Remarkably Low KIR and HLA Diversity in Amerindians Reveals Signatures of Strong Purifying Selection Shaping the Centromeric KIR Region

The killer-cell immunoglobulin-like receptors (KIR) recognize human leukocyte antigen (HLA) molecules to regulate the cytotoxic and inflammatory responses of natural killer cells. KIR genes are encoded by a rapidly evolving gene family on chromosome 19 and present an unusual variation of presence and absence of genes and high allelic diversity. Although many studies have associated KIR polymorphism with susceptibility to several diseases over the last decades, the high-resolution allele-level haplotypes have only recently started to be described in populations. Here, we use a highly innovative custom next-generation sequencing method that provides a state-of-art characterization of KIR and HLA diversity in 706 individuals from eight unique South American populations: five Amerindian populations from Brazil (three Guarani and two Kaingang); one Amerindian population from Paraguay (Aché); and two urban populations from Southern Brazil (European and Japanese descendants from Curitiba). For the first time, we describe complete high-resolution KIR haplotypes in South American populations, exploring copy number, linkage disequilibrium, and KIR–HLA interactions. We show that all Amerindians analyzed to date exhibit the lowest numbers of KIR–HLA interactions among all described worldwide populations, and that 83–97% of their KIR–HLA interactions rely on a few HLA-C molecules. Using multiple approaches, we found signatures of strong purifying selection on the KIR centromeric region, which codes for the strongest NK cell educator receptors, possibly driven by the limited HLA diversity in these populations. Our study expands the current knowledge of KIR genetic diversity in populations to understand KIR–HLA coevolution and its impact on human health and survival.     

Rice Resistance to Brown Spot Mediated by Nitrogen and Potassium

This study was undertaken to investigate the effects of both nitrogen (N) and potassium (K) rates on rice resistance to brown spot, caused by the fungus Bipolaris oryzae. Rice plants (cultivar ‘Metica 1’) were grown in soil corrected with 0, 25, 50, 75 and 100 mg of N / kg (as NH₄NO₃) of soil as well as with 25, 50, 75, 125 and 150 mg of K / kg (as KCl) of soil. Thirty‐three‐day‐old plants were inoculated with a suspension of Bipolaris oryzae conidia and the incubation period (IP), number of lesions (NL) per cm² of leaf area and disease severity was evaluated. Disease severity was scored at 24, 48, 72, 96, 120 and 144 h after inoculation and data were used to obtain the area under brown spot progress curve (AUBSPC). Soil plant analysis development (SPAD) index, plant dry weight and concentration of N and K in leaf tissues were also determined for both non‐inoculated (NI) and inoculated (IN) plants. Concentration of N in leaf tissue increased as the N rates in the soil increased. Concentration of K in leaf tissue increased sharply as the K rates in the soil increased for both NI and IN plants. Concentration of K in leaf tissue was not affected by N rates. The IP increased as the N rates increased, but was somewhat less impacted by increasing K rates. The NL decreased as the N rates increased. The NL dramatically declined at the highest K rates. The AUBSPC dramatically declined as the N and K rates in the soil increased. SPAD index values increased as the N and K rates in the soil increased for both NI and IN plants. Plant dry weight increased as the N and K rates in the soil increased for both NI and IN plants. Results from this study suggest that combining high N and K rates may contribute to reducing the intensity of brown spot in rice while improving plant development.     

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.     

DNA methylation by tert-butyl hydroperoxide-iron (II): a role for the transition metal ion in the production of DNA base adducts.

Metabolic degradation of both endogenous and exogenous peroxides is associated with the etiology of several diseases including cancer. Tert-butyl hydroperoxide (TBHP) has been widely employed as a model compound to study the cytotoxicity and promoting effects of organic peroxides. Recently, we reported that incubations of TBHP with iron (II) and calf thymus DNA led to generation of high yields of methyl radicals and to DNA methylation. Interestingly, DNA was methylated to products expected from both free radical and ionic mechanisms such as 8-methylguanine (C8-MeGua) and 7-methylguanine (N7-MeGua), respectively. To elucidate the mechanisms by which methyl radicals can produce different types of DNA adducts, we examined the effects of transition metal ions (iron (II), iron (III) and copper (I)) and metal ion chelators (ethylenediamine-N,N,N",N"-tetraacetate (EDTA) and desferal) on the nature and the yields of the DNA adducts produced during TBHP decomposition. The results led us to propose that a direct methyl radical attack on DNA guanine residues produces C8-MeGua whereas N7-MeGua and 3-methyladenine (N3-MeAde) are likely to be produced by attack of nucleophilic DNA centers on methyl radical generated in situ by the assistance of transition metal ions bound to DNA.     

Extraocular photoreception and circadian entrainment in nonmammalian vertebrates

In mammals both the regulation of circadian rhythms and photoperiodic responses depend exclusively upon photic information provided by the lateral eyes; however, nonmammalian vertebrates can also rely on multiple extraocular photoreceptors to perform the same tasks. Extraocular photoreceptors include deep brain photoreceptors located in several distinct brain sites and the pineal complex, involving intracranial (pineal and parapineal) and extracranial (frontal organ and parietal eye) components. This review updates the research field of the most recent acquisitions concerning the roles of extraocular photoreceptors on circadian physiology and behavior, particularly photic entrainment and sun compass orientation.     

In silico analysis of nonribosomal peptide synthetases of Xanthomonas axonopodis pv. citri: identification of putative siderophore and lipopeptide biosynthetic genes

The genomes of the plant pathogens Xanthomonas axonopodis (Xac) and Xanthomonas campestris (Xcc) were analysed with the aim of deducing their ability to produce nonribosomal peptides. Nonribosomal peptide synthetase (NRPS) genes were identified in two separate loci of Xac. While the genes of locus 1 are common to both strains, locus 2 was only found in Xac. Dissection and phylogenetic analysis of the condensation and thioesterase domains of the NRPSs of loci 1 and 2 of Xac revealed homology, respectively, with siderophore and lipopeptide synthetases. Further analysis of locus 1 revealed genes related to polyketide and polyamine biosynthesis that could be involved in the assembly of substrates for siderophore biosynthesis in both strains. In vitro production of siderophores by both Xac and Xcc was confirmed. Since bacterial siderophores and lipopeptides can be pathogenic and are typically produced nonribosomally, these results suggest that the identified genes could be involved in phytotoxin production.     

Distribution of Chitinases in the Entomopathogen <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis> and Effect of <Emphasis Type="Italic">N</Emphasis>-Acetylglucosamine in Protein Secretion

For a long time, fungi have been characterized by their ability to secrete enzymes, mostly hydrolytic in function, and thus are defined as extracellular degraders. Chitin and chitinolytic enzymes are gaining importance for their biotechnological applications. Particularly, chitinases are used in agriculture to control plant pathogens. Metarhizium anisopliae produces an extracellular chitinase when grown on a medium containing chitin, indicating that synthesis is subject to induction by the substrate. Various sugar combinations were investigated for induction and repression of chitinase. N-acetylglucosamine (GlcNAc) shows a special dual regulation on chitinase production. M. anisopliae has at least two distinct, cell-bound, chitinolytic enzymes when cultured with GlcNAc as one of the carbon sources, and we suggest that this carbohydrate has an important role in protein secretion.     

The '3Rs' model and the concept of alternatives in animal research: a questionnaire survey

It has been 45 years since Russell and Burch first proposed the concept of the '3Rs', yet it remains unclear how those individuals involved in animal research view and implement these concepts. The authors used a questionnaire survey to determine how well-known experts judged issues related to the 3Rs.     

A methylcellulose microculture assay for the in vitro assessment of drug toxicity on granulocyte/macrophage progenitors (CFU-GM)

In a recent prevalidation study, the use of a methylcellulose colony-forming unit-granulocyte/macrophage (CFU-GM) macroassay for two independent in vitro tests (human and murine cell based) was suggested for quantifying the potential haematotoxicity of xenobiotics. In this paper, we describe the transfer of the macroassay to a 96-well plate microassay, in which the linearity of the response was studied (both in terms of CFU-GM and optical density [OD] versus the number of cells cultured), and the inhibitory concentration (IC) values for doxorubicin, 5-fluorouracil and taxol were determined and compared with those obtained by using the original macroassay. Fresh murine bone marrow and human umbilical cord blood mononuclear cells were used as a source of myeloid progenitors. The cells were cultured in methylcellulose containing granulocyte/macrophage-colony-stimulating factor, and in the presence of increasing drug concentrations. The cloning capacity of the progenitors was measured both as the number of colonies counted manually (CFU-GM), and as OD evaluated with an automated plate reader in an MTT test. Our results show that, in the microassay, up to 20 colonies/well could be easily counted, and that this range (20 to zero) gave a regression line from which IC values were calculated, which were very close to those obtained by using the macroassay (where the range of colony numbers was from 100 to zero). The test did not give good results when the OD (instead of the colony count) was used as the endpoint, because, although a high coefficient of determination was obtained, the OD values ranged from 0.6 to zero and the IC values determined were not comparable to those obtained by manual counts. The use of the microassay dramatically reduces the quantity of methylcellulose needed, and permits hundreds of cultures to be processed in the same experiment, contributing to significant reductions in both the work involved and the cost. A further important benefit is a reduction of the amount of drug needed for testing, which is crucial for screening new molecules, when many different toxicological tests have to be carried out. The microassay is therefore a useful and reproducible tool for screening compounds (chemicals, drugs and xenobiotics) for potential haematotoxicity directly on human myeloid progenitors, and could contribute significantly to reducing the use of animals in toxicity testing.     

Vascular endothelial growth factor inhibits programmed cell death of endothelial cells induced by clinorotation

The principal aim of this study was to investigate short- and long-term effects of clinorotation on human endothelial cells (EA hy 926 cell line) using a three-dimensional random positioning machine. Moreover, the impact of vascular endothelial growth factor (VEGF) was addressed. Immediately, within one hour and after four and twenty-four hours an increase of apoptotic cells was detected. VEGF significantly inhibited the amount of apoptotic endothelial cells (EC). VEGF reduced the amount of fas-positive EC. Moreover, after 24 hours, proliferating EC grew in form of three-dimensional multicellular spheroids and also as monolayers. The initially formed spheroids (maximum diameter 3 mm) remained stable up to the 15th day of clinorotation. Some spheroids revealed tubular structures. In addition, a clear increase of extracellular matrix proteins such as osteopontin and fibronectin was measured. The three-dimensional clinostat represents an important tool for cell biological experiments. VEGF significantly attenuated the changes of endothelial cells induced by simulated weightlessness in a cell protective manner.