Caffeine and accumbens shell dopamine

It has been reported that caffeine (1.5-30 mg/kg i.p.) as well as specific A1 (DPCPX, 8-cyclopentyl-1,3-dipropylxanthine) receptor antagonists fail to increase extracellular dopamine (DA) in the shell of the nucleus accumbens (NAc). However, it has also been reported that caffeine (10 and 30 mg/kg i.p.) and the A1 antagonist 8-cyclopentyl-1,3-dimethylxanthine (CPT) increases NAc shell DA. To clarify this issue rats were implanted with microdialysis probes at different sites in the NAc shell, in the medial prefrontal cortex (PFCX, infralimbic cortex), and at the border between those areas. Irrespective of probe placement within the NAc shell and of the use of different surgical anesthetics (chloral hydrate and ketamine), we failed to observe changes in dialysate DA after 10 and 30 mg/kg i.p. of caffeine. Similarly negative results were obtained with DPCPX and CPFPX, two potent and selective A1 receptor antagonists. A significant increase of DA was obtained after caffeine when probes were located at the border between the NAc shell and the PFCX (10 and 30 mg/kg) or in the PFCX (10 mg/kg). In view of this and of our previous report that caffeine increases dialysate DA in the medial PFCX, we conclude that the increase in dialysate DA by caffeine observed by others arises from the medial PFCX rather than from the NAc shell as a result of placement of microdialysis probes at the border between the NAc shell and the PFCX.     

Unique human tumor antigens: immunobiology and use in clinical trials

The individual, unique tumor Ags, which characterize each single tumor, were described 50 years ago in rodents but their molecular characterization was limited to few of them and obtained during the last 20 years. Here we summarize the evidence for the existence and the biological role of such Ags in human tumors, although such evidence was provided only during the last 10 years and by a limited number of studies, a fact leading to a misrepresentation of unique Ags in human tumor immunology. This was also due to the increasing knowledge on the shared, self-human tumor Ags, which have been extensively used as cancer vaccines. In this review, we highlight the biological and clinical importance of unique Ags and suggest how they could be used in clinical studies aimed at assessing their immunogenic and clinical potential both in active and adoptive immunotherapy of human tumors.     

Messinian Prolagus (Ochotonidae, Lagomorpha) of Italy

A systematic revision of Italian Messinian Prolagus findings has been performed, evidencing the presence of four different species. Comparative analyses allowed their separation in two groups corresponding to two different palaeobioprovinces characterized by distinct faunal affinities, source areas and tempos and modes of colonization. Such considerations allowed to cast a new light on the palaeobiogeography and the migration pathways of continental vertebrate faunas in the Mediterranean area during Messinian, and to give a new importance to the genus Prolagus as a palaeobiogeographical marker.     

Myofilament calcium sensitivity does not affect cross-bridge activation-relaxation kinetics

We employed single myofibril techniques to test whether the presence of slow skeletal troponin-I (ssTnI) is sufficient to induce increased myofilament calcium sensitivity (EC(50)) and whether modulation of EC(50) affects the dynamics of force development. Studies were performed using rabbit psoas myofibrils activated by rapid solution switch and in which Tn was partially replaced for either recombinant cardiac Tn(cTn) or Tn composed of recombinant cTn-T (cTnT) and cTn-C (cTnC), and recombinant ssTnI (ssTnI-chimera Tn). Tn exchange was performed in rigor solution (0.5 mg/ml Tn; 20 degrees C; 2 h) and confirmed by SDS-PAGE. cTnI exchange induced a decrease in EC(50); ssTnI-chimera Tn exchange induced a further decrease in EC(50) (in microM: endogenous Tn, 1.35 +/- 0.08; cTnI, 1.04 +/- 0.13; ssTnI-chimera Tn, 0.47 +/- 0.03). EC(50) was also decreased by application of 100 microM bepridil (control: 2.04 +/- 0.03 microM; bepridil 1.35 +/- 0.03 microM). Maximum tension was not different between any groups. Despite marked alterations in EC(50), none of the dynamic activation-relaxation parameters were affected under any condition. Our results show that 1) incorporation of ssTnI into the fast skeletal sarcomere is sufficient to induce increased myofilament Ca(2+) sensitivity, and 2) the dynamics of actin-myosin interaction do not correlate with EC(50). This result suggests that intrinsic cross-bridge cycling rate is not altered by the dynamics of thin-filament activation.     

Long-term study on symptomless human metapneumovirus infection in hematopoietic stem cell transplant recipients

From October 2004 through October 2006 a study was performed to evaluate the prevalence of human Metapneumovirus (hMPV) infection in adult hematopoietic stem cell transplant (HSCT) recipients. Sequential nasopharyngeal aspirates (NPA) were collected independently from respiratory symptoms and evaluated for hMPV-RNA by polymerase chain reaction (PCR) and sequence analysis. Results indicate epidemiological and molecular differences between the 2004-2005 and 2005-2006 periods and that hMPV seems not to symptomatically affect HSCT patients or cause late respiratory sequelae. In addition, data collected suggest a hospital origin of hMPV infection in most HSCT patients during the 2004-2005 period.     

Combretastatin CA-4 and combretastatin derivative induce mitotic catastrophe dependent on spindle checkpoint and caspase-3 activation in non-small cell lung cancer cells

Combretastatin A-4 (CA-4), a natural stilbenoid isolated from Combretum caffrum, is a new vascular targeting agent (VTA) known for its antitumor activity due to its anti-tubulin properties. We investigated the molecular mechanisms leading to cell death in non-small cell lung cancer H460 cells induced by natural (CA-4) and synthetic stilbenoids (ST2151) structurally related to CA-4. We found that both compounds induced depolymerization and rearrangement of spindle microtubules, as well as an increasingly aberrant organization of metaphase chromosomes in a dose- and time-dependent manner. Prolonged exposition to ST2151 led cells to organize multiple sites of tubulin repolymerization, whereas tubulin repolymerization was observed only after CA-4 washout. H460 cells were arrested at a pro-metaphase stage, with condensed chromosomes and a triggered spindle assembly checkpoint, as evaluated by kinetochore localization of Bub1 and Mad1 antibodies. Persistent checkpoint activation led to mitochondrial membrane permeabilization (MMP) alterations, cytochrome c release, activation of caspase-9 and -3, PARP cleavage and DNA fragmentation. On the other hand, caspase-2, and -8 were not activated by the drug treatment. The ability of cells to reassemble tubulin in the presence of an activated checkpoint may be responsible for ST2151-induced multinucleation, a recognized sign of mitotic catastrophe. In conclusion, we believe that discovery of new agents able to trigger mitotic catastrophe cell death as a result of mitotic block and prolonged spindle checkpoint activation is particularly worthwhile, considering that tumor cells have a high proliferative rate and mitotic failure occurs irrespective of p53 status. Electronic Supplementary Material Supplementary material is available in the online version of this article at . Ilio Vitale and Antonio Antoccia contribuited equally to this work.     

Caloric restrictions affect some factors involved in age-related hypercholesterolemia

Ageing has been defined as a progressive decrease in physiological capacity and a reduced ability to respond to environmental stresses. It has been observed that diet-restricted animals show a minor morbidity in age-related disease. Among these age-related diseases, hypercholesterolemia is the most recurring one and it is often associated with cardiac failure. Several studies have been published indicating age-dependent changes in circulating levels of cholesterol in both humans and in rodents; recently changes have also been reported in the proteins involved in cholesterol homeostasis, that is, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoAR), Insig-induced gene (Insig) protein, SREBP cleavage activating protein (SCAP), sterol regulatory element binding protein (SREBP), and low density lipoprotein receptor (LDLr). Most age-related modifications of biochemical parameters are normalized or very improved in food-restricted animals, so the aim of this work is to examine whether or not alterations of the factors involved in cholesterol homeostasis which occur during ageing could be counteracted by caloric restriction (CR). The data show that the diet restrictions used attenuate the age-related effects on the factors involved in the synthesis and the degradation rate of HMG-CoAR; in spite of this, CRs have a good effect on the age-related hypercholesterolemia whose reduction seems to depend both on the correct membrane LDLr localization and on the proper restored HMG-CoAR activity.     

Self-eating and self-killing: crosstalk between autophagy and apoptosis

The functional relationship between apoptosis ('self-killing') and autophagy ('self-eating') is complex in the sense that, under certain circumstances, autophagy constitutes a stress adaptation that avoids cell death (and suppresses apoptosis), whereas in other cellular settings, it constitutes an alternative cell-death pathway. Autophagy and apoptosis may be triggered by common upstream signals, and sometimes this results in combined autophagy and apoptosis; in other instances, the cell switches between the two responses in a mutually exclusive manner. On a molecular level, this means that the apoptotic and autophagic response machineries share common pathways that either link or polarize the cellular responses.     

Structural and functional changes in the zebrafish (<Emphasis Type="Italic">Danio rerio</Emphasis>) skeletal muscle after cadmium exposure

This report describes the alterations induced by an environmentally realistic concentration of cadmium in skeletal muscle fibre organization, composition, and function in the teleost zebrafish. Results demonstrate that the ion induces a significant quantitative and qualitative deterioration, disrupting sarcomeric pattern and altering glycoprotein composition. These events, together with a mitochondrial damage, result in a significant reduction in swimming performance. In conclusion, the evidence here collected indicate that in presence of an environmental cadmium contamination, important economic (yields in fisheries/aquaculture), consumer health (fish is an important source of proteins), and ecological (reduced fitness due to reduced swimming performance) consequences can be expected.     

Selective observation of the disordered import signal of a globular protein by in-cell NMR: The example of frataxins

We have exploited the capability of in-cell NMR to selectively observe flexible regions within folded proteins to carry out a comparative study of two members of the highly conserved frataxin family which are found both in prokaryotes and in eukaryotes. They all contain a globular domain which shares more than 50% identity, which in eukaryotes is preceded by an N-terminal tail containing the mitochondrial import signal. We demonstrate that the NMR spectrum of the bacterial ortholog CyaY cannot be observed in the homologous E. coli system, although it becomes fully observable as soon as the cells are lysed. This behavior has been observed for several other compact globular proteins as seems to be the rule rather than the exception. The NMR spectrum of the yeast ortholog Yfh1 contains instead visible signals from the protein. We demonstrate that they correspond to the flexible N-terminal tail indicating that this is flexible and unfolded. This flexibility of the N-terminus agrees with previous studies of human frataxin, despite the extensive sequence diversity of this region in the two proteins. Interestingly, the residues that we observe in in-cell experiments are not visible in the crystal structure of a Yfh1 mutant designed to destabilize the first helix. More importantly, our results show that, in cell, the protein is predominantly present not as an aggregate but as a monomeric species.