Physical map of phage 2 C DNA: evidence for the existence of large redundant ends

The chromosome of the Bacillus subtilis phage 2C, a linear molecule of double-stranded DNA of about 10(8) Da, in which thymine is completely replaced by hydroxymethyluracil, was cleaved by different endonucleases. In some cases restriction segments were much fewer than expected, suggesting a possible interference of the unusual base with the recognition mechanism of endonucleases. The physical map of 2C DNA was established by use of SalI and HaeIII restriction endonucleases, which yielded a limited number of fragments. The expected number of fragments was 240 for HaeIII and 23 for SalI; in reality, five segments were observed upon cleavage with HaeIII and four with SalI. The terminal fragments of the genome were first identified; the other fragments were ordered by hybridization and molecular weight determination of restriction fragments obtained by cleavage with the two endonucleases. In addition, hybridization of restriction fragments showed the presence of homologous regions at the ends of the 2C genome. The structure of these direct repetitive sequences was analyzed by cleavage with HaeIII and hybridization with EcoRI restriction fragments. Their size (9.2 MDa) was found to be about 1/11 of that of the whole chromosome.     

Human immunodeficiency virus Nef induces rapid internalization of the T-cell coreceptor CD8alphabeta

Human immunodeficiency virus (HIV) Nef is a membrane-associated protein decreasing surface expression of CD4, CD28, and major histocompatibility complex class I on infected cells. We report that Nef strongly down-modulates surface expression of the beta-chain of the CD8alphabeta receptor by accelerated endocytosis, while CD8 alpha-chain expression is less affected. By mutational analysis of the cytoplasmic tail of the CD8 beta-chain, an FMK amino acid motif was shown to be critical for Nef-induced endocytosis. Although independent of CD4, endocytosis of the CD8 beta-chain was abrogated by the same mutations in Nef that affect CD4 down-regulation, suggesting common molecular interactions. The ability to down-regulate the human CD8 beta-chain was conserved in HIV-1, HIV-2, and simian immunodeficiency virus SIVmac239 Nef and required an intact AP-2 complex. The Nef-mediated internalization of receptors, such as CD4, major histocompatibility complex class I, CD28, and CD8alphabeta, may contribute to the subversion of the host immune system and progression towards AIDS.     

Family Planning Knowledge,Attitude and Practice among Married Couples in Jimma Zone,Ethiopia

Background

Understanding why people do not use family planning is critical to address unmet needs and to increase contraceptive use. According to the Ethiopian Demographic and Health Survey 2011, most women and men had knowledge on some family planning methods but only about 29% of married women were using contraceptives. 20% women had an unmet need for family planning. We examined knowledge, attitudes and contraceptive practice as well as factors related to contraceptive use in Jimma zone, Ethiopia.

Methods

Data were collected from March to May 2010 among 854 married couples using a multi-stage sampling design. Quantitative data based on semi-structured questionnaires was triangulated with qualitative data collected during focus group discussions. We compared proportions and performed logistic regression analysis.

Result

The concept of family planning was well known in the studied population. Sex-stratified analysis showed pills and injectables were commonly known by both sexes, while long-term contraceptive methods were better known by women, and traditional methods as well as emergency contraception by men. Formal education was the most important factor associated with better knowledge about contraceptive methods (aOR = 2.07, p<0.001), in particular among women (aOR_women = 2.77 vs. aOR_men = 1.49; p<0.001). In general only 4 out of 811 men ever used contraception, while 64% and 43% females ever used and were currently using contraception respectively.

Conclusion

The high knowledge on contraceptives did not match with the high contraceptive practice in the study area. The study demonstrates that mere physical access (proximity to clinics for family planning) and awareness of contraceptives are not sufficient to ensure that contraceptive needs are met. Thus, projects aiming at increasing contraceptive use should contemplate and establish better counseling about contraceptive side effects and method switch. Furthermore in all family planning activities both wives'' and husbands'' participation should be considered.     

CC2D2A is mutated in Joubert syndrome and interacts with the ciliopathy-associated basal body protein CEP290

Joubert syndrome and related disorders (JSRD) are primarily autosomal-recessive conditions characterized by hypotonia, ataxia, abnormal eye movements, and intellectual disability with a distinctive mid-hindbrain malformation. Variable features include retinal dystrophy, cystic kidney disease, and liver fibrosis. JSRD are included in the rapidly expanding group of disorders called ciliopathies, because all six gene products implicated in JSRD (NPHP1, AHI1, CEP290, RPGRIP1L, TMEM67, and ARL13B) function in the primary cilium/basal body organelle. By using homozygosity mapping in consanguineous families, we identify loss-of-function mutations in CC2D2A in JSRD patients with and without retinal, kidney, and liver disease. CC2D2A is expressed in all fetal and adult tissues tested. In ciliated cells, we observe localization of recombinant CC2D2A at the basal body and colocalization with CEP290, whose cognate gene is mutated in multiple hereditary ciliopathies. In addition, the proteins can physically interact in vitro, as shown by yeast two-hybrid and GST pull-down experiments. A nonsense mutation in the zebrafish CC2D2A ortholog (sentinel) results in pronephric cysts, a hallmark of ciliary dysfunction analogous to human cystic kidney disease. Knockdown of cep290 function in sentinel fish results in a synergistic pronephric cyst phenotype, revealing a genetic interaction between CC2D2A and CEP290 and implicating CC2D2A in cilium/basal body function. These observations extend the genetic spectrum of JSRD and provide a model system for studying extragenic modifiers in JSRD and other ciliopathies.     

Quantitative analysis of DNA hybridization in a flowthrough microarray for molecular testing

Quantitative information about the nucleic acids hybridization reaction on microarrays is fundamental to designing optimized assays for molecular diagnostics. This study presents the kinetic, equilibrium, and thermodynamic analyses of DNA hybridization in a microarray system designed for fast molecular testing of pathogenic bacteria. Our microarray setup uses a porous, nylon membrane for probe immobilization and flowthrough incubation. The Langmuir model was used to determine the reaction rate constants of hybridization with antisense targets specific to Staphylococcus epidermidis and Staphylococcus aureus strains. The kinetic analysis revealed a sequence-dependent reaction rate, with association rate constants on the order of 10⁵ M⁻¹ s⁻¹ and dissociation rate constants of 10⁻⁴ s⁻¹. We found that by increasing the probe surface density from 10¹¹ to 10¹² molecules/cm², the hybridization rate and efficiency are suppressed while the melting temperature of the DNA duplex increases. The maximum fraction of hybridized capture probes at equilibrium did not exceed 50% for hybridization with antisense sequences and was below 6% for hybridization with long targets obtained from PCR. The van’t Hoff analysis of the temperature denaturation data showed that the DNA hybridization in our porous, flowthrough microarray is thermodynamically less favorable than the hybridization of the same sequences in solution.     

Neurofibromin is actively transported to the nucleus

Mutations in the neurofibromatosis type 1 (NF1) tumor suppressor gene predispose individuals to a variety of benign and malignant tumors. Many tumor suppressors ‘shuttle’ between the nucleus and the cytoplasm, thus regulating their function. By expressing different NF1 constructs in COS-7 cells (encompassing exons 28–49 and fused to the green fluorescent protein), we identified a functional nuclear localization signal (NLS) in exon 43. Mutation of the NLS completely abolishes the nuclear entry of the NF1-derivative fusion protein. A highly expressed splice variant that lacks this NLS controls the localization and hence the function of neurofibromin. The localization of neurofibromin in the nucleus may provide novel clues to unknown functions for NF1.     

Biodistribution of [77Br]-5-Bromo-2-Deoxyuridine in Rats

Abstract

The feasibility of 5-bromo-2′-deoxyuridine as tumour imaging agent for PET studies in comparison with thymidine is discussed.     

Cognitive deficits and increases in creatine precursors in a brain‐specific knockout of the creatine transporter gene Slc6a8

Creatine transporter (CrT; SLC6A8) deficiency (CTD) is an X‐linked disorder characterized by severe cognitive deficits, impairments in language and an absence of brain creatine (Cr). In a previous study, we generated floxed Slc6a8 (Slc6a8 ^flox) mice to create ubiquitous Slc6a8 knockout (Slc6a8^?/y) mice. Slc6a8^?/y mice lacked whole body Cr and exhibited cognitive deficits. While Slc6a8^?/y mice have a similar biochemical phenotype to CTD patients, they also showed a reduction in size and reductions in swim speed that may have contributed to the observed deficits. To address this, we created brain‐specific Slc6a8 knockout (bKO) mice by crossing Slc6a8^flox mice with Nestin‐cre mice. bKO mice had reduced cerebral Cr levels while maintaining normal Cr levels in peripheral tissue. Interestingly, brain concentrations of the Cr synthesis precursor guanidinoacetic acid were increased in bKO mice. bKO mice had longer latencies and path lengths in the Morris water maze, without reductions in swim speed. In accordance with data from Slc6a8 ^?/y mice, bKO mice showed deficits in novel object recognition as well as contextual and cued fear conditioning. bKO mice were also hyperactive, in contrast with data from the Slc6a8 ^?/y mice. The results show that the loss of cerebral Cr is responsible for the learning and memory deficits seen in ubiquitous Slc6a8^?/y mice.     

Molecular Evidence for Lessepsian Invasion of Soritids (Larger Symbiont Bearing Benthic Foraminifera)

The Mediterranean Sea is considered as one of the hotspots of marine bioinvasions, largely due to the influx of tropical species migrating through the Suez Canal, so-called Lessepsian migrants. Several cases of Lessepsian migration have been documented recently, however, little is known about the ecological characteristics of the migrating species and their aptitude to colonize the new areas. This study focused on Red Sea soritids, larger symbiont-bearing benthic foraminifera (LBF) that are indicative of tropical and subtropical environments and were recently found in the Israeli coast of the Eastern Mediterranean. We combined molecular phylogenetic analyses of soritids and their algal symbionts as well as network analysis of Sorites orbiculus Forskål to compare populations from the Gulf of Elat (northern Red Sea) and from a known hotspot in Shikmona (northern Israel) that consists of a single population of S. orbiculus. Our phylogenetic analyses show that all specimens found in Shikmona are genetically identical to a population of S. orbiculus living on a similar shallow water pebbles habitat in the Gulf of Elat. Our analyses also show that the symbionts found in Shikmona and Elat soritids belong to the Symbiodinium clade F5, which is common in the Red Sea and also present in the Indian Ocean and Caribbean Sea. Our study therefore provides the first genetic and ecological evidences that indicate that modern population of soritids found on the Mediterranean coast of Israel is probably Lessepsian, and is less likely the descendant of a native ancient Mediterranean species.     

Chromatography and Characterization of Phenylglycidyl Ether Nucleoside Adducts

Abstract

The HPLC separation and structure elucidation (UV, NMR, MS) of the nucleoside adducts formed between 2′—deoxyadenosine, thymidine, 2′—deoxycytidine and 2′—deoxyguanosine with phenylglycidyl ether is described.