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.     

PV1 is a key structural component for the formation of the stomatal and fenestral diaphragms

PV1 is an endothelial-specific integral membrane glycoprotein associated with the stomatal diaphragms of caveolae, transendothelial channels, and vesiculo-vacuolar organelles and the diaphragms of endothelial fenestrae. Multiple PV1 homodimers are found within each stomatal and fenestral diaphragm. We investigated the function of PV1 within these diaphragms and their regulation and found that treatment of endothelial cells in culture with phorbol myristate acetate (PMA) led to upregulation of PV1. This correlated with de novo formation of stomatal diaphragms of caveolae and transendothelial channels as well as fenestrae upon PMA treatment. The newly formed diaphragms could be labeled with anti-PV1 antibodies. The upregulation of PV1 and formation of stomatal and fenestral diaphragms by PMA was endothelium specific and was the highest in microvascular endothelial cells compared with their large vessel counterparts. By using a siRNA approach, PV1 mRNA silencing prevented the de novo formation of the diaphragms of caveolae as well as fenestrae and transendothelial channels. Overexpression of PV1 in endothelial cells as well as in cell types that do not harbor caveolar diaphragms in situ induced de novo formation of caveolar stomatal diaphragms. Lastly, PV1 upregulation by PMA required the activation of Erk1/2 MAP kinase pathway and was protein kinase C independent. Taken together, these data show that PV1 is a key structural component, necessary for the biogenesis of the stomatal and fenestral diaphragms.     

Disruption of axonal transport by loss of huntingtin or expression of pathogenic polyQ proteins in Drosophila

We tested whether proteins implicated in Huntington's and other polyglutamine (polyQ) expansion diseases can cause axonal transport defects. Reduction of Drosophila huntingtin and expression of proteins containing pathogenic polyQ repeats disrupt axonal transport. Pathogenic polyQ proteins accumulate in axonal and nuclear inclusions, titrate soluble motor proteins, and cause neuronal apoptosis and organismal death. Expression of a cytoplasmic polyQ repeat protein causes adult retinal degeneration, axonal blockages in larval neurons, and larval lethality, but not neuronal apoptosis or nuclear inclusions. A nuclear polyQ repeat protein induces neuronal apoptosis and larval lethality but no axonal blockages. We suggest that pathogenic polyQ proteins cause neuronal dysfunction and organismal death by two non-mutually exclusive mechanisms. One mechanism requires nuclear accumulation and induces apoptosis; the other interferes with axonal transport. Thus, disruption of axonal transport by pathogenic polyQ proteins could contribute to early neuropathology in Huntington's and other polyQ expansion diseases.     

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.     

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.     

GIPC is recruited by APPL to peripheral TrkA endosomes and regulates TrkA trafficking and signaling

GIPC is a PDZ protein located on peripheral endosomes that binds to the juxtamembrane region of the TrkA nerve growth factor (NGF) receptor and has been implicated in NGF signaling. We establish here that endogenous GIPC binds to the C terminus of APPL, a Rab5 binding protein, which is a marker for signaling endosomes. When PC12(615) cells are treated with either NGF or antibody agonists to activate TrkA, GIPC and APPL translocate from the cytoplasm and bind to incoming, endocytic vesicles carrying TrkA concentrated at the tips of the cell processes. GIPC, but not APPL, dissociates from these peripheral endosomes prior to or during their trafficking from the cell periphery to the juxtanuclear region, where they acquire EEA1. GIPC's interaction with APPL is essential for recruitment of GIPC to peripheral endosomes and for TrkA signaling, because a GIPC PDZ domain mutant that cannot bind APPL or APPL knockdown with small interfering RNA inhibits NGF-induced GIPC recruitment, mitogen-activated protein kinase activation, and neurite outgrowth. GIPC is also required for efficient endocytosis and trafficking of TrkA because depletion of GIPC slows down endocytosis and trafficking of TrkA and APPL to the early EEA1 endosomes in the juxtanuclear region. We conclude that GIPC, following its recruitment to TrkA by APPL, plays a key role in TrkA trafficking and signaling from endosomes.     

Microtubules and actin microfilaments regulate lipid raft/caveolae localization of adenylyl cyclase signaling components

Microtubules and actin filaments regulate plasma membrane topography, but their role in compartmentation of caveolae-resident signaling components, in particular G protein-coupled receptors (GPCR) and their stimulation of cAMP production, has not been defined. We hypothesized that the microtubular and actin cytoskeletons influence the expression and function of lipid rafts/caveolae, thereby regulating the distribution of GPCR signaling components that promote cAMP formation. Depolymerization of microtubules with colchicine (Colch) or actin microfilaments with cytochalasin D (CD) dramatically reduced the amount of caveolin-3 in buoyant (sucrose density) fractions of adult rat cardiac myocytes. Colch or CD treatment led to the exclusion of caveolin-1, caveolin-2, beta1-adrenergic receptors (beta1-AR), beta2-AR, Galpha(s), and adenylyl cyclase (AC)5/6 from buoyant fractions, decreasing AC5/6 and tyrosine-phosphorylated caveolin-1 in caveolin-1 immunoprecipitates but in parallel increased isoproterenol (beta-AR agonist)-stimulated cAMP production. Incubation with Colch decreased co-localization (by immunofluorescence microscopy) of caveolin-3 and alpha-tubulin; both Colch and CD decreased co-localization of caveolin-3 and filamin (an F-actin cross-linking protein), decreased phosphorylation of caveolin-1, Src, and p38 MAPK, and reduced the number of caveolae/mum of sarcolemma (determined by electron microscopy). Treatment of S49 T-lymphoma cells (which possess lipid rafts but lack caveolae) with CD or Colch redistributed a lipid raft marker (linker for activation of T cells (LAT)) and Galpha(s) from lipid raft domains. We conclude that microtubules and actin filaments restrict cAMP formation by regulating the localization and interaction of GPCR-G(s)-AC in lipid rafts/caveolae.     

Comparative analysis of the zeta-crystallin/quinone reductase gene in guinea pig and mouse

zeta-Crystallin is a novel nicotinamide adenine dinucleotide phosphate:quinone reductase, present at enzymatic levels in various tissues of different species, which is highly expressed in the lens of some hystricomorph rodents and camelids. We report here the complementary DNA (cDNA) cloning of zeta-crystallin from liver libraries in guinea pig (Cavia porcellus), where zeta-crystallin is highly expressed in the lens, and in the laboratory mouse (Mus musculus), where expression in the lens occurs only at enzymatic levels. A 5' untranslated sequence different from the one previously reported for the guinea pig lens cDNA was found in these clones. We also report the isolation of genomic clones including the complete guinea pig zeta-crystallin gene and the 5' region of this gene in mouse. These results show the presence of two promoters in the guinea pig zeta-crystallin gene, one responsible for expression at enzymatic levels and the other responsible for the high expression in the lens. The guinea pig lens promoter is not present in the mouse gene. This is the first example in which the recruitment of an enzyme as a lens crystallin can be explained by the acquisition of an alternative lens- specific promoter.