Assessment of the association between the frequency of micronucleus and p16INK4a/Ki-67 co-expression in patients with cervical intraepithelial lesions

Human papilloma virus (HPV) infection is the main etiological factor for cervical intraepithelial lesions (CIN). An important characteristic of this process is the loss of genome stability. Therefore, it is imperative to use biomarkers of DNA damage caused by genomic instability to identify high risk individuals. We investigated the frequency of micronuclei (MN) in peripheral blood lymphocytes (PBL) of 20 patients, diagnosed as histologically CIN 1 and 10 healthy controls. We also examined the frequency of other nuclear anomalies including nucleoplasmic bridges (NPBs) and nuclear buds (NBUDs) in PBL of patients with CIN 1 and healthy controls, and evaluated the benefits of p16^INK4a and Ki-67 (p16^INK4a/Ki-67) immunohistochemical double staining for identifying cervical squamous cells that express HPV E6/E7 oncogenes. We analyzed the association between the frequency of MN in PBL and the amount of p16^INK4a/Ki-67 co-expression in CIN 1 patients to establish genomic instability. Among CIN 1 subjects, 15% exhibited diffuse p16^INK4a/Ki-67 co-expression and were considered high positive, 25% of the CIN 1 cases exhibited p16^INK4a/Ki-67 co-expression restricted to the lower part of the epithelium and were considered low positive and the remaining 60% of cases were negative. The frequency of MN, NPBs and NBUDs differed significantly among groups. We found a statistically significant positive correlation between p16^INK4a/Ki-67 co-expression and the frequency of MN, NPBs and NBUDs in PBL. Our findings demonstrate the efficacy of p16^INK4a/Ki-67 double immunostaining for histological samples with CIN 1. MN frequency in PBL might be useful for detecting genomic instability in cases of HPV infection and CIN.     

shRNA knockdown of Bmi-1 reveals a critical role for p21-Rb pathway in NSC self-renewal during development

Knockout studies have shown that the polycomb gene Bmi-1 is important for postnatal, but not embryonic, neural stem cell (NSC) self-renewal and have identified the cell-cycle inhibitors p16/p19 as molecular targets. Here, using lentiviral-delivered shRNAs in vitro and in vivo, we determined that Bmi-1 is also important for NSC self-renewal in the embryo. We found that neural progenitors depend increasingly on Bmi-1 for proliferation as development proceeds from embryonic through adult stages. Acute shRNA-mediated Bmi-1 reduction causes defects in embryonic and adult NSC proliferation and self-renewal that, unexpectedly, are mediated by a different cell-cycle inhibitor, p21. Gene array analyses revealed developmental differences in Bmi-1-controlled expression of genes in the p21-Rb cell cycle regulatory pathway. Our data therefore implicate p21 as an important Bmi-1 target in NSCs, potentially with stage-related differences. Understanding stage-related mechanisms underlying NSC self-renewal has important implications for development of stem cell-based therapies.     

"Tie-ing" down the hematopoietic niche

Interaction of hematopoietic stem cells (HSCs) with their particular microenvironment, or niche, is critical for adult hematopoiesis in the bone marrow (BM). Arai et al. (this issue of Cell) demonstrate that HSCs that express the receptor tyrosine kinase Tie2 are quiescent. Ang-1, the ligand for Tie2, enhanced the ability of HSCs to become quiescent and also induced their adhesion to bone, protecting them from stresses that suppress hematopoiesis. These data suggest that the Ang-1/Tie2 signaling pathway plays a crucial role in the maintenance of HSCs in a quiescent state in the BM niche.     

Effect of modification of histidine residues on organization of the pigment--protein complexes of chromatium minutissimum

The effect of diethyl pyrocarbonate on chromatophores and isolated pigment--protein complexes of Chromatium minutissimum was studied. It is shown that modification of histidine residues results in the destruction of the core antenna LHI (B880) and in a spectral shift from 850 to 830 nm in the peripheral antenna LHII (B800-850). In the purple sulfur bacterium Chromatium minutissimum the pigment--protein complexes B800-B850 (peripheral antenna, LHII) and B880 (core antenna, LHI) collect and transmit the absorbed light energy to the reaction centers. The composition of pigments and proteins as well as primary structure of the majority of polypeptides in both types of complexes from various photosynthetic bacteria have been determined.     

Nucleotide sequence and evolution of a mammalian α-Tubulin messenger RNA

Bacterial clones containing complementary DNA sequences specific for rat brain α-tubulin messenger RNA were constructed. One plasmid, pILαTl, contains >95% of the sequences found in the mRNA: the entire coding sequence as well as extensive 5′ and 3′ untranslated sequences. Comparison of the rat amino acid sequence with the known chicken α-tubulin sequence (Valenzuela et al., 1981) reveals the extraordinary evolutionary stability of α-tubulin protein. The presence of only two interspecies amino acid differences within analogous 411 amino acid sequences predicts that amino acid substitutions in this protein are fixed with a unit evolutionary period (Wilson et al., 1977) of 550 million years (i.e. the time required for a 1% difference to arise within a specific protein in two diverging evolutionary lineages). An analysis of the silent nucleotide differences, permissible because of the degeneracy of the genetic code, demonstrates that these might not occur in a random fashion. The high guanine-cytosine bias in silent codon positions within the chicken α-tubulin sequence, previously noted by Valenzuela et al. (1981), is not conserved within the rat sequence. This decrease in guanine-cytosine bias is accompanied by a selective loss of CpG dinucleotides in the rat sequence.     

Mitogenic signalling and substrate specificity of the Flk2/Flt3 receptor tyrosine kinase in fibroblasts and interleukin 3-dependent hematopoietic cells.

Flk2/Flt3 is a recently identified receptor tyrosine kinase expressed in brain, placenta, testis, and primitive hematopoietic cells. The mitogenic signalling potential and biochemical properties of Flk2/Flt3 have been analyzed by using a chimeric receptor composed of the extracellular domain of the human colony-stimulating factor 1 receptor and the transmembrane and cytoplasmic domains of murine Flk2/Flt3. We demonstrate that colony-stimulating factor 1 stimulation of the Flk2/Flt3 kinase in transfected NIH 3T3 fibroblasts leads to a transformed phenotype and generates a full proliferative response in the absence of other growth factors. In transfected interleukin 3 (IL-3)-dependent Ba/F3 lymphoid cells, activation of the chimeric receptor can abrogate IL-3 requirement and sustain long-term proliferation. We show that phospholipase C-gamma 1, Ras GTPase-activating protein, the p85 subunit of phosphatidylinositol 3'-kinase, Shc, Grb2, Vav, Fyn, and Src are components of the Flk2/Flt3 signal transduction pathway. In addition, we demonstrate that phospholipase C-gamma 1, the p85 subunit of phosphatidylinositol 3'-kinase, Shc, Grb2, and Src family tyrosine kinases, but not Ras GTPase-activating protein, Vav, or Nck, physically associate with the Flk2/Flt3 cytoplasmic domain. Cell-type-specific differences in tyrosine phosphorylation of p85 and Shc are observed. A comparative analysis of the Flk2/Flt3 signal cascade with those of the endogenous platelet-derived growth factor and IL-3 receptors indicates that Flk2/Flt3 displays specific substrate preferences. Furthermore, tyrosine phosphorylation of p85 and Shc is similarly affected by totally different growth factors in the same cellular background.     

How can malaria rapid diagnostic tests achieve their potential? A qualitative study of a trial at health facilities in Ghana

Malaria is still a major public health problem in Brazil, with approximately 306 000 registered cases in 2009, but it is estimated that in the early 1940s, around six million cases of malaria occurred each year. As a result of the fight against the disease, the number of malaria cases decreased over the years and the smallest numbers of cases to-date were recorded in the 1960s. From the mid-1960s onwards, Brazil underwent a rapid and disorganized settlement process in the Amazon and this migratory movement led to a progressive increase in the number of reported cases. Although the main mosquito vector (Anopheles darlingi) is present in about 80% of the country, currently the incidence of malaria in Brazil is almost exclusively (99,8% of the cases) restricted to the region of the Amazon Basin, where a number of combined factors favors disease transmission and impair the use of standard control procedures. Plasmodium vivax accounts for 83,7% of registered cases, while Plasmodium falciparum is responsible for 16,3% and Plasmodium malariae is seldom observed. Although vivax malaria is thought to cause little mortality, compared to falciparum malaria, it accounts for much of the morbidity and for huge burdens on the prosperity of endemic communities. However, in the last few years a pattern of unusual clinical complications with fatal cases associated with P. vivax have been reported in Brazil and this is a matter of concern for Brazilian malariologists. In addition, the emergence of P. vivax strains resistant to chloroquine in some reports needs to be further investigated. In contrast, asymptomatic infection by P. falciparum and P. vivax has been detected in epidemiological studies in the states of Rondonia and Amazonas, indicating probably a pattern of clinical immunity in both autochthonous and migrant populations. Seropidemiological studies investigating the type of immune responses elicited in naturally-exposed populations to several malaria vaccine candidates in Brazilian populations have also been providing important information on whether immune responses specific to these antigens are generated in natural infections and their immunogenic potential as vaccine candidates. The present difficulties in reducing economic and social risk factors that determine the incidence of malaria in the Amazon Region render impracticable its elimination in the region. As a result, a malaria-integrated control effort - as a joint action on the part of the government and the population - directed towards the elimination or reduction of the risks of death or illness, is the direction adopted by the Brazilian government in the fight against the disease.