Carbohydrate receptor-mediated gene transfer to human T leukaemic cells

The mucin-type carbohydrate Tn cryptantigen (GalNAc1-O-Ser/Thr,where GalNAc is N-acetyl-D-galactosamine) is expressed in manycarcinomas, in haemopoietic disorders including the Tn syndrome,and on human immunodeficiency virus (HIV) coat glycoproteins,but is not expressed on normal, differentiated cells becauseof the expression of a Tn-processing galactosyltransferase.Using Jurkat T leukaemic cells which express high levels ofTn antigen due to deficient Tn galactosylation, we have establishedthe Tn antigen-mediated gene transfer and demonstrate the considerableefficiency of this approach. We used poly(L-lysine) conjugatesof the monoclonal antibody 1E3 directed against the Tn antigento deliver the luciferase and ß-galactosidase reportergenes to Jurkat cells by receptor-mediated endocytosis. Additionof unconjugated 1E3 reduced transfection efficiency in a concentration-dependentmanner and incubation with free GalNAc abolished DNA transfercompletely, indicating that gene delivery is indeed mediatedby the Tn antigen. Pre-treatment of Jurkat cells with Vibriocholerae sialidase, which uncovers additional Tn antigens, resultedin an improvement of gene transfection. Both human and chickenadenovirus particles attached to the DNA/polylysine complexstrongly augmented transgene expression. When the ß-galactosidase(lacZ) gene was delivered to Jurkat cells by Tn-mediated endocytosis,up to 60% of the cells were positive in the cytochemical stainusing 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside(X-gal) as a chromogenic substrate. The efficiency of the transferrinreceptor-mediated DNA uptake into Jurkat cells was comparativelylow, although these cells were shown to express considerableamounts of transferrin receptor. We show here that a mucin-typecarbohydrate antigen mediates highly efficient DNA uptake byendocytosis into Jurkat T cells. This method represents a 50-foldimprovement of Jurkat cell transfection efficiency over otherphysical gene transfer techniques. Specific gene delivery toprimary cancer cells exhibiting Tn epitopes may especially bedesirable in immunotherapy protocols. adenovirus endocytosis gene transfer T cell Tn antigen     

Vascular redox imbalance in rats submitted to chronic exercise

Exercise training has been used for treatment/prevention of many cardiovascular diseases, but the mechanisms need to be clarified. Thus, our aim was to compare oxidative stress parameters between rats submitted to a swimming training and sedentary rats (control). Twelve male rats were divided into two groups: control and exercise training. The exercise training had daily 1 h swimming sessions for 8 weeks and a load (5% of its body mass) was placed in rat's tail. Thereafter the animals were killed, aorta and heart were surgically removed and blood was collected. Body mass gain, thiobarbituric acid reactive species (TBARS), carbonyl content, total reactive antioxidant potential (TRAP), total antioxidant reactivity (TAR), superoxide dismutase (SOD) activity and catalase (CAT) activity were evaluted. The trained rats showed a lower body mass gain and no modifications on heart. An increased SOD activity was observed on aorta after the training, but no changes were seen for CAT activity, which led to an increased SOD/CAT ratio. The arterial TBARS was also increased for trained rats. The decrease in TRAP in exercise training was the single modification on plasma. Our findings suggest that the increased SOD activity could play a role in vascular adaptations to exercise training. Copyright © 2010 John Wiley & Sons, Ltd.     

Prevalence and seasonal variations of six bee viruses in Apis mellifera L. and Varroa destructor mite populations in France

A survey of six bee viruses on a large geographic scale was undertaken by using seemingly healthy bee colonies and the PCR technique. Samples of adult bees and pupae were collected from 36 apiaries in the spring, summer, and autumn during 2002. Varroa destructor samples were collected at the end of summer following acaricide treatment. In adult bees, during the year deformed wing virus (DWV) was found at least once in 97% of the apiaries, sacbrood virus (SBV) was found in 86% of the apiaries, chronic bee paralysis virus (CBPV) was found in 28% of the apiaries, acute bee paralysis virus (ABPV) was found in 58% of the apiaries, black queen cell virus (BQCV) was found in 86% of the apiaries, and Kashmir bee virus (KBV) was found in 17% of the apiaries. For pupae, the following frequencies were obtained: DWV, 94% of the apiaries; SBV, 80% of the apiaries; CBPV, none of the apiaries; ABPV, 23% of the apiaries; BQCV, 23% of the apiaries; and KBV, 6% of the apiaries. In Varroa samples, the following four viruses were identified: DWV (100% of the apiaries), SBV (45% of the apiaries), ABPV (36% of the apiaries), and KBV (5% of the apiaries). The latter findings support the putative role of mites in transmitting these viruses. Taken together, these data indicate that bee virus infections occur persistently in bee populations despite the lack of clinical signs, suggesting that colony disease outbreaks might result from environmental factors that lead to activation of viral replication in bees.     

Repeated horizontal gene transfer of GALactose metabolism genes violates Dollo’s law of irreversible loss

Dollo’s law posits that evolutionary losses are irreversible, thereby narrowing the potential paths of evolutionary change. While phenotypic reversals to ancestral states have been observed, little is known about their underlying genetic causes. The genomes of budding yeasts have been shaped by extensive reductive evolution, such as reduced genome sizes and the losses of metabolic capabilities. However, the extent and mechanisms of trait reacquisition after gene loss in yeasts have not been thoroughly studied. Here, through phylogenomic analyses, we reconstructed the evolutionary history of the yeast galactose utilization pathway and observed widespread and repeated losses of the ability to utilize galactose, which occurred concurrently with the losses of GALactose (GAL) utilization genes. Unexpectedly, we detected multiple galactose-utilizing lineages that were deeply embedded within clades that underwent ancient losses of galactose utilization. We show that at least two, and possibly three, lineages reacquired the GAL pathway via yeast-to-yeast horizontal gene transfer. Our results show how trait reacquisition can occur tens of millions of years after an initial loss via horizontal gene transfer from distant relatives. These findings demonstrate that the losses of complex traits and even whole pathways are not always evolutionary dead-ends, highlighting how reversals to ancestral states can occur.     

Countrywide Survey for MERS-Coronavirus Antibodies in Dromedaries and Humans in Pakistan

Middle East Respiratory Syndrome Coronavirus (MERS-CoV) is a zoonotic pathogen capable of causing severe respiratory disease in humans. Although dromedary camels are considered as a major reservoir host, the MERS-CoV infection dynamics in camels are not fully understood. Through surveillance in Pakistan, nasal (n = 776) and serum (n = 1050)samples were collected from camels between November 2015 and February 2018. Samples were collected from animal markets, free-roaming herds and abattoirs. An in-house ELISA was developed to detect IgG against MERS-CoV. A total of 794 camels were found seropositive for MERS-CoV. Prevalence increased with the age and the highest seroprevalence was recorded in camels aged [ 10 years (81.37%) followed by those aged 3.1–10 years (78.65%) and B 3 years (58.19%).Higher prevalence was observed in female (78.13%) as compared to male (70.70%). Of the camel nasal swabs, 22 were found to be positive by RT-qPCR though with high Ct values. Moreover, 2,409 human serum samples were also collected from four provinces of Pakistan during 2016–2017. Among the sampled population, 840 humans were camel herders.Although we found a high rate of MERS-CoV antibody positive dromedaries (75.62%) in Pakistan, no neutralizing antibodies were detected in humans with and without contact to camels.     

High-Resolution Genetic Map of the Human Glutamyl Aminopeptidase Gene (ENPEP)

The murine B-lymphocyte differentiation antigen BP-1/6C3 has been identified as glutamyl aminopeptidase (EAP), the gene symbol for which isENPEP.Using genomic DNA encoding for human EAP as a probe, we identified theENPEPgene location on human chromosome 4q25 by polymerase chain reaction analysis of a human/rodent somatic cell hybrid mapping panel and by fluorescencein situhybridization. Using a radiation hybrid panel, the gene order aroundENPEPwas determined to be centromere–D4S1236–(570 kb)–ENPEP–(210 kb)–D4S262–(270 kb)–D4S953–(270 kb)–D4S474–(570 kb)–IF. The linkage ofENPEPto complement factor I (IF) confirms the human chromosome band 4q25 localization predicted from the chromosomal location of murineENPEP.HumanENPEPthus provides an additional marker for the long arm of chromosome 4 that should facilitate studies of this genomic region.     

Tests of candidate genes in breed cross populations for QTL mapping in livestock

In recent years, several F₂ crosses between outbred lines of livestock have been developed to identify quantitative trait loci (QTL). These populations are valuable for further genetic analysis, including positional candidate gene loci (CGL). Analysis of CGL in F₂ populations is, however, hindered by extensive between-breed linkage disequilibrium (LD). The objectives here were to develop and evaluate three tests for CGL in simulated F₂ breed-cross populations. 1) A standard association test, based on the fixed effect of CGL genotype. This test was significant for CGL at considerable distances from the QTL. 2) A marker-assisted association test, based on a test at the CGL of the fixed effect of CGL genotype in a breed-cross QTL interval mapping model. This removed the impact of between-breed LD, but was not powerful in detecting CGL closely linked to the QTL, unless the CGL was the QTL. 3) An F-drop test, comparing F ratios for a QTL at the CGL with and without the CGL included as fixed effect. It had low power to distinguish close from distant CGL. Power to distinguish two CGL within 10 cM from the QTL was limited and little improved by including QTL effects associated with markers to remove between-breed LD, although the power was greater when one of the CGL was the causative mutation. Therefore, while we conclude that candidate gene tests in QTL mapping populations must be interpreted with caution, we now have a clearer picture of the value of candidate gene tests in these populations.