Mechanisms of haplotype divergence at the <Emphasis Type="Italic">RGA08</Emphasis> nucleotide-binding leucine-rich repeat gene locus in wild banana <Emphasis Type="Italic">(Musa balbisiana)</Emphasis>

Background  

Comparative sequence analysis of complex loci such as resistance gene analog clusters allows estimating the degree of sequence conservation and mechanisms of divergence at the intraspecies level. In banana (Musa sp.), two diploid wild species Musa acuminata (A genome) and Musa balbisiana (B genome) contribute to the polyploid genome of many cultivars. The M. balbisiana species is associated with vigour and tolerance to pests and disease and little is known on the genome structure and haplotype diversity within this species. Here, we compare two genomic sequences of 253 and 223 kb corresponding to two haplotypes of the RGA08 resistance gene analog locus in M. balbisiana "Pisang Klutuk Wulung" (PKW).     

Post-translational amino acid racemization in the frog skin peptide deltorphin I in the secretion granules of cutaneous serous glands

The dermal glands of the South American hylid frog Phyllomedusa bicolor synthesize and expel huge amounts of cationic, alpha-helical, 24- to 33-residue antimicrobial peptides, the dermaseptins B. These glands also produce a wide array of peptides that are similar to mammalian hormones and neuropeptides, including a heptapeptide opioid containing a D-amino acid, deltorphin I (Tyr-DAla-Phe-Asp-Val-Val-Gly NH2). Its biological activity is due to the racemization of L-Ala2 to D-Ala. The dermaseptins B and deltorphins are all derived from a single family of precursor polypeptides that have an N-terminal preprosequence that is remarkably well conserved, although the progenitor sequences giving rise to mature opioid or antimicrobial peptides are markedly different. Monoclonal and polyclonal antibodies were used to examine the cellular and ultrastructural distributions of deltorphin I and dermaseptin B in the serous glands by immunofluoresence confocal microscopy and immunogold-electron microscopy. Preprodeltorphin I and preprodermaseptins B are sorted into the regulated pathway of secretion, where they are processed to give the mature products. Deltorphin I, [l-Ala2]-deltorphin I and dermaseptin B are all stored together in secretion granules which accumulate in the cytoplasm of all serous glands. We conclude that the L- to D-amino acid isomerization of the deltorphin I occurs in the secretory granules as a post-translational event. Thus the specificity of isomerization depends on the presence of structural and/or conformational determinants in the peptide N-terminus surrounding the isomerization site.     

Rapid generation of new powdery mildew resistance genes after wheat domestication

Plant defence against pathogens is controlled by disease resistance (R) gene products that directly or indirectly detect specific pathogen effectors. Plant-pathogen interactions have been proposed to follow a co-evolutionary arms-race model where R genes are recent and evolve rapidly in response to structural changes in matching pathogen effectors. However, the longevity and extensive polymorphism of R genes studied were more consistent with balancing selection maintaining ancient and diverse R genes or alleles. In bread wheat (Triticum aestivum), the Pm3 locus confers race-specific resistance to wheat powdery mildew (Blumeria graminis f.sp. triticii). Here we describe recently generated Pm3 resistance alleles that all derive from one susceptible allele, Pm3CS, which is widespread among hexaploid bread-wheat lines. One group of four Pm3 resistance alleles shows few, clearly delimited, polymorphic sequence blocks of ancient origin, embedded in sequences identical to Pm3CS and possibly derived from gene conversion. A second group of three alleles differs from Pm3CS by only two to five mutations, all non-synonymous, and all in the leucine-rich repeat-encoding region. Transient transformation experiments confirmed that Pm3 resistance specificities are based on one or few amino acid changes. The Pm3CS allele was found in wild tetraploid wheat, the ancestor of hexaploid bread wheat, specifically from southern Turkey, a region proposed to be the site of wheat domestication. Based on these data, we propose that the Pm3 resistance alleles were generated in agricultural ecosystems after domestication of wheat 10,000 years ago. The evolution of Pm3 alleles in wheat is best described by the model of evolved recycling, where novel genetic variation is integrated in plant populations together with recycling of old variation.     

Regulatory T Cells Negatively Affect IL-2 Production of Effector T Cells through CD39/Adenosine Pathway in HIV Infection

The mechanisms by which Regulatory T cells suppress IL-2 production of effector CD4+ T cells in pathological conditions are unclear. A subpopulation of human Treg expresses the ectoenzyme CD39, which in association with CD73 converts ATP/ADP/AMP to adenosine. We show here that Treg/CD39+ suppress IL-2 expression of activated CD4+ T-cells more efficiently than Treg/CD39−. This inhibition is due to the demethylation of an essential CpG site of the il-2 gene promoter, which was reversed by an anti-CD39 mAb. By recapitulating the events downstream CD39/adenosine receptor (A2AR) axis, we show that A2AR agonist and soluble cAMP inhibit CpG site demethylation of the il-2 gene promoter. A high frequency of Treg/CD39+ is associated with a low clinical outcome in HIV infection. We show here that CD4+ T-cells from HIV-1 infected individuals express high levels of A2AR and intracellular cAMP. Following in vitro stimulation, these cells exhibit a lower degree of demethylation of il-2 gene promoter associated with a lower expression of IL-2, compared to healthy individuals. These results extend previous data on the role of Treg in HIV infection by filling the gap between expansion of Treg/CD39+ in HIV infection and the suppression of CD4+ T-cell function through inhibition of IL-2 production.     

Carbonic anhydrase inhibitors: Benzenesulfonamides incorporating cyanoacrylamide moieties are low nanomolar/subnanomolar inhibitors of the tumor-associated isoforms IX and XII

A series of benzenesulfonamides incorporating cyanoacrylamide moieties (tyrphostine analogues) have been obtained by reaction of sulfanilamide with ethylcyanoacetate followed by condensation with aromatic/heterocyclic aldehydes, isothiocyanates or diazonium salts. The new compounds have been investigated as inhibitors of the metalloenzyme carbonic anhydrase (CA, EC 4. 2.1.1), and more specifically against the cytosolic human (h) isoforms hCA I and II, as well as the transmembrane, tumor-associated ones CA IX and XII, which are validated antitumor targets. Most of the new benzenesulfonamides were low nanomolar or subnanomolar CA IX/XII inhibitors whereas they were less effective as inhibitors of CA I and II. The structure–activity relationship for this class of effective CA inhibitors is also discussed. Generally, electron donating groups in the starting aldehyde reagent favored CA IX and XII inhibition, whereas halogeno, methoxy and dimethylamino moieties led to very potent CA XII inhibitors.     

The Mycobacterium tuberculosis FAS-II dehydratases and methyltransferases define the specificity of the mycolic acid elongation complexes

Background

The human pathogen Mycobacterium tuberculosis (Mtb) has the originality of possessing a multifunctional mega-enzyme FAS-I (Fatty Acid Synthase-I), together with a multi-protein FAS-II system, to carry out the biosynthesis of common and of specific long chain fatty acids: the mycolic acids (MA). MA are the main constituents of the external mycomembrane that represents a tight permeability barrier involved in the pathogenicity of Mtb. The MA biosynthesis pathway is essential and contains targets for efficient antibiotics. We have demonstrated previously that proteins of FAS-II interact specifically to form specialized and interconnected complexes. This finding suggested that the organization of FAS-II resemble to the architecture of multifunctional mega-enzyme like the mammalian mFAS-I, which is devoted to the fatty acid biosynthesis.

Principal Findings

Based on conventional and reliable studies using yeast-two hybrid, yeast-three-hybrid and in vitro Co-immunoprecipitation, we completed here the analysis of the composition and architecture of the interactome between the known components of the Mtb FAS-II complexes. We showed that the recently identified dehydratases HadAB and HadBC are part of the FAS-II elongation complexes and may represent a specific link between the core of FAS-II and the condensing enzymes of the system. By testing four additional methyltransferases involved in the biosynthesis of mycolic acids, we demonstrated that they display specific interactions with each type of complexes suggesting their coordinated action during MA elongation.

Significance

These results provide a global update of the architecture and organization of a FAS-II system. The FAS-II system of Mtb is organized in specialized interconnected complexes and the specificity of each elongation complex is given by preferential interactions between condensing enzymes and dehydratase heterodimers. This study will probably allow defining essential and specific interactions that correspond to promising targets for Mtb FAS-II inhibitors.     

Exploring first-year undergraduate medical students' self-directed learning readiness to physiology

Medical students are expected to possess self-directed learning skills to pursue lifelong learning. Previous studies have reported that the readiness for self-directed learning depends on personal attributes as well as the curriculum followed in institutions. Melaka Manipal Medical College of Manipal University (Karnataka, India) offers a Bachelor of Medicine and Bachelor of Surgery (MBBS) twinning program that is of 5 yr in duration. Keeping in mind the amount of time that the curriculum has devoted for self-directed learning, we explored the self-directed learning readiness of first-year MBBS students (n = 130) using a self-directed learning readiness scale (SDLRS) and explored the correlation between SDLRS scores of high achievers, medium achievers, and low achievers with their academic performance in physiology examinations. Students were requested to respond to each item of the SDLRS on a Likert scale. Median scores of the three scales of the SDLRS were compared across the three groups of students using a Kruskall-Wallis test. SDLRS scores of the students (n = 130) were correlated with their marks in theory papers of first, second, and third block-end examinations using Spearmann's correlation coefficient. The mean item score for desire for learning was found to be higher followed by self-control and self-management. Data analyses showed significantly high (P < 0.03) median scores for self-control for high achievers compared with medium and low achievers. Between the groups, high achievers had a higher score for all the three scales of the SDLRS followed by low and medium achievers. SDLRS scores and academic performance of the three groups of students were found to exhibit a weak correlation. This study threw light on the fact that despite having a high desire for learning and ability of self-control, students need to be supported in their self-management skills.     

Isoelectric point (pI)-based phase separation (pI-BPS) purification of elastin-like polypeptides (ELPs) containing charged,biologically active fusion proteins (ELP-FPs)

Elastin-like polypeptides (ELPs) are peptide-based biomaterials with residue sequence (VPGXG)n where X is any residue except proline. ELPs are a useful modality for delivering biologically active proteins (growth factors, protease inhibitors, anti-inflammatory peptides, etc.) as fusion proteins (ELP-FP). ELP-FPs are particularly cost-effective because they can be rapidly purified using Inverse Temperature Cycling (ITC) via the reversible formation and precipitation of entropically driven aggregates above a transition temperature (T_t). When ELP fusion proteins (ELP-FPs) contain significant charge density at physiological pH, electrostatic repulsion between them severely inhibits aggregate formation. The literature does not currently describe methods for purifying ELP-FPs containing charged proteins on either side of the ELP sequence as fusion partners without organic solvents. Here, the isoelectric point (pI) of ELP-FPs is discussed as a means of neutralizing surface charges on ELP-FPs and increasing ITC yield to dramatically high levels. We use pI-based phase separation (pI-BPS) to purify ELP-FPs containing cationic and anionic fusion proteins. We report a dramatic increase in protein yield when using pI-BPS for purification of ELP-FPs. Proteins purified by this method also retain the functional activity of the protein present in the ELP-FP. Techniques developed here enable significant diversification of possible fusion proteins delivered by ELPs as ELP-FPs by allowing them to be produced and purified at higher quantities and yields.     

A genome-wide association study identifies rs2000999 as a strong genetic determinant of circulating haptoglobin levels

Haptoglobin is an acute phase inflammatory marker. Its main function is to bind hemoglobin released from erythrocytes to aid its elimination, and thereby haptoglobin prevents the generation of reactive oxygen species in the blood. Haptoglobin levels have been repeatedly associated with a variety of inflammation-linked infectious and non-infectious diseases, including malaria, tuberculosis, human immunodeficiency virus, hepatitis C, diabetes, carotid atherosclerosis, and acute myocardial infarction. However, a comprehensive genetic assessment of the inter-individual variability of circulating haptoglobin levels has not been conducted so far.We used a genome-wide association study initially conducted in 631 French children followed by a replication in three additional European sample sets and we identified a common single nucleotide polymorphism (SNP), rs2000999 located in the Haptoglobin gene (HP) as a strong genetic predictor of circulating Haptoglobin levels (P(overall) = 8.1 × 10(-59)), explaining 45.4% of its genetic variability (11.8% of Hp global variance). The functional relevance of rs2000999 was further demonstrated by its specific association with HP mRNA levels (β = 0.23 ± 0.08, P = 0.007). Finally, SNP rs2000999 was associated with decreased total and low-density lipoprotein cholesterol in 8,789 European children (P(total cholesterol) = 0.002 and P(LDL) = 0.0008).Given the central position of haptoglobin in many inflammation-related metabolic pathways, the relevance of rs2000999 genotyping when evaluating haptoglobin concentration should be further investigated in order to improve its diagnostic/therapeutic and/or prevention impact.