embryonic expression of these transcripts. The possible involvement of maternal mRNAs for glucocorticoid and sex hormone receptors in the programming of early zebrafish development is intriguing, since they mainly occur at stages in which gene replication predominates over transcription.

Aldosterone and the conquest of land.

The sequence of the phylogenetic events that preceded the appearance of aldosterone in vertebrates is described, starting from the ancestral conversion of cytochrome P450s from oxygen detoxification to xenobiotic detoxification and synthesis of oxygenated endobiotics with useful functions in intercellular signalling, such as steroid hormones. At the end of the Silurian period [438-408 million yr ago, (Mya)], a complete set of cytochrome P450s for corticoid synthesis was presumably already available, except for mitochondrial cytochrome P450c18 or aldosterone synthase encoded by CYP11B2. This gene arose by duplication of the CYP11B gene in the sarcopterygian or lobe-finned fish/tetrapod line after its divergence from the actinopterygian or ray-finned fish line 420 Mya, but before the beginning of the colonization of land by tetrapods in the late Devonian period, around 370 Mya. The fact that aldosterone is already present in Dipnoi, which occupy an evolutionary transition between water- and air-breathing but are fully aquatic, suggests that the role of this steroid was to potentiate the corticoid response to hypoxia, rather than to prevent dehydration out of the water. In terrestrial amphibians, there is no differentiation between the secretion rates and gluco- and mineralocorticoid effects of aldosterone and corticosterone. In sauropsids, plasma aldosterone concentrations are much lower than in amphibians, but regulation of salt/water balance is dependent upon both aldosterone and corticosterone, though sometimes with opposed actions. In terrestrial mammals, aldosterone acquires a specific mineralocorticoid function, because its interaction with the mineralocorticoid receptor is protected by the coexpression of the enzyme 11beta-hydroxysteroid dehydrogenase type 2, which inactivates both cortisol and corticosterone. There is evidence that aldosterone can be also synthesized extra-adrenally in brain neurons and cardiac myocytes, which lack this protection and where the effects of aldosterone oppose those of glucocorticoids. In conclusion, the phylogenetic history of aldosterone documents the erratic progression of evolutionary changes in the course of the strenuous struggle for environmental resources and survival.

Development of a sensitive and quantitative diagnostic assay for fish nervous necrosis virus based on two-target real-time PCR.

The aim of the present work was to develop two new independent SYBR Green I-based real-time PCR assays for both detection and quantification of betanodavirus, an RNA virus that infects several species of marine teleost fish causing massive mortalities in larvae and juveniles. The assays utilized two pairs of primers targeting highly conserved regions of both the RNA molecules forming the betanodavirus genome: RNA1 encoding the RNA-dependent RNA polymerase (RdRP) and RNA2 encoding the coat protein (CP). The specificity of amplifications was monitored by the melting analysis and agarose gel electrophoresis of the amplified products. The applicability of these assays was confirmed with 21 betanodavirus strains, covering all the four main clades. In addition, a BLAST (NCBI) search with the primer sequences showed no genomic cross-reactivity with other viruses. The new assays were able to quantify concentrations of betanodavirus genes ranging from 10(1) to 10(8) copies per reaction. The intra-assay coefficients of variation (CV) of threshold cycle (Ct) values of the assays were 1.5% and 1.4% for CP and RdRP RNAs, respectively. The inter-assay CVs of Ct values were 2.3% and 2.4% for CP and RdRP RNAs, respectively. Moreover, regression analysis showed a significant correlation (R2>0.97) between genome number, as determined by real-time PCR assays and the corresponding virus titer expressed as TCID50/ml of two different betanodavirus strains propagated in cell culture. The two assays were compared with a previously established one-step RT-PCR assay and with the classical virus isolation test and found to be more sensitive. In conclusion, the developed real-time RT-PCR assays are a reliable, specific and sensitive tool for the quantitative diagnosis of betanodavirus.

Genomic organization of the CYP19b genes in the rainbow trout (Oncorhynchus mykiss Walbaum).

We report the occurrence of two CYP19b genes, namely CYP19b-I and CYP19b-II, encoding forms I and II of cytochrome P450aromB, the prevalently cerebral variant of aromatase in fish, in the nuclear genome of the rainbow trout. The CYP19b-I gene is 7.6 kbp-long, more than double the size of the known fish CYP19a and b genes, owing to the presence of three introns (1, 4 and 5) that enclose repeated sequences and are longer than 1 kbp. Unlike the CYP19a genes, but similarly to the CYP19b gene of the Nile tilapia, it contains 10, and not 9, exons, including an untranslated exon 1 (83 bp), as found also in the 5' non-coding region of mammalian CYP19 genes. The 5'-UTR is composed by exon 1 and the first 41 bp of exon 2 (150 bp), whose coding region covers the first 36 amino acid residues that incorporate the transmembrane domain. The CYP19b-II gene is only 2.5 kbp-long, because it contains only one intron, corresponding to the third intron of CYP19b-I, and lacks also its first two exons. Thus, it encodes for a presumably soluble protein. Apart from this difference, the rest of the coding region is virtually the same as that of the CYP19b-I gene. The 5'-UTR corresponds in part to the 3'-end (132 bp) of the second intron of the CYP19b-I gene, while the remaining portion (208 bp) bears no homology. CYP19b-II could be regarded as a pseudogene of the CYP19b-I gene, though it is unclear whether it is a processed or a duplicated pseudogene. Moreover, since it is transcriptionally active, it may retain a functional role for the overall brain aromatase activity in the rainbow trout.

Myostatin expression during development and chronic stress in zebrafish (Danio rerio).

Myostatin, a member of the transforming growth factor-beta superfamily, is a negative regulator of skeletal muscle mass in mammals. We have studied myostatin expression during embryonic and post-hatching development in zebrafish by semiquantitative RT-PCR. The transcript is present in just-fertilized eggs and declines at 8 h post-fertilization (hpf), suggesting a maternal origin. A secondary rise occurs at 16 hpf, indicating the onset of embryonic transcription at the time of muscle cell differentiation. The level of myostatin mRNA decreases slightly at 24 hpf, when somitogenesis is almost concluded, and rises again at and after hatching, during the period of limited muscle hyperplastic growth that is typical of slow-growing, small fish. In the adult muscle, we found the highest expression of myostatin mRNA and protein, which were detectable by Northern and Western blot analyses respectively. Although only the precursor protein form was revealed in the adult lateral muscle, we demonstrated that zebrafish myostatin is proteolytically processed and secreted in cultured cells, as is its mammalian counterpart. These results suggest that myostatin may play an important regulatory role during myogenesis and muscle growth in fish, as it does in mammals. In chronically stressed fish, grown from 16 days post-fertilization to adulthood in an overcrowded environment, we observed both depression of body growth and a diminished level of myostatin mRNA in the adult muscle, as compared with controls. We propose that chronic stunting in fish brings about a general depression of muscle protein synthesis which does not spare myostatin.

European sea bass (Dicentrarchus labrax L.) cytochrome P450arom: cDNA cloning, expression and genomic organization.

Cytochrome P450arom, a key enzyme in the hormonal steroidogenic pathway, mediates the conversion of androgens to estrogens. This work describes the molecular cloning of the cDNA encoding the European sea bass (Dicentrarchus labrax L.) cytochrome P450arom by means of reverse transcriptase and polymerase chain reaction (RT-PCR) and 5' and 3'-rapid amplification of cDNA ends (RACE) analyses. The cDNA is 1822bp in length and encodes a putative protein of 517 amino acids. Northern blot analysis revealed that the ovary expressed a transcript of about 2.2kb in size. Analysis of the deduced amino acid sequence indicated 62-86% identity with ovarian P450arom of other teleost fish, the highest identity being found with the Japanese flounder, Paralichthys olivaceous. Identity was lower (56-65%) with the P450arom forms first reported in teleost brain. Only 52% identity was observed with the corresponding fragment of the cartilaginous fish, Dasyatis sabina. RT-PCR revealed that the sea bass P450arom mRNA was also expressed, at low levels, in testis and brain. Between the 5' and 3'-untranslated terminal regions (UTR), the sea bass CYP19 gene contains eight introns. All introns conform to the GT/AG rule for RNA splicing and are inserted in exactly the same positions as those found in Oryzias latipes and the human CYP19 gene.

Hormonal steroidogenesis in liver and small intestine of the green frog, Rana esculenta L.

We have investigated the potential of autonomous hormonal steroidogenesis in liver and small intestine of male and female frogs, Rana esculenta, during the recovery phase. After incubation of mitochondrial fractions with [4-14C]cholesterol, female liver and intestine formed pregnenolone at a rate of 0.63 and 2.3 pmol/mg protein/h, respectively, whereas conversion by male organs was only c. 0.03 pmol/mg protein/h. Minced tissues preparations transformed [4-14C]pregnenolone into progesterone and 17alpha-hydroxypregnenolone, the former prevailing in the liver, the latter in the intestine. Moreover, both tissues produced 20alpha-dihydropregnenolone, 20alpha-dihydroprogesterone and dehydroepiandrosterone. From incubates with [4-14C]dehydroepiandrosterone, androstenedione and androst-5-ene-3beta, 17beta-diol were identified, the former being more abundant in the liver, the latter in the intestine. These results indicate that both liver and intestine in frog can be independent sources of hormonally active steroids in both sexes.

Geriatric problem solving with adhesive dentistry, using the Contour Strip and direct composite placement.

The ability to create periodontally sound gingival and non-staining or plaque retaining interproximal margins on spacious carious lesions is a daunting challenge in many situations. The case as presented will illustrate the use of a unique matrix system, The Contour Strip from Vivadent, which will encompass the entire outline margins of the upper right lateral incisor of an elderly patient. The age of the patient has no bearing on the use of the technique, but merely reenforces the value of the system in all other situations mimicking a similar restorative obstacle. The use of this custom shaped matrix band, eliminate the free-hand placement and then the laborious creation and finishing of the restoration. LEARNING OBJECTIVES: After reading this paper the reader will understand the manipulation and use of a new labial matrix band which can totally surround the gingival and proximal margins of any anterior and pre-molar teeth. TECHNIQUE CONSIDERATIONS: There are four rules to follow when using adhesive dentistry: Proper preparation of tooth surfaces, both dentin and enamel through mechanical and/or chemical means. Creation of matrix formed molds. Injecting all resins, from the lowest to the highest viscosity. Polymerizing thoroughly through trans-enamel polymerization thus controlling shrinkage.

Contemporary posterior direct composites using state-of-the-art techniques.

The dental team that wishes to increase the use of posterior composite restorations will benefit greatly by the information and step-by-step techniques found in this article (Fig. 7A and B). Starting with a clean slate and analyzing the chemistries, the physical nature of resin-based composites, and the systems and devices available to use them most efficiently, the author presents a state-of-the-art, evidence-based technique. By cutting out many of the steps that follow traditional methods with traditional materials and instruments for the placement of silver amalgam, sophisticated practitioners will readily understand the reasoning behind this successful, contemporary, direct posterior composite technique (Fig. 8A-J).

Potential for estrogen synthesis and action in human normal and neoplastic thyroid tissues.

To investigate the potential of intracrine or paracrine estrogen synthesis and action in the human thyroid gland and thyroid tumors, the presence of the messenger ribonucleic acids (mRNAs) of both cytochrome P450 aromatase (P450arom) and estrogen receptor (ER) was investigated by RT-PCR with primers designed on the respective coding regions and Southern hybridization analysis with specific probes in neoplastic (n = 42), hyperplastic (n = 7), and adjacent histologically normal thyroid tissues (n = 33) obtained from 43 female and 7 male patients. Most thyroid tissues were positive for both mRNAs, but 2 normal and 3 neoplastic tissues were negative for P450arom mRNA only, 3 normal and 1 hyperplastic tissues were negative for ER mRNA only, and 2 normal tissues were double negative. In some patients, P450arom mRNA was absent in either the neoplastic tissue or the normal one. Single and double negative samples were relatively more frequent in men (n = 4) than in women (n = 7). All negative samples were positive for beta-actin mRNA. RT-PCR amplification and Southern blotting of promoter-specific untranslated 5'-termini revealed that the human thyroid gland and tumors mainly use the ovarian-type promoter, promoter II, for CYP19 expression. Transcripts with either exon I.4 or I.1 were present only in some samples and in very low copy number. When 18 neoplastic samples with their surrounding normal tissues were analyzed immunohistochemically, 57% of those that were positive for P450arom mRNA also had a positive immunoresponse for the corresponding protein. In the case of ER, the percentage was 58%. Immunostaining for P450arom was often particularly intense in neoplastic samples. When 3 adenomata and 1 papillary cancer were incubated with [1,2,6,7-3H]testosterone, 17beta-estradiol could be radiochemically identified with a maximal yield of 10.5 fmol/mg x h. In conclusion, the human thyroid gland appears to have the potential for both estrogen synthesis and intracrine or paracrine estrogen responsiveness, which seem to be greater in women than men and may become enhanced with the process of tumorigenesis.

Single-sitting, fiber-reinforced fixed bridges for the missing lateral or central incisors in adolescent patients.

Many materials, methods, and techniques for the reinforcing of composites to bond a pontic onto abutment teeth have been tried and promoted. In this article, the author examines the use of fiber reinforcement in fixed bridges, describing the various steps performed by the dentist during the procedure.

Occurrence of steroidogenic enzymes in the bovine mammary gland at different functional stages.

After the incubation of minced mammary tissues from non-lactating/non-pregnant (NL/NP), nonlactating/pregnant (NL/P), fully lactating (FL) and late-lactating (LL) cows with [14C]-labelled pregnenolone or progesterone and dehydroepiandrosterone (DHEA), the following metabolites were identified at all stages: 20alpha-dihydropregnenolone, progesterone (from pregnenolone), 5alpha-pregnanedione, 5alpha-pregnan-3beta-ol-20-one, 20alpha- and 20beta-dihydroprogesterone (from progesterone), 5-androstene-3beta,17beta-diol, 5alpha-androstanedione, 5alpha-androstan-3beta-ol-17-one, androstenedione, testosterone and DHEA acyl ester (from DHEA). These products indicate the occurrence of 3beta-hydroxysteroid dehydrogenase/delta5-delta4 isomerase, 17beta-hydroxysteroid oxidoreductase (17beta-HOR), 20alpha- and 20beta-hydroxysteroid dehydrogenases, steroid 5alpha-reductase and acyl transferase activities. Incubation of mammary tissue homogenates with [1,2,6,7-(3)H]androstenedione and testosterone confirmed the presence of a 17beta-HOR acting prevalently in a reductive way but failed to show evidence of any aromatase activity beyond background level. When total RNA from mammary tissues of NL/NP and LL cows was reverse-transcribed and amplified by polymerase chain reaction (PCR) with three sets of primers specific for bovine P450scc, P450c17 and P450arom cDNAs, no fragment of the expected size could be detected on gel. Southern analysis with corresponding digoxigenin-labelled ovarian probes, however, gave a positive signal for P450arom cDNA in five out of eight samples of LL mammary tissue. These data indicate that the bovine mammary gland has very limited steroidogenic capabilities that are essentially compatible with the terminal activation of circulating steroids from steroidogenic endocrines. It is uncertain, however, whether this conclusion applies to anestrous or ovariectomized lactating cows as well.

Kinetic analysis of duodenal and testicular cytochrome P450c17 in the rat.

In the adult rat, the duodenal tissue of both sexes can convert progesterone to 17alpha-hydroxyprogesterone, androstenedione and testosterone. The transition from C21 to C19 steroids is apparently controlled by the same cytochrome P450c17 expressed in the testis, which catalyzes both 17alpha-hydroxylation and C-17,20 bond scission at a single bifunctional active site. The kinetic parameters of this enzyme were measured at the steady state for both reactions using [1,2-3H]progesterone and [1,2-3H]17alpha-hydroxyprogesterone as substrates. In the testis and male and female duodena, the Km values for progesterone 17alpha-hydroxylation were 14.2, 23.8 and 23.2 nM, whereas the Vmax values were 105, 3.5 and 3.1 pmol/mg protein/min, respectively. With respect to C-17,20 lyase activity, the Km values for exogenous 17alpha-hydroxyprogesterone were 525, 675 and 637 nM, whereas the Vmax values were 283, 7.8 and 7.8 pmol/mg protein/min, respectively. However, when the Km values were calculated with respect to intermediate 17alpha-hydroxyprogesterone formed from progesterone, they were similar to the Km values for 17alpha-hydroxylase, being 15, 31.4 and 24.8 nM, whereas the Vmax values were 26.3, 2 and 1.8 pmol/mg protein/min, respectively. The similarity of Km values is due to the fact that the relative androgen formation efficiency (bond scission events/total 17alpha-hydroxylation events ratio) was remarkably constant in both testicular and duodenal incubates, irrespective of progesterone concentration. Efficiency values were 2-fold higher in duodenal tissue (0.54) than in testis (0.25). Estradiol-17beta inhibited 17alpha-hydroxylation but not bond scission on intermediate 17alpha-hydroxyprogesterone, because it did not affect the efficiency value. Rat duodenal P450c17 has the same substrate affinity, a lower specific activity and a higher androgen formation efficiency than testicular P450c17.