IN SITU LOCALIZATION OF GLOBIN MESSENGER RNA FORMATION : I. During Mouse Fetal Liver Development

Globin mRNA levels in 11–15-day mouse fetal liver cells have been estimated by in situ hybridization of a highly labeled DNA copy (cDNA) of adult globin messenger RNAs (mRNAs) (globin cDNA) to fixed preparations of cells. Under the conditions employed, no significant in situ hybridization occurred to lymphoma cells (L 51787), mouse L cells, or hepatocytes; whereas reticulocytes from phenyl hydrazine-treated mice showed extensive in situ hybridization. The proportion of fetal liver cells showing predominantly cytoplasmic in situ hybridization increased from about 30% at the 11th day of development to 80–85% by days 13–15. Unlike more mature cells, proerythroblasts did not show in situ hybridization, except to a slight extent at later stages of development. These studies therefore indicate that globin mRNAs begin to accumulate during or shortly after the proerythroblastbasophilic erythroblast transition. The fact that certain immature erythroid cells from 14-day fetal liver contain substantial amounts of globin mRNAs has been confirmed by comparing the hybridization in solution of globin cDNA to cytoplasmic RNA extracted from total fetal liver cells or from immature erythroid cells obtained by treatment of fetal liver cells with an antiserum raised against erythrocytes

IN SITU LOCALIZATION OF GLOBIN MESSENGER RNA FORMATION : II. After Treatment of Friend Virus-Transformed Mouse Cells with Dimethyl Sulfoxide

Globin messenger RNA (mRNA) levels in Friend virus-transformed mouse cells have been estimated by in situ hybridization of DNA copy (cDNA) to fixed preparations of cells and by hybridization of cDNA to extracted cytoplasmic RNA in true solution. The results obtained by both methods agree in showing that a low level of globin mRNA can be detected in untreated Friend cells. The levels of hemoglobin and globin mRNA have also been correlated after treatment of Friend cells with dimethyl sulfoxide (DMSO). The results obtained by both experimental approaches show that there is a minimum period of treatment with DMSO required in order that Friend cells may become hemoglobinized, and that this period coincides with the time when globin mRNA accumulates. Moreover, bromodeoxyuridine prevents both hemoglobin and globin mRNA accumulation

The human RPS4 paralogue on Yq11.223 encodes a structurally conserved ribosomal protein and is preferentially expressed during spermatogenesis.

BACKGROUND: The Y chromosome of mammals is particularly prone to accumulate genes related to male fertility. However, the high rate of molecular evolution on this chromosome predicts reduced power to the across-species comparative approach in identifying male-specific genes that are essential for sperm production in humans. We performed a comprehensive analysis of expression of Y-linked transcripts and their X homologues in several human tissues, and in biopsies of infertile patients, in an attempt to identify new testis-specific genes involved in human spermatogenesis. RESULTS: We present evidence that one of the primate-specific Y-linked ribosomal protein genes, RPS4Y2, has restricted expression in testis and prostate, in contrast with its X-linked homologue, which is ubiquitously expressed. Moreover, we have determined by highly specific quantitative real time PCR that RPS4Y2 is more highly expressed in testis biopsies containing germ cells. The in silico analysis of the promoter region of RPS4Y2 revealed several differences relative to RPS4Y1, the more widely expressed paralogue from which Y2 has originated through duplication. Finally, through comparative modelling we obtained the three dimensional models of the human S4 proteins, revealing a conserved structure. Interestingly, RPS4Y2 shows different inter-domain contacts and the potential to establish specific interactions. CONCLUSIONS: These results suggest that one of the Y-linked copies of the ribosomal protein S4 is preferentially expressed during spermatogenesis and might be important for germ cell development. Even though RPS4Y2 has accumulated several amino acid changes following its duplication from RPS4Y1, approximately 35 million years ago, the evolution of the Y-encoded RPS4 proteins is structurally constrained. However, the exclusive expression pattern of RPS4Y2 and the novelties acquired at the C-terminus of the protein may indicate some degree of functional specialisation of this protein in spermatogenesis.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

Deficiency in the Multicopy Sycp3-Like X-Linked Genes Slx and Slxl1 Causes Major Defects in Spermatid Differentiation

Slx and Slxl1 are genes present in multiple copies on the mouse X chromosome. Using transgenically-delivered small interfering RNAs to disrupt their function, we show that Slx and Slxl1 are important for normal sperm differentiation and male fertility

Serology describes a profile of declining malaria transmission in Farafenni, The Gambia

BACKGROUND: Malaria morbidity and mortality has declined in recent years in a number of settings. The ability to describe changes in malaria transmission associated with these declines is important in terms of assessing the potential effects of control interventions, and for monitoring and evaluation purposes. METHODS: Data from five cross-sectional surveys conducted in Farafenni and surrounding villages on the north bank of River Gambia between 1988 and 2011 were compiled. Antibody responses to MSP-119 were measured in samples from all surveys, data were normalized and expressed as seroprevalence and seroconversion rates (SCR) using different mathematical models. RESULTS: Results showed declines in serological metrics with seroprevalence in children aged one to 5 years dropping from 19 % (95 % CI 15-23 %) in 1988 to 1 % (0-2 %) in 2011 (p value for trend in proportions < 0.001) and the SCR dropping from 0.069 year(-1) (0.059-0.080) to 0.022 year(-1) (0.017-0.028; p = 0.004). The serological data were consistent with previously described drops in both parasite prevalence in children aged 1-5 years (62 %, 57-66 %, in 1988 to 2 %, 0-4 %, in 2011; p < 0.001), and all-cause under five mortality rates (37 per 1000 person-years, 34-41, in 1990 to 17, 15-19, in 2006; p = 0.059). CONCLUSIONS: This analysis shows accurate reconstruction of historical malaria transmission patterns in the Farafenni area using anti-malarial antibody responses. Demonstrating congruence between serological measures, and conventional clinical and parasitological measures suggests broader utility for serology in monitoring and evaluation of malaria transmission

Phenotypic Overlap between MMP-13 and the Plasminogen Activation System during Wound Healing in Mice

BACKGROUND: Proteolytic degradation of extracellular matrix is a crucial step in the healing of incisional skin wounds. Thus, healing of skin wounds is delayed by either plasminogen-deficiency or by treatment with the broad-spectrum metalloproteinase (MP) inhibitor Galardin alone, while the two perturbations combined completely prevent wound healing. Both urokinase-type plasminogen activator and several matrix metallo proteinases (MMPs), such as MMP-3, -9 and -13, are expressed in the leading-edge keratinocytes of skin wounds, which may account for this phenotypic overlap between these classes of proteases. METHODOLOGY: To further test that hypothesis we generated Mmp13;Plau and Mmp13;Plg double-deficient mice in a cross between Mmp13- and Plau-deficient mice as well as Mmp13- and Plg-deficient mice. These mice were examined for normal physiology in a large cohort study and in a well-characterized skin wound healing model, in which we made incisional 20 mm-long full-thickness skin wounds. PRINCIPAL FINDINGS: While mice that are deficient in Mmp13 have a mean healing time indistinguishable to wild-type mice, wound healing in both Plau- and Plg-deficient mice is significantly delayed. Histological analysis of healed wounds revealed a significant increase in keratin 10/14 immunoreactive layers of kerationcytes in the skin surface in Mmp13;Plau double-deficient mice. Furthermore, we observe, by immunohistological analysis, an aberrant angiogenic pattern during wound healing induced by Plau-deficiency, which has not previously been described. CONCLUSIONS: We demonstrate a phenotypic overlap, defined as an additional delay in wound healing in the double-deficient mice compared to the individual single-deficient mice, between MMP-13 and the plasminogen activation system in the process of wound healing, but not during gestation and in postnatal development. Thus, a dual targeting of uPA and MMP-13 might be a possible future strategy in designing therapies aimed at tissue repair or other pathological processes, such as cancer invasion, where proteolytic degradation is a hallmark

Detecting Foci of Malaria Transmission with School Surveys: A Pilot Study in the Gambia.

BACKGROUND: In areas of declining malaria transmission such as in The Gambia, the identification of malaria infected individuals becomes increasingly harder. School surveys may be used to identify foci of malaria transmission in the community. METHODS: The survey was carried out in May-June 2011, before the beginning of the malaria transmission season. Thirty two schools in the Upper River Region of The Gambia were selected with probability proportional to size; in each school approximately 100 children were randomly chosen for inclusion in the study. Each child had a finger prick blood sample collected for the determination of antimalarial antibodies by ELISA, malaria infection by microscopy and PCR, and for haemoglobin measurement. In addition, a simple questionnaire on socio-demographic variables and the use of insecticide-treated bed nets was completed. The cut-off for positivity for antimalarial antibodies was obtained using finite mixture models. The clustered nature of the data was taken into account in the analyses. RESULTS: A total of 3,277 children were included in the survey. The mean age was 10 years (SD = 2.7) [range 4-21], with males and females evenly distributed. The prevalence of malaria infection as determined by PCR was 13.6% (426/3124) [95% CI = 12.2-16.3] with marked variation between schools (range 3-25%, p<0.001), while the seroprevalence was 7.8% (234/2994) [95%CI = 6.4-9.8] for MSP119, 11.6% (364/2997) [95%CI = 9.4-14.5] for MSP2, and 20.0% (593/2973) [95% CI = 16.5-23.2) for AMA1. The prevalence of all the three antimalarial antibodies positive was 2.7% (79/2920). CONCLUSIONS: This survey shows that malaria prevalence and seroprevalence before the transmission season were highly heterogeneous

Malaria Prevalence among Young Infants in Different Transmission Settings, Africa.

The prevalence and consequences of malaria among infants are not well characterized and may be underestimated. A better understanding of the risk for malaria in early infancy is critical for drug development and informed decision making. In a cross-sectional survey in Guinea, The Gambia, and Benin, countries with different malaria transmission intensities, the overall prevalence of malaria among infants <6 months of age was 11.8% (Guinea, 21.7%; The Gambia, 3.7%; and Benin, 10.2%). Seroprevalence ranged from 5.7% in The Gambia to 41.6% in Guinea. Mean parasite densities in infants were significantly lower than those in children 1-9 years of age in The Gambia (p<0.0001) and Benin (p = 0.0021). Malaria in infants was significantly associated with fever or recent history of fever (p = 0.007) and anemia (p = 0.001). Targeted preventive interventions, adequate drug formulations, and treatment guidelines are needed to address the sizeable prevalence of malaria among young infants in malaria-endemic countries

Emerging Roles of PAR-1 and PAFR in Melanoma Metastasis

Melanoma growth, angiogenesis and metastatic progression are strongly promoted by the inflammatory tumor microenvironment due to high levels of cytokine and chemokine secretion by the recruited inflammatory and stromal cells. In addition, platelets and molecular components of procoagulant pathways have been recently emerging as critical players of tumor growth and metastasis. In particular, thrombin, through the activity of its receptor protease-activated receptor-1 (PAR-1), regulates tumor cell adhesion to platelets and endothelial cells, stimulates tumor angiogenesis, and promotes tumor growth and metastasis. Notably, in many tumor types including melanoma, PAR-1 expression directly correlates with their metastatic phenotype and is directly responsible for the expression of interleukin-8, matrix metalloproteinase-2 (MMP-2), vascular endothelial growth factor, platelet-derived growth factor, and integrins. Another proinflammatory receptor–ligand pair, platelet-activating factor (PAF) and its receptor (PAFR), have been shown to act as important modulators of tumor cell adhesion to endothelial cells, angiogenesis, tumor growth and metastasis. PAF is a bioactive lipid produced by a variety of cells from membrane glycerophospholipids in the same reaction that releases arachidonic acid, and can be secreted by platelets, inflammatory cells, keratinocytes and endothelial cells. We have demonstrated that in metastatic melanoma cells, PAF stimulates the phosphorylation of cyclic adenosine monophosphate response element-binding protein (CREB) and activating transcription factor 1 (ATF-1), which results in overexpression of MMP-2 and membrane type 1-MMP (membrane type 1-MMP). Since only metastatic melanoma cells overexpress CREB/ATF-1, we propose that metastatic melanoma cells are better equipped than their non-metastatic counterparts to respond to PAF within the tumor microenvironment. The evidence supporting the hypothesis that the two G-protein coupled receptors, PAR-1 and PAFR, contribute to the acquisition of the metastatic phenotype of melanoma is presented and discussed