Essential amino acid supplements increase muscle weight, bone mass and bone strength in adult osteoporotic rats

Protein undernutrition is known to play an important role in the pathogenesis of osteoporotic fracture in elderly. The mechanisms underlying the bone loss in protein undernutrition appeared to be related to an uncoupling between increased bone resorption and bone formation. This was associated with decreased plasma IGF-I levels, with anoestrus and decreased muscle mass. Reversibility of protein undernutrition-induced bone loss was investigated in ovariectomized adult rats, which were fed isocaloric 2.5 % casein diet (OVX2.5) for 16 weeks. Then, the animals were given a supplement of essential amino-acids in similar proportion to that of casein at doses of 2.5% (EAA2.5) or 5% (EAA5) of total food intake for an additional 16 weeks. Essential amino acid supplements increased bone mineral mass and strength in ovariectomized protein-deprived rats. EAA supplements were associated with stimulated bone formation and reduced bone resorption, with increment of plasma IGF-I and of limb muscle mass weight. These results suggest that nutritional intervention with essential amino acid supplements can increase bone mineral mass, bone strength and muscle mass in osteoporotic rats possibly by correcting IGFI status.     

The universal ancestor lived in a thermophilic or hyperthermophilic environment

Galtier et al. (Science 1999, 283, 220-221) exploit the correlation between the optimal growth temperature in prokaryotes and the G+C content of rRNAs and establish that the last universal common ancestor (LUCA) lived in a mesophilic environment. This result was achieved by estimating the G+C content of the ancestral sequences of the rRNAs of the LUCA through use of a complex Markov model. I have re-analysed their alignments of the rDNAs with maximum parsimony and I have found that their result is not robust and is, in all likelihood, incorrect. In particular, the rRNA ancestral sequences reconstructed with maximum parsimony from these rDNA alignments as well as those reconstructed after eliminating all the sites that turn out to be ambiguous to the parsimony algorithm and to a site-by-site inspection of these alignments, are such as to suggest that the LUCA lived in a thermophilic or hyperthermophilic environment. This finding is also supported by some tRNA ancestral sequences. The main conclusion of this analysis is that if the LUCA was a progenote then the origin of life might have taken place at a high temperature.     

Titanium-carbon functionalities on an oxo surface defined by a calix [4] arene moiety and its redox chemistry

Lithiation of [p-Bu^t-calix[4]-(OMe)₂(OH)₂] (1), followed by reaction with TiCl₃(thf)₃ or TiCl₄(thf)₂, led to the corresponding titanium-calix[4]arene complexes [p-Bu^t-calix[4]-(OMe)₂(O)₂]TiCl] (2) and [p-Bu^t-calix[4]-(OMe)₂(O)₂]TiCl₂] (3), respectively. Reaction of 1 with TiCl₄(thf)₂ results in demethylation of the calix[4]arene and the obtention of [p-Bu^t-calix[4]-(OMe)₂(O)₃]TiCl] (4), whose hydrolysis led to [p-Bu^t-calix[4]-(OMe)(OH)₃] (6). The preparation of 6 can be carried out as a one-pot synthesis. Both 2 and 4 undergo alkylation reactions using conventional procedures, thus forming surprisingly stable organometallic species, namely [p-Bu^t-calix[4]-(OMe)₂(O)₂Ti(R)] (R = Me (7); CH₂Ph (8), p-MeC₆H₄ (9) and [p-Bu^t-calix[4]-(OMe)(O)₃Ti(R)] (R = Me (10); CH₂Ph (11); p-MeC₆H₄ (12)). Complexes 7 and 9 undergo a thermal oxidative conversion into 10 and 12, occurring with the demethylation of one of the methoxy groups. A solid state structural property of 9 and 12 has been revealed by X-ray analysis showing a self-assembly of the monomeric units into a columnar polymer, where the p-tolyl substituent at the metal functions as a guest group for an adjacent titanium-calixarene. Reductive alkylation of 3 with Mg(CH₂Ph)₂ gave 8 instead of forming the corresponding dialkyl derivative. Two synthetic routes have been devised for the synthesis of the Ti(III)-Ti(III) dimer [p-Bu^t-calix[4]-(OMe)(O)₃Ti]₂] (13): the reduction of 4 and the reaction of TiCl₃(thf)₃ with the lithiated form of 6. A very strong antiferromagnetic coupling is responsible for the peculiar magnetic behavior of 13. The proposed structures have been supported by the X-ray analyses of 4, 9, 12 and 13.     

The Fibronectin Domain ED-A Is Crucial for Myofibroblastic Phenotype Induction by Transforming Growth Factor-β1

Transforming growth factor-β1 (TGFβ1), a major promoter of myofibroblast differentiation, induces α-smooth muscle (sn) actin, modulates the expression of adhesive receptors, and enhances the synthesis of extracellular matrix (ECM) molecules including ED-A fibronectin (FN), an isoform de novo expressed during wound healing and fibrotic changes. We report here that ED-A FN deposition precedes α-SM actin expression by fibroblasts during granulation tissue evolution in vivo and after TGFβ1 stimulation in vitro. Moreover, there is a correlation between in vitro expression of α-SM actin and ED-A FN in different fibroblastic populations. Seeding fibroblasts on ED-A FN does not elicit per se α-SM actin expression; however, incubation of fibroblasts with the anti-ED-A monoclonal antibody IST-9 specifically blocks the TGFβ1-triggered enhancement of α-SM actin and collagen type I, but not that of plasminogen activator inhibitor-1 mRNA. Interestingly, the same inhibiting action is exerted by the soluble recombinant domain ED-A, but neither of these inhibitory agents alter FN matrix assembly. Our findings indicate that ED-A–containing polymerized FN is necessary for the induction of the myofibroblastic phenotype by TGFβ1 and identify a hitherto unknown mechanism of cytokine-determined gene stimulation based on the generation of an ECM-derived permissive outside in signaling, under the control of the cytokine itself.     

Somatic embryogenesis and plant regeneration from undeveloped ovules and stigma/style explants of sweet orange navel group [ Citrus sinensis (L.) Osb.]

Somatic embryogenesis was induced and plant regeneration was obtained in 11 different genotypes of sweet orange navel group [Citrus sinensis (L.) Osb.] from cultures of stigma/style explants and undeveloped ovules. Explants were cultured on 3 different modifications of Murashige and Skoog medium: 500 mg l-1 malt extract; 500 mg l-1 malt extract and 4.6 μM kinetin; and 500 mg l-1 malt extract and 13.3 μM 6-benzylaminopurine. Sucrose (146 mM) was used as carbon source. Somatic embryogenesis occurred 1–3 months after culture initiation from undeveloped ovule and stigma/style cultures of all the genotypes tested. Somatic embryos developed into plantlets with a high frequency (74%) after transfer to Murashige and Skoog medium supplemented with 146 mM sucrose and 500 mg l-1 malt extract. Plants were successfully transferred to soil. This revised version was published online in June 2006 with corrections to the Cover Date.     

Environmental tuning of an insect ensemble: The tenebrionid beetles inhabiting a Mediterranean coastal dune zonation

Few studies have investigated insect ensembles, i.e. phylogenetically bounded groups of species that use a similar set of resources within a community. The zonation of dune vegetation makes these ecosystems ideal for the study of insect ensembles in a short space. In this study, we investigated if the tenebrionid beetles forming an ensemble on a dune zonation showed variations in community organization (relative abundances and species diversity) in different but spatially associated biotopes defined by different plant communities. Three biotopes (corresponding to European Commission habitat 2110, 2120 and 2230) of a well-preserved Mediterranean dune were sampled using square plots of 2 × 2 m at three places. To investigate if there was some association between species and habitat we applied a χ² test. Variations in community structure parameters were investigated using Shannon index. The three biotopes host tenebrionid communities with similar species composition and overall abundances, confirming that they form a single ensemble. However, tenebrionid species are differently associated with different biotopes along the zonation, with some species occurring with different proportions among the biotopes. A local selection process can be postulated as a mechanism responsible for these differences.     

Identification of pH-sensing Sites in the Light Harvesting Complex Stress-related 3 Protein Essential for Triggering Non-photochemical Quenching in Chlamydomonas reinhardtii

Light harvesting complex stress-related 3 (LHCSR3) is the protein essential for photoprotective excess energy dissipation (non-photochemical quenching, NPQ) in the model green alga Chlamydomonas reinhardtii. Activation of NPQ requires low pH in the thylakoid lumen, which is induced in excess light conditions and sensed by lumen-exposed acidic residues. In this work we have used site-specific mutagenesis in vivo and in vitro for identification of the residues in LHCSR3 that are responsible for sensing lumen pH. Lumen-exposed protonatable residues, aspartate and glutamate, were mutated to asparagine and glutamine, respectively. By expression in a mutant lacking all LHCSR isoforms, residues Asp¹¹⁷, Glu²²¹, and Glu²²⁴ were shown to be essential for LHCSR3-dependent NPQ induction in C. reinhardtii. Analysis of recombinant proteins carrying the same mutations refolded in vitro with pigments showed that the capacity of responding to low pH by decreasing the fluorescence lifetime, present in the wild-type protein, was lost. Consistent with a role in pH sensing, the mutations led to a substantial reduction in binding the NPQ inhibitor dicyclohexylcarbodiimide.     

Liver peroxisomes in newborns from clofibrate-treated rats. II. A biochemical study of the recovery period.

The fatty-acyl-CoA beta-oxidation (FAO) and catalase activities, as well as membrane fluidity of liver peroxisomes of newborns from normal and clofibrate-treated rats were studied during the recovery period, ie, throughout the first week of postnatal life. In the test animals the enzyme activities, which are significantly higher than controls at birth return to normal levels showing a somewhat different time course with FAO rapidly decreasing to control values within three days but with catalase still higher than controls at day 6. The half-life and degradation rate (Kd) of FAO are identical to those calculated by us for the whole organelles and to those reported by others for total catalase in normal or clofibrate-treated adult animals in the presence of catalase inhibitors. Soluble catalase shows turnover values which are similar though not identical to those of FAO, while total catalase has a very long half-life and a low Kd. Peroxisomal membrane fluidity, as determined by fluorescence anisotropy of 1-anilinonaphthalene-8-sulfonate (ANS) bound to purified peroxisomal fractions is higher in tests than in controls, recovering normal values within 6 days. Our results demonstrate that liver peroxisomes of rats prenatally exposed to clofibrate return to control conditions within about 1 week. The turnover parameters of enzymes and the membrane fluidity values are discussed in terms of disposal mechanism(s) for the excess of induced peroxisomes.