Localization of HSP70, Cdc2, and cyclin B in sea urchin oocytes in non-stressed conditions

In Paracentrotus lividus embryos, a Mediterranean sea urchin species, HSP70 is present in all the cells. During cell division it localizes under normal growth conditions on the centrosomes and on the whole isolated mitotic apparatus. Now, in situ hybridization, Western blot analyses, and immunohistochemistry show that the HSP70 mRNA is present in both small and large P. lividus oocytes, that all four isoforms of HSP70 can be found also in the oocytes, and that a certain amount of HSP70 localizes on asters and spindles during polar body formation. Moreover, two representative cell-cycle related proteins, cyclin B, and Cdc2, are present both in small and large oocytes, concentrating in the germinal vesicles before its breaking down. Cdc2 has been found in the cytoplasm of small oocytes and in the germinal vesicles of the large ones and then together with HSP70 on the mitotic apparatus of the dividing oocytes.     

Antimalarial and antiproliferative evaluation of bis-steroidal tetraoxanes

Several cis and trans bis-steroidal 1,2,4,5-tetraoxanes possessing amide terminus were synthesised and evaluated as antimalarials and antiproliferatives. The compounds exhibited submicromolar antimalarial activity against Plasmodium falciparum D6 and W2 strains. The existence of HN-C(O) moiety was found necessary for pronounced antimalarial and antiproliferative activity. In antiproliferative screen, the trans tetraoxane 6 was found to exhibit a pronounced cytotoxicity on 14 cancer cell lines. In addition, tetraoxanes 11 and 12 exhibited significant cytotoxic activity too; microscopic examination of treated HeLa cells showed morphological appearance reminiscent for apoptosis (condensed and/or fragmented nuclei).     

Citron-N is a neuronal Rho-associated protein involved in Golgi organization through actin cytoskeleton regulation

The actin cytoskeleton is best known for its role during cellular morphogenesis. However, other evidence suggests that actin is also crucial for the organization and dynamics of membrane organelles such as endosomes and the Golgi complex. As in morphogenesis, the Rho family of small GTPases are key mediators of organelle actin-driven events, although it is unclear how these ubiquitously distributed proteins are activated to regulate actin dynamics in an organelle-specific manner. Here we show that the brain-specific Rho-binding protein Citron-N is enriched at, and associates with, the Golgi apparatus of hippocampal neurons in culture. Suppression of the whole protein or expression of a mutant form lacking the Rho-binding activity results in dispersion of the Golgi apparatus. In contrast, high intracellular levels induce localized accumulation of RhoA and filamentous actin, protecting the Golgi from the rupture normally produced by actin depolymerization. Biochemical and functional analyses indicate that Citron-N controls actin locally by assembling together the Rho effector ROCK-II and the actin-binding, neuron-specific, protein Profilin-IIa (PIIa). Together with recent data on endosomal dynamics, our results highlight the importance of organelle-specific Rho modulators for actin-dependent organelle organization and dynamics.     

Effect of Environmental Pollution on the Chromosomal Variability of Chironomus Riparius

Different frequencies of chromosomal alterations in salivary gland polytene chromosomes AB, CD and EF were described in larvae of Chironomus riparius (syn. Chironomus thummi) from the trace metal-polluted station of Santena on the river Banna, near Turin, and from the unpolluted station of Corio (40 Km from Turin) which was taken as a reference area. In a sample of 56 larvae from Santena, no specimen with the standard karyotype in all cells of the salivary glands was found. Different types of aberrations were found: 33 paracentric and five pericentric inversions, three deficiencies, four amplified sections and one chromatid break. Fifteen out of the 38 inversions and two amplified sections appeared to be inherited, while all the other aberrations were somatic. Most of the aberrations' breakpoints were located on both sides of the centromere regions, where constitutive heterochromatin is present. Also functional alterations were observed (mainly telomere and centromere decondensations and nine novel puffs). In a sample of 49 larvae of a population from the well-preserved area of Corio only six somatic and one inherited paracentric inversions were found. These results suggest that the strong destabilization of the genomes of C. riparius larvae from Santena could be a reaction to the activity of the toxic substances present in the polluted sediments of the river Banna. This revised version was published online in August 2006 with corrections to the Cover Date.     

Neuronal glycolytic pathway impairment induced by HIV envenlope glyocprotein gp120

Neurological impairment is a common feature of Acquired Immunodeficiency Syndrome (AIDS); functional alterations have been reported both in central and peripheral nervous system and the Human Immunodeficiency Virus (HIV) envelope glycoprotein gp120 has been proposed as a neurotoxin acting through a calcium-dependent mechanism. On the other hand it has been reported that gp120 treatment also induce about a 20% decrease in the cerebral glucose utilization and in the cellular ATP levels. The reported observations were performed on experimental system where also non-neuronal cells where present; in order to evaluate whether a direct interaction between HIV proteins and neuronal cells takes place, we used a neuroblastoma cultures where only neuronal cells are present.We analysed the effects of gp120 on the N18TG2 neuroblastoma clone. Treatments were performed both on growing and confluent cultures. Short time treatment with gp120 of confluent cultures causes a 25% reduction in the level of neuron-specific enolase, resulting in a similar decrease of oxygen consumption. Long time exposure of growing cells also causes a reduction in cell survival. Furthermore, using a membrane-specific fluorescent probe we observed that gp120 produces an increase of membrane trafficking. These observations suggest a direct interaction between the viral envelope protein and neuronal cells, which results in an alteration of glycolytic metabolism. This alteration may be related to the neurologic impairments observed in AIDS patients.     

A recombinant human monoclonal antibody to human metapneumovirus fusion protein that neutralizes virus in vitro and is effective therapeutically in vivo

Human metapneumovirus (hMPV) is a recently discovered paramyxovirus that is a major cause of lower-respiratory-tract disease. hMPV is associated with more severe disease in infants and persons with underlying medical conditions. Animal studies have shown that the hMPV fusion (F) protein alone is capable of inducing protective immunity. Here, we report the use of phage display technology to generate a fully human monoclonal antibody fragment (Fab) with biological activity against hMPV. Phage antibody libraries prepared from human donor tissues were selected against recombinant hMPV F protein with multiple rounds of panning. Recombinant Fabs then were expressed in bacteria, and supernatants were screened by enzyme-linked immunosorbent assay and immunofluorescent assays. A number of Fabs that bound to hMPV F were isolated, and several of these exhibited neutralizing activity in vitro. Fab DS7 neutralized the parent strain of hMPV with a 60% plaque reduction activity of 1.1 mug/ml and bound to hMPV F with an affinity of 9.8 x10(-10) M, as measured by surface plasmon resonance. To test the in vivo activity of Fab DS7, groups of cotton rats were infected with hMPV and given Fab intranasally 3 days after infection. Nasal turbinates and lungs were harvested on day 4 postinfection and virus titers determined. Animals treated with Fab DS7 exhibited a >1,500-fold reduction in viral titer in the lungs, with a modest 4-fold reduction in the nasal tissues. There was a dose-response relationship between the dose of DS7 and virus titer. Human Fab DS7 may have prophylactic or therapeutic potential against severe hMPV infection.     

Modulation of heme and myristate binding to human serum albumin by anti-HIV drugs. An optical and NMR spectroscopic study

Human serum albumin (HSA) has an extraordinary ligand-binding capacity, and transports Fe(III)heme and medium- and long-chain fatty acids. In human immunodeficiency virus-infected patients the administered drugs bind to HSA and act as allosteric effectors. Here, the binding of Fe(III)heme to HSA in the presence of three representative anti-HIV drugs and myristate is investigated. Values of the dissociation equilibrium constant K(d) for Fe(III)heme binding to HSA were determined at different myristate concentrations, in the absence and presence of anti-HIV drugs. Nuclear magnetic relaxation dispersion profiles of HSA-Fe(III)heme were measured, at different myristate concentrations, in the absence and presence of anti-HIV drugs. Structural bases for anti-HIV drug binding to HSA are provided by automatic docking simulation. Abacavir and nevirapine bind to HSA with K(d) values of 1 x 10(-6) and 2 x 10(-6) M, respectively. Therefore, at concentrations used in therapy (in the 1-5 x 10(-6) M range) abacavir and nevirapine bind to HSA and increase the affinity of heme for HSA. In the presence of abacavir or nevirapine, the affinity is not lowered by myristate. FA7 should therefore be intended as a secondary binding site for abacavir and nevirapine. Binding of atazanavir is limited by the large size of the drug, although preferential binding may be envisaged to a site positively coupled with FA1 and FA2, and negatively coupled to FA7. As a whole, these results provide a foundation for the comprehension of the complex network of links modulating HSA-binding properties.     

Evidence for selective mitochondrial autophagy and failure in aging

Autophagy is a major intracellular degradation/recycling system ubiquitous in eukaryotic cells. It contributes to the turnover of cellular components by delivering portions of the cytoplasm and organelles to lysosomes, where they are digested. Starvation-induced autophagy is required for maintaining an amino acid pool for gluconeogenesis and for the synthesis of proteins essential to survival under starvation conditions. In addition, autophagy plays an important role in the degradation of excess or injured organelles, including mitochondria. To test the hypothesis of an involvement of a decrease in autophagy in the process of aging, we explored the antiaging effects of pharmacological stimulation of autophagy on the age-dependent accumulation of 8-OHdG-rich mitochondria in rat liver. Male 3-month and 16-month-old 24 hours-fasted Sprague Dawley rats were injected with the antilipolytic agent [3,5-dimethylpyrazole (DMP)] intraperitoneally. Results showed that drug injection rescued older cells from the accumulation of 8-OHdG in the mtDNA in less than 6 hours, but no significant decrease in the level of cytochrome c oxidase activity was observed. Together, these data provide indirect evidence that 8-OHdG might accumulate in a small pool of mitochondria with increasing age rather than be degraded by the autophagic machinery selectively.