Degradation of the immunogenic peptide gp100(280-288) by the monocyte-like U937 cell line

The possible degradation of the tumor antigen epitope gp100(280-288) (YLEPGPVTA) in the presence of the monocyte-like line U937, and the effect of degradation on the in vitro-measured immune recognition, were investigated by chromatographic techniques and immunological assays. Results indicate a rapid hydrolysis of the substrate in the presence of the model cells, which is consistent with the hypothesis that degradation of gp100(280-288) is caused by the activity of U937-expressed enzymes, specifically amino- and carboxypeptidases. On the other hand, these results do not support the involvement of other enzymes known to be expressed by U937 cells. From a functional point of view, these data indicate that the degradation of gp100(280-288) severely hampered recognition by specific CTL clones. The results obtained may provide a model for epitope degradation by the antigen-presenting cells found in defined anatomical compartments and may, at least in part, account for the low activity of peptide-based antineoplastic vaccines, as well as for the transience of the effects of subcutaneously administered peptides in general.     

Myogenic potential of mouse primordial germ cells

Primordial germ cells are the only stem cells that retain true developmental totipotency after gastrulation, express markers typical of totipotent/pluripotent status and are able both in vivo and in vitro to give rise to pluripotent stem cells as EC and EG cells. We have therefore explored the possibility of the trans-differentiation of mouse PGCs to a myogenic lineage by transplanting them directly or after in vitro culture into a regenerating muscle and by culturing them on monolayers of differentianting muscle cells. The results obtained suggest that mouse PGCs may trans-differentiate into myogenic cells, provided that their somatic environment is preserved. This occurs at an estimated frequency of 0.01%, which is no higher than that reported for stem cells of adult tissues.     

Excited state charge-transfer dynamics study of poplar plastocyanin by ultrafast pump-probe spectroscopy and molecular dynamics simulation

We have applied ultrafast pump-probe spectroscopy to investigate the excited state dynamics of the blue copper protein poplar plastocyanin, by exciting in the blue side of its 600-nm absorption band. The decay of the charge-transfer excited state occurs exponentially with a time constant of approximately 280 fs and is modulated by well visible oscillations. The Fourier transform of the oscillatory component, besides providing most of the vibrational modes found by conventional resonance Raman, presents additional bands in the low frequency region modes, which are reminiscent of collective motions of biological relevance. Notably, a high frequency mode at approximately 508 cm(-1), whose dynamics are consistent with that of the excited state and already observed for other blue copper proteins, is shown to be present also in poplar plastocyanin. This vibrational mode is reproduced by a molecular dynamics simulation involving the excited state of the copper site.     

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.