Fixation of conserved sequences shapes human intron size and influences transposon-insertion dynamics

The basis for intron expansion in humans is largely unexplored. In this article, we demonstrate that intron expansion has primarily been determined by fixation of multispecies conserved sequences (MCSs) over time. The presence of MCSs has shaped intron features: the insertion of transposable elements (TEs) has been constrained as more MCSs were fixed. Analysis of TE and MCS distribution suggested an unprecedented estimate of information requirements for proper splicing of long introns with indication of sequence constraints extending up to >3 kb downstream 5' splice sites.     

Neuropeptide neurotensin stimulates intestinal wound healing following chronic intestinal inflammation

Because neurotensin (NT) and its high-affinity receptor (NTR1) modulate immune responses, chloride secretion, and epithelial cell proliferation, we sought to investigate their role in the repair process that follows the development of mucosal injuries during a persistent inflammation. Colonic NT and NTR1, mRNA, and protein significantly increased only after dextran sodium sulfate (DSS)-induced inflammatory damage developed. Colitis-induced body weight loss, colonic myeloperoxidase activity, and histological damage were significantly enhanced by SR-48642 administration, a nonpeptide NTR1 antagonist, whereas continuous NT infusion ameliorated colitis outcome. To evaluate the NT and NTR1 role in tissue healing, mucosal inflammatory injury was established administering 3% DSS for 5 days. After DSS discontinuation, mice rapidly gained weight, ulcers were healed, and colonic NT, NTR1, and cyclooxygenase (COX)-2 mRNA levels were upregulated, whereas SR-48642 treatment caused a further body weight loss, ulcer enlargement, and a blunted colonic COX-2 mRNA upregulation. In a wound-healing model in vitro, NT-induced cell migration in the denuded area was inhibited by indomethacin but not by an antitransforming growth factor-beta neutralizing antibody. Furthermore, NT significantly increased COX-2 mRNA levels by 2.4-fold and stimulated PGE(2) release in HT-29 cells. These findings suggest that NT and NTR1 are part of the network activated after mucosal injuries and that NT stimulates epithelial restitution at least, in part, through a COX-2 dependent pathway.     

Study of the Villin headpiece folding dynamics by combining coarse-grained Monte Carlo evolution and all-atom molecular dynamics

The folding mechanism of the Villin headpiece (HP36) is studied by means of a novel approach which entails an initial coarse-grained Monte Carlo (MC) scheme followed by all-atom molecular dynamics (MD) simulations in explicit solvent. The MC evolution occurs in a simplified free-energy landscape and allows an efficient selection of marginally-compact structures which are taken as viable initial conformations for the MD. The coarse-grained MC structural representation is connected to the one with atomic resolution through a "fine-graining" reconstruction algorithm. This two-stage strategy is used to select and follow the dynamics of seven different unrelated conformations of HP36. In a notable case the MD trajectory rapidly evolves towards the folded state, yielding a typical root-mean-square deviation (RMSD) of the core region of only 2.4 A from the closest NMR model (the typical RMSD over the whole structure being 4.0 A). The analysis of the various MC-MD trajectories provides valuable insight into the details of the folding and mis-folding mechanisms and particularly about the delicate influence of local and nonlocal interactions in steering the folding process.     

Expression of signal transduction proteins during the differentiation of primary human erythroblasts

The high number (>10(8-10)) of primary human pro-erythroblasts (CD36high/CD235alow) obtainable in HEMA culture (Migliaccio et al., 2002) is exploited here to analyse the expression of proteins implicated in erythropoietin (EPO)-signalling (STATs, PI-3K, and PLCs) during the process of erythroid maturation. Human pro-erythroblasts progressed in 4 days of culture with EPO into basophilic- (CD36high/CD235amedium, 24 h), polychromatic-(CD36high/CD235ahigh, 48 h), and, finally, orthochromatic-(CD36low/CD235ahigh, 72-96 h) erythroblasts. During this maturation, STAT-1 was expressed up to the orthochromatic stage, expression of STAT-5, as well as of its target proteins BclxL and IRF1, remained constant up to 48 h (polychromatic-erythroblasts) but decreased by 96 h (orthochromatic-erythroblasts), while that of STAT-3 decreased constantly from 24 h on and became undetectable by 96 h. Expression of PI-3K rapidly decreased with differentiation since only 50% of original protein levels were detected by 48 h. On the other hand, among the members of PLC families investigated, PLC beta4 was not expressed, PLC beta2, delta1, and gamma2 were expressed at constant levels throughout the maturation process, while expression of PLC beta3 and of PLC gamma1 decreased, as PI-3K, by 24 h and that of PLC beta1 was induced by 6 h and became undetectable by 24 h. In conclusion, these data depict the dynamic signalling scenario associated with the maturation of erythroid cells and provide the first indication that members of PLC families (PLC beta1, beta3, and gamma1) might be involved in the control of erythroid differentiation in humans.     

Surveillance of different recombination defects in mouse spermatocytes yields distinct responses despite elimination at an identical developmental stage

Fundamentally different recombination defects cause apoptosis of mouse spermatocytes at the same stage in development, stage IV of the seminiferous epithelium cycle, equivalent to mid-pachynema in normal males. To understand the cellular response(s) that triggers apoptosis, we examined markers of spermatocyte development in mice with different recombination defects. In Spo11(-)(/)(-) mutants, which lack the double-strand breaks (DSBs) that initiate recombination, spermatocytes express markers of early to mid-pachynema, forming chromatin domains that contain sex body-associated proteins but that rarely encompass the sex chromosomes. Dmc1(-)(/)(-) spermatocytes, impaired in DSB repair, appear to arrest at or about late zygonema. Epistasis analysis reveals that this earlier arrest is a response to unrepaired DSBs, and cytological analysis implicates the BRCT-containing checkpoint protein TOPBP1. Atm(-)(/)(-) spermatocytes show similarities to Dmc1(-)(/)(-) spermatocytes, suggesting that ATM promotes meiotic DSB repair. Msh5(-)(/)(-) mutants display a set of characteristics distinct from these other mutants. Thus, despite equivalent stages of spermatocyte elimination, different recombination-defective mutants manifest distinct responses, providing insight into surveillance mechanisms in male meiosis.     

Tempo and mode of speciation in the Baja California disjunct fish species Anisotremus davidsonii

The Baja California region provides a natural setting for studying the early mechanisms of allopatric speciation in marine systems. Disjunct fish populations from several species that occur in the northern Gulf of California and northern Pacific coast of Baja California, but are absent from its southern shores, were previously shown to be genetically isolated, making them excellent candidates for studying allopatry. In addition, one of these species, the sargo Anisotremus davidsonii, has two pairs of congeneric Panamic trans-isthmian geminate species that allow for internal molecular clock calibration. Phylogeographic and demographic approaches based on mitochondrial (cytochrome b) and nuclear (S7 ribosomal protein) sequences showed that A. davidsonii entered the gulf from the south, and later colonized the Pacific coast, approximately 0.6-0.16 million years ago. Pacific coast colonization may have used a route either around the southern cape of Baja California or across the peninsula through a natural seaway. However, while several seaways have been described from different geological times, none matches the dates of population disjunction, yet much geological work remains to be done in that area. At the present time, there is no evidence for dispersal around the southern tip of the Baja California Peninsula. Signatures of incipient allopatric speciation were observed, such as the reciprocal monophyly of disjunct populations for the mitochondrial marker. However, other characteristics were lacking, such as a strong difference in divergence and coalescence times. Taken together, these results suggest that disjunct populations of A. davidsonii may be consistent with the earliest stages of allopatric speciation.     

Outbreak of avian polyomavirus infection with high mortality in recently captured Crimson's seedcrackers (Pyrenestes sanguineus)

Avian polyomavirus (APV) infection of recently imported Crimson's seedcrackers (Pyrenestes sanguineus) resulted in mortality in 56 of 70 (80%) birds in January 2000. Viral infection in these birds was characterized by diarrhea, anorexia, and lethargy, and death usually ensued within 48 to 72 hr of initial clinical signs. Bacteriologic testing resulted in consistently negative results. Histologic examination of tissues from dead birds revealed large intranuclear inclusion bodies, which at electron microscopy examination, contained 42- to 49-nm viral particles. The diagnosis of APV infection was based on immunohistochemistry and immunoelectronmicroscopy, using a monoclonal antibody specific for VP-1 major capsidic APV protein. This is the first report of an acute APV outbreak in wild, recently imported, Crimson's seedcrackers.     

Structure-activity relationships of linear and cyclic peptides containing the NGR tumor-homing motif

Cyclic and linear peptides containing the Asn-Gly-Arg (NGR) motif have proven useful for delivering various anti-tumor compounds and viral particles to tumor vessels. We have investigated the role of cyclic constraints on the structure and tumor-homing properties of NGR peptides using tumor necrosis factor-alpha (TNF) derivatives containing disulfide-bridged (CNGRC-TNF) and linear (GNGRG-TNF) NGR domains. Experiments carried out in animal models showed that both GNGRG and CNGRC can target TNF to tumors. However, the anti-tumor activity of CNGRC-TNF was >10-fold higher than that of GNGRG-TNF. Molecular dynamic simulation of cyclic CNGRC showed the presence of a bend geometry involving residues Gly(3)-Arg(4). Molecular dynamic simulation of the same peptide without disulfide constraints showed that the most populated and thermodynamically favored configuration is characterized by the presence of a beta-turn involving residues Gly(3)-Arg(4) and hydrogen bonding interactions between the backbone atoms of Asn(2) and Cys(5). These results suggest that the NGR motif has a strong propensity to form beta-turn in linear peptides and may explain the finding that GNGRG peptide can target TNF to tumors, albeit to a lower extent than CNGRC. The disulfide bridge constraint is critical for stabilizing the bent conformation and for increasing the tumor targeting efficiency.     

Modeling enzyme reactivity in organic solvents and water through computer simulations

In this article, we review how molecular modeling techniques can be used to shed light on how water and organic solvents influence the reactivity of enzymes. The application of thermodynamics-based models allowed the first qualitative predictions on the selectivity of many reaction types. However, it was with the application of quantum mechanical/molecular mechanical (QM/MM) methods that quantitative models of actual reactivity patterns could be realistically formulated.