Accumulation and effects of sulfadimethoxine in Salix fragilis L. plants: a preliminary study to phytoremediation purposes

The application of manure to fertilize arable lands is one of the major means through which veterinary sulfonamides (SAs) enter the environment. Little is known about the capacity of woody plants to phytoremediate this class of antibiotics. To this purpose we performed preliminary studies to evaluate Salix fragilis L. response to sulfadimethoxine (SDM) by investigating both its ability to absorb and tolerate doses of SDM found in fresh faeces of treated calves. Forty cuttings were exposed to either 0, 0.5, 1, or 2 mM of SDM for one month. Decreases in photosynthetic electron transport rate and net CO2 assimilation after 25 days for the higher SDM concentrations were noticed. Moreover, alterations in root morphology of treated plants were observed and further investigated through electron microscopy. However, collected data revealed high root accumulation potential. These preliminary results are promising as they demonstrate that Salix fragilis L. can both absorb and tolerate high concentrations of SAs.     

Generation of skeletal muscle cells from embryonic and induced pluripotent stem cells as an in vitro model and for therapy of muscular dystrophies

Muscular dystrophies (MDs) are a heterogeneous group of inherited disorders characterized by progressive muscle wasting and weakness likely associated with exhaustion of muscle regeneration potential. At present, no cures or efficacious treatments are available for these diseases, but cell transplantation could be a potential therapeutic strategy. Transplantation of myoblasts using satellite cells or other myogenic cell populations has been attempted to promote muscle regeneration, based on the hypothesis that the donor cells repopulate the muscle and contribute to its regeneration. Embryonic stem cells (ESCs) and more recently induced pluripotent stem cells (iPSCs) could generate an unlimited source of differentiated cell types, including myogenic cells. Here we review the literature regarding the generation of myogenic cells considering the main techniques employed to date to elicit efficient differentiation of human and murine ESCs or iPSCs into skeletal muscle. We also critically analyse the possibility of using these cellular populations as an alternative source of myogenic cells for cell therapy of MDs.     

Radical sites in Mycobacterium tuberculosis KatG identified using electron paramagnetic resonance spectroscopy, the three-dimensional crystal structure, and electron transfer couplings

Catalase-peroxidase (KatG) from Mycobacterium tuberculosis, a Class I peroxidase, exhibits high catalase activity and peroxidase activity with various substrates and is responsible for activation of the commonly used antitubercular drug, isoniazid (INH). KatG readily forms amino acid-based radicals during turnover with alkyl peroxides, and this work focuses on extending the identification and characterization of radicals forming on the millisecond to second time scale. Rapid freeze-quench electron paramagnetic resonance spectroscopy (RFQ-EPR) reveals a change in the structure of the initially formed radical in the presence of INH. Heme pocket binding of the drug and knowledge that KatG[Y229F] lacks this signal provides evidence for radical formation on residue Tyr(229). High field RFQ-EPR spectroscopy confirmed a tryptophanyl radical signal, and new analyses of X-band RFQ-EPR spectra also established its presence. High field EPR spectroscopy also confirmed that the majority radical species is a tyrosyl radical. Site-directed mutagenesis, along with simulations of EPR spectra based on x-ray structural data for particular tyrosine and tryptophan residues, enabled assignments based on predicted hyperfine coupling parameters. KatG mutants W107F, Y229F, and the double mutant W107F/Y229F showed alteration in type and yield of radical species. Results are consistent with formation of a tyrosyl radical reasonably assigned to residue Tyr(229) within the first few milliseconds of turnover. This is followed by a mixture of tyrosyl and tryptophanyl radical species and finally to only a tyrosyl radical on residue Tyr(353), which lies more distant from the heme. The radical processing of enzyme lacking the Trp(107)-Tyr(229)-Met(255) adduct (found as a unique structural feature of catalase-peroxidases) is suggested to be a reasonable assignment of the phenomena.     

Patterns of contraceptive use in Australia: analysis of the 2001 National Health Survey

The purpose of this paper is to review the patterns of contraceptive use in Australia, using data from a nationally representative sample of 5872 women aged 18 to 49. This survey was conducted by the Australian Bureau of Statistics in 2001 as part of the National Health Survey. Results of the analysis indicate that the oral contraceptive pill and condom were the two most frequently used methods. More than 76% of the respondents reported having ever used the pill. Over 23% of women were currently using condoms; of these 80% of the condom users used them for contraception - this included 36% who used condoms for both protection against infection and for contraception - and the remainder used them only for protection. Withdrawal was the third most popular non-surgical method up to age 40. Few women used IUDs, injections or diaphragms. Just over 3% of the respondents were using natural methods with the highest rate reported among those in their 30s. The 'morning-after pill' was reported mostly by women aged 18-24; however, there was no evidence to suggest that it was being used as a primary method of birth control. Contraceptive use declined in older women who turned to sterilization for themselves and/or their partners. Use of the contraceptive pill was somewhat higher among better-educated women, but lower among less-educated women and those from non-English-speaking backgrounds.     

Purinoceptor-mediated calcium signaling in primary neuron-glia trigeminal cultures

Receptors for extracellular nucleotides (the P2X-calcium channels and the phospholipase C-coupled P2Y receptors) play key roles in pain signaling, but little is known on their function in trigeminal ganglia, whose hyperactivation leads to the development of migraine pain. Here we characterize calcium signaling via P2X(3) and P2Y receptors in primary mouse neuron-glia trigeminal cultures. Comparison with intact ganglion showed that, in dissociated cultures, sensory neurons retain, at least in part, their physical relationships with satellite glia. RT-PCR indicated expression of P2X(2)/P2X(3) (confirmed by immunocytochemistry) and of all cloned P2Y receptors. Single-cell calcium imaging with subtype-selective P2-agonists/antagonists revealed presence of functional neuronal P2X(3), as well as of ADP-sensitive P2Y(1,12,13) and UTP-activated P2Y(2)/P2Y(4) receptors on both neurons and glia. Calcium responses were much higher in glia, that also responded to UDP, suggesting functional P2Y(6) receptors. To study whether trigeminal ganglia P2 receptors are modulated upon treatment with pro-inflammatory agents, cultures were acutely (up to 3 min) or chronically (24 h) exposed to bradykinin. This resulted in potentiation of algogenic P2X(3) receptor-mediated calcium responses followed by their down-regulation at 24 h. At this exposure time, P2Y receptors responses in satellite glia were instead upregulated, suggesting a complex modulation of P2 receptors in pain signaling.     

Development of the digestive system of Argentine hake,Merluccius hubbsi,larvae

The Argentine hake Merluccius hubbsi is an important fishery resource of the Southwestern Atlantic Ocean and it is also a potential species for cultivation. In this work, the digestive system development in field-caught hake larvae was studied using histological and histochemical approaches. The digestive tract of larvae was divided into: oropharyngeal cavity (OPC), esophagus, stomach (that develops in the preflexion stage), and intestine. The annexed digestive glands consisted of the liver and the exocrine pancreas. At the beginning of the preflexion stage, teeth were developed in the OPC. There were mucous cells in the esophagus secreting different glycoconjugates from hatching. The enterocytes in the posterior intestine exhibited supranuclear vesicles associated with protein absorption. Mucous cells were observed in the posterior intestine in the preflexion stage and, in the anterior region, ending the flexion stage. Each type of glycoconjugates has a specific role. Acidic mucins lubricate and protect from mechanical damage, sialomucines protect from bacterial infections and neutral mucins regulate the acidity of mucus secretion, protect against abrasion and participate in the formation of the chyme, indicating a pregastric digestion. The liver was present since hatching with pancreatic tissue inside and increased in size acquiring the typical structure with hepatocyte cords, sinusoids, vacuoles, and hepatic duct. The hepatocytes vacuolization increased with larval development. The pancreas became extra-hepatic, with basophilic acinar cells and acidophilic zymogen granules. Throughout the ontogeny, the increased structural and functional complexity of the digestive system reflected the transition to exogenous feeding and nutritional increasing needs.     

Structure of Pyridoxal Kinase from Sheep Brain and Role of the Tryptophanyl Residues

The primary structure of sheep brain pyridoxal kinase has been determined by direct chemical and physical methods. The enzyme contains 312 amino acid residues with an acetylated methionine at the N-terminus, yielding a molecular mass of 34,861 Da. The functional role played by the two tryptophanyl residues in positions 52 and 244 of the polypeptide chain has been investigated by fluorescence spectroscopy. The tryptophanyl residues are not completely exposed to the rapidly relaxing solvent and they are poorly accessible to collisional quenchers. Chemical modification with NBS abolishes the catalytic activity of the kinase. The amino acid sequence of the sheep brain enzyme shows high similarity (86.2% identity) with the human pyridoxal kinase recently reported [Hanna, Turner, and Kirkness, (1997), J. Biol. Chem. 272, 10756–10760]. Comparison of the mammalian proteins with bacterial and yeast putative pyridoxal kinases retrieved from the Swiss-Prot data bank shows a low degree of overall similarity. In particular, the putative ATP-binding domain is conserved, whereas the region that appears to be crucial in the binding of the pyridoxal substrate is not. Thus, the assignment of the bacterial and yeast cDNA-deduced proteins as pyridoxal kinases should be taken with caution.     

Heterologous expression of bovine and porcine beta-lactoglobulins in Pichia pastoris: towards a comparative functional characterisation

Bovine and porcine beta-lactoglobulins were cloned and expressed in host cells with the aim of developing the tools necessary for their structural, functional and conformational characterisation by NMR techniques. Both lipocalins were expressed in Pichia pastoris, where the use of a constitutive promoter turned out to allow the highest productivity. The yield of recombinant proteins was further improved through multiple integration of the encoding genes and by increasing aeration of the transformed cultures. Both proteins were obtained in the culture medium at the concentration of 200 microg/ml. Recombinant lipocalins were purified by ion-exchange chromatography from the culture medium. A preliminary NMR characterisation showed that both proteins were correctly folded.