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1.
Opt Lett ; 45(14): 3925-3928, 2020 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-32667320

RESUMO

We demonstrate a novel dispersion-scan (d-scan) scheme for single-shot temporal characterization of ultrashort laser pulses. The novelty of this method relies on the use of a highly dispersive crystal featuring antiparallel nonlinear domains with a random distribution and size. This crystal, capable of generating a transverse second-harmonic signal, acts simultaneously as the dispersive element and the nonlinear medium of the d-scan device. The resulting in-line architecture makes the technique very simple and robust, allowing the acquisition of single-shot d-scan traces in real time. The retrieved pulses are in very good agreement with independent frequency-resolved optical grating measurements. We also apply the new single-shot d-scan to a terawatt-class laser equipped with a programmable pulse shaper, obtaining an excellent agreement between the applied and the d-scan retrieved dispersions.

2.
J Fish Biol ; 90(5): 2148-2156, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-28345209

RESUMO

The present study shows that the olfactory potency of intestinal and bile fluids taken from dominant male chameleon cichlids Australoheros facetus is greater than those from subordinate males. Thus, dominant status may be communicated by odorants released in the intestinal fluid and bile acids may contribute towards this.


Assuntos
Líquidos Corporais/química , Ciclídeos/fisiologia , Fezes/química , Percepção Olfatória/fisiologia , Predomínio Social , Animais , Ácidos e Sais Biliares/química , Ácidos e Sais Biliares/metabolismo , Intestinos , Masculino
3.
Mol Hum Reprod ; 19(9): 581-9, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23615721

RESUMO

Sperm undergo maturation acquiring progressive motility and the ability to fertilize oocytes through exposure to the components of the epididymal fluid (EF). Although the establishment of a calcium (Ca(2+)) gradient along the epididymis has been described, its direct effects on epididymal function remain poorly explored. Regucalcin (RGN) is a Ca(2+)-binding protein, regulating the activity of Ca(2+)-channels and Ca(2+)-ATPase, for which a role in male reproductive function has been suggested. This study aimed at comparing the morphology, assessed by histological analysis, and function of epididymis, by analysis of sperm parameters, antioxidant potential and Ca(2+) fluxes, between transgenic rats overexpressing RGN (Tg-RGN) and their wild-type littermates. Tg-RGN animals displayed an altered morphology of epididymis and lower sperm counts and motility. Tissue incubation with (45)Ca(2+) showed also that epididymis of Tg-RGN displayed a diminished rate of Ca(2+)-influx, indicating unbalanced Ca(2+) concentrations in the epididymal lumen. Sperm viability and the frequency of normal sperm, determined by the one-step eosin-nigrosin staining technique and the Diff-Quik staining method, respectively, were higher in Tg-RGN. Moreover, sperm of Tg-RGN rats showed a diminished incidence of tail defects. Western blot analysis demonstrated the presence of RGN in EF as well as its higher expression in the corpus region. The results presented herein demonstrated the importance of maintaining Ca(2+)-levels in the epididymal lumen and suggest a role for RGN in sperm maturation. Overall, a new insight into the molecular mechanisms driving epididymal sperm maturation was obtained, which could be relevant to development of better approaches in male infertility treatment and contraception.


Assuntos
Proteínas de Ligação ao Cálcio/genética , Cálcio/metabolismo , Epididimo/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Maturação do Esperma/genética , Motilidade dos Espermatozoides/genética , Espermatozoides/metabolismo , Animais , Radioisótopos de Cálcio , Proteínas de Ligação ao Cálcio/metabolismo , Hidrolases de Éster Carboxílico , Sobrevivência Celular , Epididimo/ultraestrutura , Regulação da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Transporte de Íons , Masculino , Ratos , Ratos Sprague-Dawley , Ratos Transgênicos , Transdução de Sinais , Contagem de Espermatozoides , Espermatozoides/citologia
4.
Gen Comp Endocrinol ; 179(3): 421-7, 2012 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-23036732

RESUMO

In teleosts the regulation of skeletal homeostasis and turnover by estrogen is poorly understood. For this reason raloxifene, a selective estrogen-receptor modulator (SERM), was administered to sea bream (Sparus auratus) and its effect on plasma calcium balance and transcript expression in dentary (dermal bone) and vertebra (perichondral bone) was studied. The concentration of total calcium or phosphorus in plasma was unchanged by raloxifene treatment for 6days. The activity of alkaline phosphatase (ALP) in dentary bone of raloxifene treated fish was significantly (p<0.05) higher than control fish but it was not changed in vertebral bone. Transcripts for estrogen receptor (ER) α were in very low abundance in the sea bream dentary and vertebra and were unchanged by raloxifene treatment. In contrast, raloxifene caused significant (p<0.05) up-regulation of the duplicate ERß transcripts in the dentary but did not affect specific transcripts for osteoclast (TRAP), osteoblast (ALP, Runx2, osteonectin) or cartilage (IGF1, CILP2, FN1a). In the vertebra ERßb was not changed by raloxifene but ERßa was significantly (p<0.05) down-regulated as was the skeletal specific transcripts, TRAP, ALP, CILP2, FN1a. In summary, ERßs regulate estrogen sensitivity of the skeleton in sea bream, which responds in a non uniform manner. In common with mammals raloxifene appears to have an anti-resorptive role (in sea bream vertebra), but also an osteoblast stimulatory role, inducing ALP activity in the dentary of sea bream. Overall, the results indicate bone specific responsiveness to raloxifene in the sea bream. Further work will be required to understand the basis of bone responsiveness and the role of E(2) and ERs in teleost bone homeostasis.


Assuntos
Moduladores Seletivos de Receptor Estrogênico/farmacologia , Coluna Vertebral/efeitos dos fármacos , Coluna Vertebral/metabolismo , Fosfatase Alcalina/metabolismo , Animais , Receptor alfa de Estrogênio/metabolismo , Receptor beta de Estrogênio/metabolismo , Cloridrato de Raloxifeno/farmacologia , Dourada/metabolismo
5.
J Endocrinol ; 193(3): 473-80, 2007 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-17535884

RESUMO

Gilthead sea bream (Sparus auratus L.) were fed a vitamin D-deficient diet for 22 weeks. Growth rate, whole body mineral pools and calcium balance were determined. Plasma parathyroid hormone-related protein (PTHrP) and calcitriol levels were assessed. Expression of mRNA for pthrp and pth1r was quantified in gills and hypophysis. Fish on vitamin D-deficient diet (D- fish) showed reduced growth and lower calcium turnover (calcium influx, efflux and accumulation rates decreased) and unaltered plasma calcium levels. Plasma calcitriol levels became undetectable, PTHrP levels decreased in the D- fish. In controls, a significant increase in plasma PTHrP level over time was seen, i.e. it increased with body mass. Relationships were found between plasma PTHrP and the whole body pools of calcium, phosphorus and magnesium, indicative of a role for PTHrP in bone development. Expression of pthrp and pth1r mRNA was down-regulated in the hypophysis of D-fish, whereas in gill tissue, pthrp and pth1r mRNA were up-regulated. We conclude that lower pthrp mRNA expression and plasma values in D- fish reflect lower turnover of PTHrP under conditions of hampered growth; up-regulation of pthrp mRNA in gills indicate compensatory paracrine activity of PTHrP during calcitriol deficiency to guarantee well-regulated branchial calcium uptake. This is the first report to document a relation between PTHrP and calcitriol in fish.


Assuntos
Cálcio/metabolismo , Regulação da Expressão Gênica , Proteína Relacionada ao Hormônio Paratireóideo/sangue , Dourada/metabolismo , Deficiência de Vitamina D/metabolismo , Ração Animal , Animais , Calcitriol/sangue , Brânquias/metabolismo , Proteína Relacionada ao Hormônio Paratireóideo/genética , Hipófise/metabolismo , RNA Mensageiro/análise , Receptor Tipo 1 de Hormônio Paratireóideo/genética , Receptor Tipo 1 de Hormônio Paratireóideo/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Dourada/crescimento & desenvolvimento
6.
Biochim Biophys Acta ; 1216(1): 137-9, 1993 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-8218405

RESUMO

Here, we report the molecular analysis of two independent 5S rRNA clusters found in the intergenic region of two ubiquitin genomic clones isolated from Tetrahymena pyriformis. Each cluster contains two 120-bp-long coding regions organized in tandem with 142/145-bp-long spacers.


Assuntos
Genes de Protozoários , RNA Ribossômico 5S/química , Tetrahymena pyriformis/genética , Ubiquitinas/genética , Animais , Sequência de Bases , Dados de Sequência Molecular , Alinhamento de Sequência
7.
Endocrinology ; 146(1): 71-6, 2005 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-15459121

RESUMO

The mode of action of PTHrP in the regulation of sea bream (Sparus auratus) interrenal cortisol production was studied in vitro using a dynamic superfusion system. Piscine (1-34)PTHrP (10(-6)-10(-11) M) stimulated cortisol production in a dose-dependent manner. The ED50 of (1-34)PTHrP was 2.8 times higher than that of (1-39)ACTH, and maximum increase in cortisol production in response to 10(-8) M of (1-34)PTHrP was approximately 7-fold lower than for 10(-8) M of (1-39)ACTH. In contrast to (1-34)PTHrP, piscine (10-20)PTHrP, (79-93)PTHrP, and (100-125)PTHrP (10(-9)-10(-7) M) did not stimulate cortisol production. The effect of piscine (1-34)PTHrP on cortisol production was abolished by N-terminal peptides in which the first amino acid (Ser) was absent and by simultaneous addition of inhibitors of the adenylyl cyclase-protein kinase A and phospholipase C-protein kinase C intracellular pathways but not by each separately. The PTHrP-induced signal transduction was further investigated by measurements of cAMP production and [H3]myo-inositol incorporation in an interrenal cell suspension. Piscine (1-34)PTHrP increased cAMP and total inositol phosphate accumulation, which is indicative that the mechanism of action of PTHrP in interrenal tissue involves the activation of both the adenylyl cyclase-cAMP and phospholipase C-inositol phosphate signaling pathways. These results, together with the expression of mRNA for PTHrP and for PTH receptor (PTHR) type 1 and PTHR type 3 receptors in sea bream interrenal tissue, suggest a specific paracrine or autocrine steroidogenic action of PTHrP mediated by the PTHRs.


Assuntos
Hidrocortisona/metabolismo , Rim/efeitos dos fármacos , Rim/metabolismo , Proteína Relacionada ao Hormônio Paratireóideo/química , Proteína Relacionada ao Hormônio Paratireóideo/farmacologia , Dourada/metabolismo , Adenilil Ciclases/metabolismo , Hormônio Adrenocorticotrópico/farmacologia , Sequência de Aminoácidos , Animais , AMP Cíclico/metabolismo , Inositol/metabolismo , Rim/citologia , Proteína Relacionada ao Hormônio Paratireóideo/genética , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/farmacologia , Transdução de Sinais/fisiologia , Fosfolipases Tipo C/metabolismo
8.
Regul Pept ; 132(1-3): 33-40, 2005 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-16181689

RESUMO

The scales of bony fish represent a significant reservoir of calcium but little is known about their contribution, as well as of bone, to calcium balance and how calcium deposition and mobilization are regulated in calcified tissues. In the present study we report the action of parathyroid hormone-related protein (PTHrP) on calcium mobilization from sea bream (Sparus auratus) scales in an in vitro bioassay. Ligand binding studies of piscine 125I-(1-35(tyr))PTHrP to the membrane fraction of isolated sea bream scales revealed the existence of a single PTH receptor (PTHR) type. RT-PCR of fish scale cDNA using specific primers for two receptor types found in teleosts, PTH1R, and PTH3R, showed expression only of PTH1R. The signalling mechanisms mediating binding of the N-terminal amino acid region of PTHrP were investigated. A synthetic peptide (10(-8) M) based on the N-terminal 1-34 amino acid residues of Fugu rubripes PTHrP strongly stimulated cAMP synthesis and [3H]myo-inositol incorporation in sea bream scales. However, peptides (10(-8) M) with N-terminal deletions, such as (2-34), (3-34) and (7-34)PTHrP, were defective in stimulating cAMP production but stimulated [3H]myo-inositol incorporation. (1-34)PTHrP induced significant osteoclastic activity in scale tissue as indicated by its stimulation of tartrate-resistant acid phosphatase. In contrast, (7-34)PTHrP failed to stimulate the activity of this enzyme. This activity could also be abolished by the adenylyl cyclase inhibitor SQ-22536, but not by the phospholipase C inhibitor U-73122. The results of the study indicate that one mechanism through which N-terminal (1-34)PTHrP stimulates osteoclastic activity of sea bream scales, is through PTH1R and via the cAMP/AC intracellular signalling pathway. It appears, therefore, that fish scales can act as calcium stores and that (1-34)PTHrP regulates calcium mobilization from them; it remains to be established if this mechanism contributes to calcium homeostasis in vivo.


Assuntos
Cálcio/metabolismo , Proteína Relacionada ao Hormônio Paratireóideo/metabolismo , Receptor Tipo 1 de Hormônio Paratireóideo/metabolismo , Dourada/metabolismo , Animais , AMP Cíclico/biossíntese , Osteoclastos/metabolismo , Ligação Proteica/fisiologia , Receptores de Hormônios Paratireóideos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais
9.
Gene ; 182(1-2): 183-8, 1996 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-8982086

RESUMO

A 5-unit polyubiquitin gene, TTU3, was isolated from a T. thermophila genomic library and sequenced. This gene presents an extra triplet coding for Phe, a AGAGA motif and a putative HSE element in its 5'-non-coding region. The ubiquitin gene expression in this ciliate was investigated by Northern blot hybridization in conjugating cells or cells under stress conditions. Exponentially growing cells express two ubiquitin mRNAs of 0.75 and 1.8 kb and a new species of 1.4 kb is induced under hyperthermic stress. During sexual reproduction of the cells (conjugation) the 1.8-kb mRNA is still transcribed whereas the steady-state population of the 0.75 mRNA transcripts is strongly diminished. Southern blot analysis suggests that ubiquitin in T. thermophila constitutes a large family of about ten members.


Assuntos
Regulação da Expressão Gênica/genética , Tetrahymena thermophila/química , Ubiquitinas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Southern Blotting , Clonagem Molecular , Dosagem de Genes , Biblioteca Gênica , Mitose/genética , Dados de Sequência Molecular , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Análise de Sequência , Temperatura
10.
FEBS Lett ; 383(3): 277-83, 1996 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-8925913

RESUMO

We report here the cloning and the characterization of the T. pyriformis CCT eta gene (TpCCT eta) and also a partial sequence of the corresponding T. thermophila gene (TtCCT eta). The TpCCt eta gene encodes a protein sharing a 60.3% identity with the mouse CCT eta. We have studied the expression of these genes in Tetrahymena exponentially growing cells, cells regenerating their cilia for different periods and during different stages of the cell sexual reproduction. These genes have similar patterns of expression to those of the previously identified TpCCt gamma gene. Indeed, the Tetrahymena CCT eta and CCT gamma genes are up-regulated at 60-120 min of cilia recovery, and in conjugation when vegetative growth was resumed and cell division took place. Our results seem to indicate that both CCT subunits play an important role in the biogenesis of the newly synthesized cilia of Tetrahymena and during its cell division.


Assuntos
Chaperoninas/biossíntese , Regulação da Expressão Gênica , Genes de Protozoários , Proteínas de Protozoários , Tetrahymena pyriformis/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Divisão Celular , Chaperonina com TCP-1 , Clonagem Molecular , Primers do DNA , Substâncias Macromoleculares , Camundongos , Dados de Sequência Molecular , Proteínas Recombinantes/biossíntese , Reprodução , Homologia de Sequência de Aminoácidos , Tetrahymena pyriformis/genética , Tetrahymena pyriformis/metabolismo , Tetrahymena thermophila/genética , Tetrahymena thermophila/fisiologia
11.
J Endocrinol ; 173(2): 377-85, 2002 May.
Artigo em Inglês | MEDLINE | ID: mdl-12010645

RESUMO

In all teleost fishes vitellogenesis is triggered and maintained by oestradiol-17beta (E2) and is accompanied by an increase of blood plasma calcium and phosphate. The action of this hormone on calcium metabolism was investigated by treating fast-growing immature juvenile sea bream (Sparus aurata) with coconut butter implants alone (control) or implants containing 10 microg/g E2. Treatment with E2 induced the production of circulating vitellogenin, a 2.5-fold increase in plasma ionic Ca2+ and a 10-fold increase in plasma total calcium, largely bound to protein. In contrast to freshwater species, which obtain most of their calcium from the environment directly through the gills, the intestinal component of calcium uptake of the salt water-living sea bream represented up to 60-70% of the total uptake. The whole body calcium uptake, expressed as the sum of calcium obtained via intestinal and extra-intestinal (likely branchial) routes increased significantly in response to E2. Combined influx and unchanged efflux rates resulted in a significant 31% increase in net calcium uptake. There was no evidence for an effect of E2 on the calcium and phosphate content of the scales or the tartrate-resistant acid phosphatase activity (an index for bone/scale osteoclast activity). While most freshwater fish appear to rely on internal stores of calcium, i.e. bone and/or scales to increase calcium availability, the marine sea bream accommodates calcium-transporting mechanisms to obtain calcium from the environment and preserve internal stores. These observations suggest that a fundamental difference may exist in the E2-dependent calcium regulation between freshwater and marine teleosts.


Assuntos
Cálcio/metabolismo , Estradiol/farmacologia , Dourada/metabolismo , Maturidade Sexual/efeitos dos fármacos , Vitelogênese/efeitos dos fármacos , Fosfatase Ácida/metabolismo , Animais , Transporte Biológico/efeitos dos fármacos , Cálcio/análise , Cálcio/sangue , Feminino , Absorção Intestinal/efeitos dos fármacos , Isoenzimas/metabolismo , Fosfatos/análise , Pele/química , Fosfatase Ácida Resistente a Tartarato
12.
DNA Seq ; 2(3): 173-80, 1991.
Artigo em Inglês | MEDLINE | ID: mdl-1667985

RESUMO

The presence of ubiquitin in ciliates was first demonstrated in Tetrahymena pyriformis. One clone--pTU2--presents two incomplete open reading frames and the putative polyubiquitin genes have been shown to be highly similar to those of other organisms. To further analyze the organization of this multigene family, several fragments of macronuclear DNA were cloned. We report here the isolation and characterization of one genomic clone (pTU20) that encodes a polyubiquitin gene (TU20) with five tandem repeats and presenting only one extra triplet CAA (Gln) upstream from the TGA. The promoter region of TU20 also presents a consensus heat shock element. The specific detection of RNA species with a synthetic oligonucleotide probe reveals that it corresponds to the 1.8 kb mRNA species whose expression is increased by temperature stress.


Assuntos
Polímeros , Tetrahymena pyriformis/genética , Ubiquitinas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Southern Blotting , Núcleo Celular/metabolismo , Clonagem Molecular , Códon , DNA de Protozoário , Éxons , Regulação da Expressão Gênica , Humanos , Dados de Sequência Molecular , Poliubiquitina , Mapeamento por Restrição , Alinhamento de Sequência , Transcrição Gênica
13.
J Air Waste Manag Assoc ; 49(1): 82-7, 1999 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-9951417

RESUMO

A series of continuous ambient tropospheric ozone measurements were taken in Mendoza, Argentina, for a period of one year starting in November 1995. The data obtained were analyzed in terms of diurnal and annual variation. Indications were found of the strong impact of the mountain-valley circulation system, which ventilates and considerably cleans the air in Mendoza. The data are discussed in comparison with air pollution in the German city of Leipzig. In Mendoza, the high concentration of precursors and the strong solar radiation contribute to high levels of ozone. In fact, monitoring reveals considerably lower concentrations than in Leipzig, owing to the diluting effect of local meteorology. The low-level jet is mainly active during the summer. It lowers the peak mid-day ozone concentration and produces a temporary concentration increase at night. The Environmental Protection Agency standard of a maximum one-hour mean concentration of 0.250 mg ozone/m3 (125 ppb) is never reached, and the World Health Organization standard of 0.113 mg ozone/m3 (56.5 ppb) is only rarely exceeded during winter.


Assuntos
Oxidantes Fotoquímicos/análise , Ozônio/análise , Argentina
14.
Gen Comp Endocrinol ; 155(1): 94-100, 2008 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-17434515

RESUMO

Brain or blood plasma melatonin was analysed as a measure for pineal melatonin production in sea bream. Access to calcium was limited by diluting the seawater to 2.5 per thousand and removing calcium from the diet or by prolonged feeding of vitamin D-deficient diet. Interactions/relations between melatonin and calcium balance and the hypercalcemic endocrines PTHrP and calcitriol were assessed. Restricting calcium availability in both water and diet had no effect on plasma melatonin, but when calcium was low in the water or absent from food, increased and decreased plasma melatonin was observed, respectively. Fish on a vitamin D-deficient diet (D- fish) showed decreased plasma calcitriol levels and remained normocalcemic. Decreased brain melatonin was found at all sampling times (10-22 weeks) in the D- fish compared to the controls. A positive correlation between plasma Ca2+ and plasma melatonin was found (R(2)=0.19; N=41; P <0.01) and brain melatonin was negatively correlated with plasma PTHrP (R(2)=0.78; N=4; P <0.05). The positive correlation between plasma levels of melatonin and Ca2+ provides evidence that melatonin synthesis is influenced by plasma Ca2+. The decreased melatonin production in the D- fish points to direct or indirect involvement of calcitriol in melatonin synthesis by the pineal organ in teleosts. The hypercalcemic factors PTHrP and calcitriol appeared to be negatively correlated with melatonin and this substantiates an involvement of melatonin in modulating the endocrine response to cope with hypocalcemia. It further points to the importance of Ca2+ in melatonin physiology.


Assuntos
Cálcio/fisiologia , Hipocalcemia/metabolismo , Melatonina/biossíntese , Dourada/metabolismo , Animais , Encéfalo/efeitos dos fármacos , Química Encefálica , Cálcio/sangue , Cálcio/farmacologia , Dieta , Melatonina/análise , Melatonina/sangue , Dourada/sangue
15.
Cell Tissue Res ; 323(2): 333-41, 2006 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-16189716

RESUMO

Whole animal studies have indicated that Ca(2+) uptake by the gastrointestinal tract is regulated by the action of parathyroid hormone-related peptide (PTHrP) in teleost fish. We have characterised PTH receptors (PTHR) in piscine enterocytes and established, by using amino-terminal PTHrP peptides, the amino acid residues important for receptor activation and for stabilising the ligand/receptor complex. Ligand binding of (125)I-(1-35(tyr)) PTHrP to the membrane fraction of isolated sea bream enterocytes revealed the existence of a single saturable high-affinity receptor (K (D)=2.59 nM; B (max)=71 fmol/mg protein). Reverse transcription/polymerase chain reaction with specific primers for sea bream PTH1R and PTH3R confirmed the mRNA expression of only the later receptor. Fugu (1-34)PTHrP increased cAMP levels in enterocytes but had no effect on total inositol phosphate accumulation. The amino-terminal peptides (2-34)PTHrP, (3-34)PTHrP and (7-34)PTHrP bound efficiently to the receptor but were severely defective in stimulating cAMP in enterocyte cells indicating that the first six residues of piscine (1-34)PTHrP, although not important for receptor binding, are essential for activation of the adenylate cyclase/phosphokinase A (AC-PKA)-receptor-coupled intracellular signalling pathway. Therefore, PTHrP in teleosts acts on the gastrointestinal tract through PTH3R and the AC-PKA intracellular signalling pathway and might regulate Ca(2+) uptake at this site. Ligand-receptor binding and activity throughout the vertebrates appears to be allocated to the same amino acid residues of the amino-terminal domain of the PTHrP molecule.


Assuntos
Sinalização do Cálcio , Enterócitos/metabolismo , Receptor Tipo 1 de Hormônio Paratireóideo/metabolismo , Dourada/fisiologia , Sequência de Aminoácidos , Animais , Sítios de Ligação , AMP Cíclico/metabolismo , Enterócitos/citologia , Expressão Gênica , Ligantes , Dados de Sequência Molecular , Proteína Relacionada ao Hormônio Paratireóideo/metabolismo , RNA Mensageiro/metabolismo , Receptor Tipo 1 de Hormônio Paratireóideo/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Takifugu/metabolismo , Peixe-Zebra/genética
16.
Gen Comp Endocrinol ; 143(1): 57-65, 2005 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-15993105

RESUMO

The production and purification of gilthead sea bream recombinant parathyroid hormone related protein [sbPTHrP(1-125)] using an Escherichia coli system and one step purification process with continuous elution gel electrophoresis is reported. The cDNA encoding sbPTHrP(1-125) was cloned into a prokaryotic expression vector pET-11a. The recombinant plasmid was used to transfect E. coli BL21(DE3) pLysS and sbPTHrP(1-125) synthesis was induced by addition of 1mM isopropyl-beta-d-thiogalactopyranoside. The rapid one step isolation method gave pure sbPTHrP(1-125) as judged by SDS-PAGE and yielded up to 40mg/L of culture medium (3.3mg protein/g of bacteria). The bioactivity of recombinant sbPTHrP(1-125) assessed using an in vitro scale bioassay was found to be equipotent to PTHrP(1-34) in stimulating cAMP accumulation. Assessment of the immunological reactivity of the isolated protein by Western blot revealed it cross-reacts with antisera specific for the N-terminal and C-terminal region of PTHrP. In a radioimmunoassay specific for piscine N-terminal (1-34aa) PTHrP, the recombinant sbPTHrP(1-125) was equipotent with PTHrP(1-34) in displacing labelled (125)I-PTHrP(1-36) PTHrP from the antisera. The availability of recombinant sbPTHrP will allow the development of region specific assays and studies aimed at defining post-secretory processing of this protein and its biological activity in fish.


Assuntos
AMP Cíclico/metabolismo , Proteína Relacionada ao Hormônio Paratireóideo/biossíntese , Proteína Relacionada ao Hormônio Paratireóideo/genética , Dourada , Sequência de Aminoácidos , Animais , Western Blotting , DNA Complementar/metabolismo , Eletroforese em Gel de Poliacrilamida , Vetores Genéticos , Dados de Sequência Molecular , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Alinhamento de Sequência
17.
Yeast ; 18(9): 781-7, 2001 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-11427960

RESUMO

Six open reading frames (ORFs) located on chromosome VII of Saccharomyces cerevisiae (YGR205w, YGR210c, YGR211w, YGR241c, YGR243w and YGR244c) were disrupted in two different genetic backgrounds using short-flanking homology (SFH) gene replacement. Sporulation and tetrad analysis showed that YGR211w, recently identified as the yeast ZPR1 gene, is an essential gene. The other five genes are non-essential, and no phenotypes could be associated to their inactivation. Two of these genes have recently been further characterized: YGR241c (YAP1802) encodes a yeast adaptor protein and YGR244c (LSC2) encodes the beta-subunit of the succinyl-CoA ligase. For each ORF, a replacement cassette with long flanking regions homologous to the target locus was cloned in pUG7, and the cognate wild-type gene was cloned in pRS416.


Assuntos
Cromossomos Fúngicos/genética , Genes Essenciais/genética , Genes Fúngicos/genética , Fases de Leitura Aberta/genética , Saccharomyces cerevisiae/genética , Deleção de Genes , Mutagênese Insercional , Fenótipo , Plasmídeos/genética , Reação em Cadeia da Polimerase , Saccharomyces cerevisiae/crescimento & desenvolvimento , Transformação Genética
18.
Yeast ; 13(3): 275-80, 1997 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-9090057

RESUMO

A 9.9 kb DNA fragment from the right arm of chromosome VII of Saccharomyces cerevisiae has been sequenced and analysed. The sequence contains four open reading frames (ORFs) longer than 100 amino acids. One gene, PFK1, has already been cloned and sequenced and the other one is the probable yeast gene coding for the beta-subunit of the succinyl-CoA synthetase. The two remaining ORFs share homology with the deduced amino acid sequence (and their physical arrangement is similar to that) of the YHR161c and YHR162w ORFs from chromosome VIII.


Assuntos
Mapeamento Cromossômico , Cromossomos Fúngicos/genética , DNA Fúngico/análise , Proteínas/genética , Saccharomyces cerevisiae/genética , Succinato-CoA Ligases/genética , Sequência de Aminoácidos , Genoma Fúngico , Peptídeos e Proteínas de Sinalização Intercelular , Dados de Sequência Molecular , Fases de Leitura Aberta , Plasmídeos , Mapeamento por Restrição , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos
19.
Chirality ; 12(5-6): 408-10, 2000 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-10824162

RESUMO

The enantioselective ruthenium promoted hydrogenation of beta-keto ester, derived from (S)- or (R)-proline and (S)-pipecolic acid, provided a new efficient route to hydroxylated pyrrolizidine or indolizidine ring systems in diastereomeric excesses up to 99%. A practical synthesis of (+)-alpha-conhydrine is also reported.


Assuntos
Aminoácidos/química , Química/métodos , Hidrogênio/metabolismo , Indolizinas/síntese química , Alcaloides de Pirrolizidina/síntese química , Rutênio/química , Alcaloides/química , Modelos Químicos
20.
Mol Gen Genet ; 230(1-2): 186-92, 1991 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-1660564

RESUMO

The genome of Tetrahymena pyriformis has been shown to contain a ubiquitin multigene family consisting of several polyubiquitin genes and at least one ubiquitin fusion gene. We report here the isolation and characterization of one genomic clone (pTU11), that encodes a ubiquitin extension protein. A comparison of the predicted amino acid sequence of the ubiquitin extension protein gene of T. pyriformis with those from other organisms indicated a high degree of homology. However, the Tetrahymena ubiquitin extension protein contains 53 and not 52 amino acids. This feature is different from all ubiquitin 52-amino-acid extension protein genes thus far sequenced. Furthermore, we found an array of four cysteine residues similar to those found in nucleic acid binding proteins. Also, the C-terminal sequence possesses a conserved motif which may represent a nuclear translocation signal. The ubiquitin 53-amino-acid extension protein gene encodes the smallest class of ubiquitin mRNAs in T. pyriformis.


Assuntos
Clonagem Molecular , DNA de Protozoário/genética , Precursores de Proteínas/genética , Proteínas de Protozoários/genética , Tetrahymena pyriformis/genética , Ubiquitinas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Southern Blotting , Dados de Sequência Molecular , Família Multigênica , RNA de Protozoário/genética , Mapeamento por Restrição , Homologia de Sequência do Ácido Nucleico
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