Unique patterns of androgen regulation of the expression of two genes in murine kidney

The kidneys of androgen stimulated mice exhibit a hypertrophic response but no hyperplasia or concomitant DNA replication. Androgens increase the expression of several genes in mouse kidney. The response of the beta-glucuronidase gene to testosterone in this tissue is characterized by a 1-2 day lag and relatively slow induction kinetics. The gene coding for kidney androgen-regulated protein (KAP) exhibits quite a different response to the hormone when compared on the basis of initial response to a given dose, dose required to produce maximal response, and apparent sensitivity to low levels of androgen-receptor complexes in renal nuclei. The analysis of the accumulation of the mRNAs produced by these two genes suggests that gene-specific differential sensitivity to androgen receptor complexes governs the development of the cellular male phenotype in this tissue.     

Myostatin precursor is present in several tissues in teleost fish: a comparative immunolocalization study

In this study, the distribution of myostatin was investigated during larval and postlarval developmental stages of Sparus aurata(sea bream), Solea solea(sole) and Brachydanio rerio(zebrafish) by immunohistochemistry using antisera raised against a synthetic peptide located within the precursor region of sea bream myostatin. All the three species examined showed the strongest immunoreactivity in red skeletal muscle in juveniles and adults. During larval development of sea bream, strong staining was detected in skin and brain. Immunoreactivity was also found in muscle, pharynx, gills, pancreas and liver. From metamorphosis, immunoreactivity was identifiable in the oesophagus, in the apical portion of the stomach epithelium, in the intestinal epithelium and in renal tubules. In larval zebrafish at hatching, the most intense myostatin immunoreactivity was evident in the skin epithelium. Immunoreactivity was also found in the retina and brain. In the adult, an intense immunostaining occurred in the gastrointestinal tract as well as in the ovary. In sole larvae, immunoreactivity was found in liver and intestine. Our results support the hypothesis suggested earlier that myostatins in fish have retained a different partition (compared with mammals) of the expression patterns and functions which characterized the ancestral gene before the duplication event that gave rise to growth differentiation factor-11 (GDF-11) and GDF-8 (myostatin).     

Enhanced levels of free and protein-bound threonine in transgenic alfalfa (Medicago sativa L.) expressing a bacterial feedback-insensitive aspartate kinase gene

Threonine, lysine, methionine, and tryptophan are essential amino acids for humans and monogastric animals. Many of the commonly used diet formulations, particularly for pigs and poultry, contain limiting amounts of these amino acids. One approach for raising the level of essential amino acids is based on altering the regulation of their biosynthetic pathways in transgenic plants. Here we describe the first production of a transgenic forage plant, alfalfa (Medicago sativa L.) with modified regulation of the aspartate-family amino acid biosynthetic pathway. This was achieved by over-expressing the Escherichia coli feedback-insensitive aspartate kinase (AK) in transgenic plants. These plants showed enhanced levels of both free and protein-bound threonine. In many transgenic plants the rise in free threonine was accompanied by a significant reduction both in aspartate and in glutamate. Our data suggest that in alfalfa, AK might not be the only limiting factor for threonine biosynthesis, and that the free threonine pool in this plant limits its incorporation into plant proteins.     

Proteome characterization of cassava (<Emphasis Type="Italic">Manihot esculenta</Emphasis> Crantz) somatic embryos,plantlets and tuberous roots

Background  

Proteomics is increasingly becoming an important tool for the study of many different aspects of plant functions, such as investigating the molecular processes underlying in plant physiology, development, differentiation and their interaction with the environments. To investigate the cassava (Manihot esculenta Crantz) proteome, we extracted proteins from somatic embryos, plantlets and tuberous roots of cultivar SC8 and separated them by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).     

Expression, purification, renaturation and activation of fish myostatin expressed in Escherichia coli: facilitation of refolding and activity inhibition by myostatin prodomain

Myostatin (growth and differentiation factor-8) is a member of the transforming growth factor-beta superfamily, is expressed mainly in skeletal muscle and acts as a negative growth regulator. Mature myostatin (C-terminal) is a homodimer that is cleaved post-translationally from the precursor myostatin, also yielding the N-terminal prodomain. We expressed in Escherichia coli three forms of fish myostatin: precursor, prodomain and mature. The three forms were over-expressed as inclusion bodies. Highly purified inclusion bodies were solubilized in a solution containing guanidine hydrochloride and the reducing agent DTT. Refolding (indicated by a dimer formation) of precursor myostatin, mature myostatin or a mixture of prodomain and mature myostatin was compared under identical refolding conditions, performed in a solution containing sodium chloride, arginine, a low concentration of guanidine hydrochloride and reduced and oxidized glutathione at 4 degrees C for 14 days. While precursor myostatin formed a reversible disulfide bond with no apparent precipitation, mature myostatin precipitated in the same refolding solution, unless CHAPS was included, and only a small proportion formed a disulfide bond. The trans presence of the prodomain in the refolding solution prevented precipitation of mature myostatin but did not promote formation of a dimer. Proteolytic cleavage of purified, refolded precursor myostatin with furin yielded a monomeric prodomain and a disulfide-linked, homodimeric mature myostatin, which remained as a latent complex. Activation of the latent complex was achieved by acidic or thermal treatments. These results demonstrate that the cis presence of the prodomain is essential for the proper refolding of fish myostatin and that the cleaved mature dimer exists as a latent form.     

Cloning and expression of insulin-like growth factors I and II in the shi drum (Umbrina cirrosa)

Insulin-like growth factors (IGFs) are evolutionarily ancient polypeptides, with potent metabolic actions, affecting cell development and growth. The IGF system consists of two ligands: IGF-I and IGF-II, several binding proteins and high-affinity transmembrane receptors. To understand growth regulation in the teleost shi drum, Umbrina cirrosa, we cloned IGF-I and IGF-II cDNAs, studied their expression and determined the cellular localization of IGF-II peptide by immunohistochemistry. A fragment of 1110 nucleotides, coding for U. cirrosa IGF-I (ucIGF-I), was cloned from liver by PCR. It includes an open reading frame of 561 nucleotides, encoding a 187 amino acid preproIGF-I. A fragment of 938 nucleotides that includes part of the coding sequence and the 3' UTR of IGF-II (ucIGF-II) was cloned as well. Sequence analysis of ucIGF-I and ucIGF-II showed a high degree of homology with known fish IGF-I and IGF-II. Real-Time PCR showed a higher expression of IGF-I and IGF-II in liver, compared to all other tissues analysed. IGF-II peptide was detected in larval liver, intestine, gills and heart musculature. After metamorphosis, reactivity was particularly evident in the kidney and in red fibres of skeletal muscle. These results add novel information on the nucleotide sequence of IGF-I and IGF-II in a marine teleost, the shi drum, that was recently introduced to the mariculture industry in southern Europe and emphasizes the conservation in the 5' UTR of IGF-I among teleosts. Furthermore, this study suggests, on the basis of a combined approach of RT-PCR, Real-Time PCR and immunohistochemistry that IGF-I and IGF-II are involved in the regulation of somatic growth in the shi drum.     

An osmotic model of the growing pollen tube

Pollen tube growth is central to the sexual reproduction of plants and is a longstanding model for cellular tip growth. For rapid tip growth, cell wall deposition and hardening must balance the rate of osmotic water uptake, and this involves the control of turgor pressure. Pressure contributes directly to both the driving force for water entry and tip expansion causing thinning of wall material. Understanding tip growth requires an analysis of the coordination of these processes and their regulation. Here we develop a quantitative physiological model which includes water entry by osmosis, the incorporation of cell wall material and the spreading of that material as a film at the tip. Parameters of the model have been determined from the literature and from measurements, by light, confocal and electron microscopy, together with results from experiments made on dye entry and plasmolysis in Lilium longiflorum. The model yields values of variables such as osmotic and turgor pressure, growth rates and wall thickness. The model and its predictive capacity were tested by comparing programmed simulations with experimental observations following perturbations of the growth medium. The model explains the role of turgor pressure and its observed constancy during oscillations; the stability of wall thickness under different conditions, without which the cell would burst; and some surprising properties such as the need for restricting osmotic permeability to a constant area near the tip, which was experimentally confirmed. To achieve both constancy of pressure and wall thickness under the range of conditions observed in steady-state growth the model reveals the need for a sensor that detects the driving potential for water entry and controls the deposition rate of wall material at the tip.     

Cellular localization of insulin-like growth factor-II protein in the sea bass (Dicentrarchus labrax) from hatching to adult

The cellular localization of IGF-II protein was investigated during larval and postlarval developmental stages of sea bass (Dicentrarchus labrax) by immunohistochemistry using antisera raised against Sparus aurata IGF-II. At hatching, IGF-II immunoreactivity was already present in the skin, developing intestine and skeletal muscle. During larval life IGF-II protein was also observed in heart musculature, in kidney and gill epithelia as well as in liver. In fry skeletal muscle a moderate IGF-II immunostaining was detected in red fibres, whereas white muscle fibres exhibited a faint immunoreactivity. In adults, a marked IGF-II immunostaining was observed in red muscle fibres. A moderate immunoreactivity was also present in white fibres as well as in heart striated myocardial fibres. These results are in agreement with previous findings on the spatial localization of IGF-II and IGF type 1 receptor in S. aurata and Umbrina cirrosa, confirming the role of IGF system during development and growth of fish.