A high-resolution map of synteny disruptions in gibbon and human genomes

Gibbons are part of the same superfamily (Hominoidea) as humans and great apes, but their karyotype has diverged faster from the common hominoid ancestor. At least 24 major chromosome rearrangements are required to convert the presumed ancestral karyotype of gibbons into that of the hominoid ancestor. Up to 28 additional rearrangements distinguish the various living species from the common gibbon ancestor. Using the northern white-cheeked gibbon (2n = 52) (Nomascus leucogenys leucogenys) as a model, we created a high-resolution map of the homologous regions between the gibbon and human. The positions of 100 synteny breakpoints relative to the assembled human genome were determined at a resolution of about 200 kb. Interestingly, 46% of the gibbon–human synteny breakpoints occur in regions that correspond to segmental duplications in the human lineage, indicating a common source of plasticity leading to a different outcome in the two species. Additionally, the full sequences of 11 gibbon BACs spanning evolutionary breakpoints reveal either segmental duplications or interspersed repeats at the exact breakpoint locations. No specific sequence element appears to be common among independent rearrangements. We speculate that the extraordinarily high level of rearrangements seen in gibbons may be due to factors that increase the incidence of chromosome breakage or fixation of the derivative chromosomes in a homozygous state.     

Counting the zinc-proteins encoded in the human genome

Metalloproteins are proteins capable of binding one or more metal ions, which may be required for their biological function, or for regulation of their activities or for structural purposes. Genome sequencing projects have provided a huge number of protein primary sequences, but, even though several different elaborate analyses and annotations have been enabled by a rich and ever-increasing portfolio of bioinformatic tools, metal-binding properties remain difficult to predict as well as to investigate experimentally. Consequently, the present knowledge about metalloproteins is only partial. The present bioinformatic research proposes a strategy to answer the question of how many and which proteins encoded in the human genome may require zinc for their physiological function. This is achieved by a combination of approaches, which include: (i) searching in the proteome for the zinc-binding patterns that, on their turn, are obtained from all available X-ray data; (ii) using libraries of metal-binding protein domains based on multiple sequence alignments of known metalloproteins obtained from the Pfam database; and (iii) mining the annotations of human gene sequences, which are based on any type of information available. It is found that 1684 proteins in the human proteome are independently identified by all three approaches as zinc-proteins, 746 are identified by two, and 777 are identified by only one method. By assuming that all proteins identified by at least two approaches are truly zinc-binding and inspecting the proteins identified by a single method, it can be proposed that ca. 2800 human proteins are potentially zinc-binding in vivo, corresponding to 10% of the human proteome, with an uncertainty of 400 sequences. Available functional information suggests that the large majority of human zinc-binding proteins are involved in the regulation of gene expression. The most abundant class of zinc-binding proteins in humans is that of zinc-fingers, with Cys4 and Cys2His2 being the most common types of coordination environment.     

Spacing behaviour of juvenile corn mice, Calomys musculinus, at the beginning of the breeding period, in absence of adult males

This research was carried out to examine the hypothesis that the absence of fathers promotes a different spacing behaviour in juveniles Calomys musculinus at the beginning of the breeding period. The study was carried out in four 0.25-ha enclosures (two control and two experimental), in a natural pasture, between November 2003 and February 2004. In this study the fathers were removed from the experimental enclosures after juveniles were born. Home-range size depended on sex of juveniles and treatment (father removal). In control and experimental enclosures, female home-range sizes were always smaller than male home-ranges. Male home-ranges were always larger in experimental enclosures than in control enclosures. Treatment and overlap type (intra- and inter-sexual) were not independent. The overlap proportions of male home-ranges were greatest in experimental enclosures than in control enclosures, in both the overlap types (male/male, males/females). The intra- (females/females) and inter-sexual (females/males) overlap proportions of female home-ranges were independent of treatment. In C. musculinus, at the beginning of the breeding period and in absence of adult males, juvenile males increase their home-range size and therefore the degree of inter- and intra-sexual home-range overlap as a mechanism for enlarging the number of receptive females that they encounter.     

The combined luminol/isoluminol chemiluminescence method for differentiating between extracellular and intracellular oxidant production by neutrophils

To address the question why isoluminol, but not luminol, failed to detect oxidants produced intracellularly, differences between these luminophores were investigated with respect to physicochemical parameters and the character of chemiluminescence signal. Our results showed the isoluminol molecule to be more polar, more hydrophilic and possessing lower ability to form intramolecular bonds than the luminol molecule. Therefore, isoluminol: (i) only slightly pervaded biological membranes; (ii) depended essentially on extracellular peroxidase; (iii) did not produce chemiluminescence in the presence of extracellular scavengers; and (iv) it could be considered a specific detector of extracellular radicals. On the other hand, the physicochemical parameters of luminol and partial resistance of its chemiluminescence to the effect of extracellular inhibitors proved the lipo/hydrophilic character of this luminophore and thus its ability to interact with radicals both outside and inside of cells. The luminol chemiluminescence measured in the presence of extracellular scavengers and the isoluminol chemiluminescence were used with the intention to differentiate the effects of two antihistamine drugs on intra- and extracellular radical formation. In activated human neutrophils, brompheniramine inhibited the extracellular and potentiated the intracellular part of chemiluminescence signal, whereas a reducing effect of loratadine was observed in both compartments.     

In vitro poly(ADP-ribosyl)ated histones H1a and H1t modulate rat testis chromatin condensation differently

Rat testis H1 proteins were poly(ADP-ribosyl)ated in vitro. The modifying product, poly(ADP-ribose), was found covalently bound to each histone variant at various extents and exhibited distinct structural features (linear and short, rather than branched and long chains). Interest was focused on the somatic H1a, particularly abundant in the testis, as compared with other tissues, and the testis-specific H1t, which appears only at the pachytene spermatocyte stage of germ cell development. These H1s were modified with poly(ADP-ribose) by means of two in vitro experimental approaches. In the first system, each variant was incubated with purified rat testis poly(ADP-ribose)polymerase in the presence of [(32)P] NAD. In parallel, poly(ADP-ribosyl)ated H1s were also prepared following incubation of intact rat testis nuclei with [(32)P] NAD. In both experiments, the poly(ADP-ribosyl)ated proteins were purified from the native forms by means of phenyl boronic agarose chromatography. The results from both analyses were in agreement and showed qualitative differences with regard to the poly(ADP-ribose) covalently associated with H1a and H1t. Comparison of the bound polymers clearly indicated that the oligomers associated with H1a were within 10-12 units long, whereas longer chains (相似文献   

Regulation of nuclear phospholipase C activity

A body of evidence, linking inositide-specific phospholipase C (PI-PLC) to the nucleus, is quite extensive. The main isoform in the nucleus is PI-PLCbeta1, whose activity is up-regulated in response to insulin-like growth factor-1 (IGF-1) or insulin stimulation. Whilst at the plasma membrane this PI-PLC is activated and regulated by Galphaq/alpha(11) and Gbetagamma subunits, there is yet no evidence that qalpha/alpha(11) is present within the nuclear compartment, neither GTP-gamma-S nor AlF4 can stimulate PI-PLCbeta1 activity in isolated nuclei. Here we review the evidence that upon occupancy of type 1 IGF receptor there is translocation to the nucleus of phosphorylated mitogen-activated protein kinase (MAPK) which phosphorylates nuclear PI-PLCbeta1 and triggers its signalling, hinting at a separate pathway of regulation depending on the subcellular location of PI-PLCbeta1. The difference in the regulation of the activity of PI-PLCbeta1mirrors the evidence that nuclear and cytoplasmatic inositides can differ markedly in their signalling capability. Indeed, we do know that agonists which affect nuclear inositol lipid cycle at the nucleus do not stimulate the one at the plasma membrane.     

RGD- and MLD-disintegrins, jarastatin and EC3, activate integrin-mediated signaling modulating the human neutrophils chemotaxis, apoptosis and IL-8 gene expression

The effects of jarastatin (JT), a monomeric RGD-disintegrin, were compared with those of the heterodimeric MLD-disintegrin, EC3, on human neutrophil activation and functions. Both disintegrins inhibited neutrophil chemotaxis induced by fMet-Leu-Phe and were also potent chemotactic agents. These effects were accompanied by an increase in actin polymerization, and both were inhibited by genistein, a tyrosine kinase inhibitor. While JT, but not other RGD-disintegrins, inhibited EC3-induced chemotaxis, EC3 was not able to inhibit JT effect. The chemotactic effect of JT was blocked by anti-alpha(M) antibody whereas anti-alpha(9)beta(1) inhibited EC3 effect. Both JT and EC3 induced focal adhesion kinase (FAK) and phosphoinositide 3-kinase (PI3K) activation. Accordingly, LY294002, a PI3K inhibitor, impaired their chemotactic effect on neutrophils. JT induced Erk-2 translocation to nucleus and a delay of the spontaneous apoptosis of neutrophils in vitro. In contrast, EC3 inhibited Erk-2 activation and had a proapoptotic effect. These effects were reverted by PD98059, an MEK 1/2 inhibitor and blocked by z-VAD-FMK, a caspase inhibitor. In addition, JT, but not EC3, increased the IL-8 mRNA levels in neutrophils. The data indicate that JT and EC3 directly activate an integrin-coupled signaling and modulate the MAPK pathway in different ways, leading the neutrophils to express different functional response.     

Distinct pools of proliferating cell nuclear antigen associated to DNA replication sites interact with the p125 subunit of DNA polymerase delta or DNA ligase I

Proliferating cell nuclear antigen (PCNA) plays an essential role in DNA replication, repair, and cell cycle control. PCNA is a homotrimeric ring that, when encircling DNA, is not easily extractable. Consequently, the dynamics of protein-protein interactions established by PCNA at DNA replication sites is not well understood. We have used DNase I to release DNA-bound PCNA together with replication proteins including the p125-catalytic subunit of DNA polymerase delta (p125-pol delta), DNA ligase I, cyclin A, and cyclin-dependent kinase 2 (CDK2). Interaction with these proteins was investigated by immunoprecipitation with antibodies binding near the interdomain connector loop or to the C-terminal domain of PCNA, respectively, or with antibodies to p125-pol delta or DNA ligase I. PCNA interaction with p125-pol delta or DNA ligase I was detected only by the latter antibodies, and found to be mutually exclusive. In contrast, antibodies to PCNA co-immunoprecipitated only CDK2. A GST-p21(waf1/cip1) C-terminal peptide displaced p125-pol delta and DNA ligase I, but not CDK2, from PCNA. These results suggest that PCNA trimers bound to DNA during the S phase are organized as distinct pools able to bind selectively different partners. Among them, p125-pol delta and DNA ligase I interact with PCNA in a mutually exclusive manner.