Conserved intron positions in ancient protein modules

Background  

The timing of the origin of introns is of crucial importance for an understanding of early genome architecture. The Exon theory of genes proposed a role for introns in the formation of multi-exon proteins by exon shuffling and predicts the presence of conserved splice sites in ancient genes. In this study, large-scale analysis of potential conserved splice sites was performed using an intron-exon database (ExInt) derived from GenBank.     

The hamster clock phase-response curve from summerlike light:dark cycles and its role in daily and seasonal timekeeping

We address the subject of entrainment of the hamster clock by the day:night cycle in summer when the sun sets after 6 PM and rises before 6 AM (nights < 12 h). Summer day:night cycles were simulated by 6 light:dark (LD) cycles with D < 12 h (summerlike, SLD) ranging from SLD 12.5 h:11.5 h (D, 6:15 PM-5:45 AM) to 18 h:6 h (D, 9 PM-3 AM). These are the near limiting SLDs for constant PM timing (entrainment) of behavioral estrus and wheel running in hamsters. The onset of estrus was observed every 4 d in the same hamsters as a phase marker of their 24 h clock. On the day before an experimental estrus, preceded and followed by control onsets, a dark period was imposed to cover a putative 6 PM-6 AM light-sensitive period (LSP). This was scanned with a light pulse (and periodic 5 sec bell alarms) lasting 5-240 min. Shifts in onset of estrus on the next day were plotted vs. the end of the light pulse for PM times ("dusk") and its onset for AM times ("dawn"). The resulting phase shifts from the six SLDs were similar, permitting their combination into a single phase-response curve (PRC) of 1605 shifts. This SLD composite PRC rose at 10:15 PM, peaked at 2 AM (81 min advanced shift), fell linearly to 5:55 AM, and then abruptly to normal at 6 AM (no shift). Peak shift was unaffected by light pulse duration or intensity, or hamster age. The SLD composite PRC lacked the 6 PM-9 PM curve of delayed shifts present in reported PRCs from LD 12 h:12 h and DD. However, a two-pulse experiment showed that all light from 6 PM to L-off was needed to block (balance) the advancing action of a 5 min morning light pulse, thereby maintaining entrainment. A working hypothesis to explain daily entrainment and seasonal fertility in the golden hamster is illustrated. A nomenclature for labeling the phases of the hamster clock (circadian time) is proposed.     

Neurobehavioural responses to hypergravity environment in the CD-1 mouse

Behavioural responses of CD-1 mice exposed to 2 g hypergravity (HG; 60 or 120 min) were investigated during adolescence and at adulthood. To characterise motion sickness (MS), pica behaviour, a reliable MS index measured through kaolin consumption, and spontaneous activity were evaluated before, during and after HG exposure. Short- and/or long-lasting effects on emotional responses, exploratory behaviour and spatial learning performances were also investigated and brain levels of nerve growth factor (NGF) and brain derived neurotrophic factor (BDNF) assessed. An increased pica behaviour during post rotational days and a reduction in spontaneous activity during rotation indicated a mild sickness associated with HG, and susceptibility to MS was sex and age related. Short- and long-lasting effects of exposure were also observed, rotated mice showing altered emotional/anxiety behavioural profiles and impaired spatial learning performances. Moreover, central NGF levels were markedly increased after rotation, while minor changes were observed in BDNF levels.     

In vitro reconstitution of the Schizosaccharomyces pombe alternative excision repair pathway

Schizosaccharomyces pombe alternative excision repair has been shown genetically and biochemically to be involved in the repair of a wide variety of DNA lesions. AER is initiated by a damage-specific endonuclease (Uve1p) that recognizes UV-induced photoproducts, base mispairs, abasic sites, and platinum G-G diadducts and cleaves the DNA phosphodiester backbone 5' to a lesion. Several models exist that employ various mechanisms for damage removal based on the activities of Rad2p, a nuclease thought to be responsible for damage excision in AER. This study represents the first report of the biochemical reconstitution of the AER pathway. A base mispair-containing substrate is repaired in a reaction requiring S. pombe Uve1p, Rad2p, DNA polymerase delta, replication factor C, proliferating cell nuclear antigen, and T4 DNA ligase. Surprisingly, damage is removed exclusively by the 5' to 3' exonuclease activity of Rad2p and not its "flap endonuclease" activity and is absolutely dependent upon the presence of the 5'-phosphoryl moiety at the Uve1p cleavage site.     

Identifying the structure of the active sites of human recombinant prolidase

In this paper we provide a detailed biochemical and structural characterization of the active site of recombinant human prolidase, a dimeric metalloenzyme, whose misfunctioning causes a recessive connective tissue disorder (prolidase deficiency) characterized by severe skin lesions, mental retardation and respiratory tract infections. It is known that the protein can host two metal ions in the active site of each constituent monomer. We prove that two different kinds of metals (Mn and Zn) can be simultaneously present in the protein active sites with the protein partially maintaining its enzymatic activity. Structural information extracted from X-ray absorption spectroscopy measurements have been used to yield a full reconstruction of the atomic environment around each one of the two monomeric active sites. In particular, as for the metal ion occupation configuration of the recombinant human prolidase, we have found that one of the two active sites is occupied by two Zn ions and the second one by one Zn and one Mn ion. In both dinuclear units a histidine residue is bound to a Zn ion.     

Sepsis progression and outcome: a dynamical model

Background  

Sepsis (bloodstream infection) is the leading cause of death in non-surgical intensive care units. It is diagnosed in 750,000 US patients per annum, and has high mortality. Current understanding of sepsis is predominately observational and correlational, with only a partial and incomplete understanding of the physiological dynamics underlying the syndrome. There exists a need for dynamical models of sepsis progression, based upon basic physiologic principles, which could eventually guide hourly treatment decisions.     

Plasma membrane of Beta vulgaris storage root shows high water channel activity regulated by cytoplasmic pH and a dual range of calcium concentrations

Plasma membrane vesicles isolated by two-phase partitioning from the storage root of Beta vulgaris show atypically high water permeability that is equivalent only to those reported for active aquaporins in tonoplast or animal red cells (Pf=542 microm s(-1)). The values were determined from the shrinking kinetics measured by stopped-flow light scattering. This high Pf was only partially inhibited by mercury (HgCl2) but showed low activation energy (Ea) consistent with water permeation through water channels. To study short-term regulation of water transport that could be the result of channel gating, the effects of pH, divalent cations, and protection against dephosphorylation were tested. The high Pf observed at pH 8.3 was dramatically reduced by medium acidification. Moreover, intra-vesicular acidification (corresponding to the cytoplasmic face of the membrane) shut down the aquaporins. De-phosphorylation was discounted as a regulatory mechanism in this preparation. On the other hand, among divalent cations, only calcium showed a clear effect on aquaporin activity, with two distinct ranges of sensitivity to free Ca2+ concentration (pCa 8 and pCa 4). Since the normal cytoplasmic free Ca2+ sits between these ranges it allows for the possibility of changes in Ca2+ to finely up- or down-regulate water channel activity. The calcium effect is predominantly on the cytoplasmic face, and inhibition corresponds to an increase in the activation energy for water transport. In conclusion, these findings establish both cytoplasmic pH and Ca2+ as important regulatory factors involved in aquaporin gating.     

Alpha-tocopheryl succinate induces DR4 and DR5 expression by a p53-dependent route: implication for sensitisation of resistant cancer cells to TRAIL apoptosis

We evaluated the ability of alpha-tocopheryl succinate (alpha-TOS) to sensitise TRAIL-resistant malignant mesothelioma (MM) cells to TRAIL-induced apoptosis. We show that alpha-TOS activates expression of DR4/DR5 in a p53-dependent manner and re-establishes sensitivity of resistant MM cells to TRAIL-mediated apoptosis, as documented in p53wt MM cells but not in their p53null counterparts. MM cells selected for TRAIL resistance expressed low cell surface levels of DR4 and DR5. Treatment with sub-lethal doses of alpha-TOS restored expression of DR4 and DR5. The ability of alpha-TOS to modulate expression of pro-apoptotic genes may play a role in sensitisation of tumour cells to immunological stimuli.     

mRNA biogenesis-related helicase eIF4AIII from <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> is an important factor for abiotic stress adaptation

Similar to other plant species, Arabidopsis has a huge repertoire of predicted helicases, including the eIF4AIII factor, a putative component of the exon junction complex related to mRNA biogenesis. In this article, we integrated evolutionary and functional approaches to have a better understanding of eIF4AIII function in plants. Phylogenetic analysis showed that the mRNA biogenesis-related helicase eIF4AIII is the ortholog of the stress-related helicases PDH45 from Pisum sativum and MH1 from Medicago sativa, suggesting evolutionary and probably functional equivalences between mRNA biogenesis and stress-related plant helicases. Molecular and genetic analyses confirmed the relevance of eIF4AIII during abiotic stress adaptation in Arabidopsis. Therefore, in addition to its function in mRNA biogenesis, eIF4AIII can play a role in abiotic stress adaptation.