The mitochondrial solute carrier SLC25A5 at Xq24 is a novel candidate gene for non-syndromic intellectual disability

Loss-of-function mutations in several different neuronal pathways have been related to intellectual disability (ID). Such mutations often are found on the X chromosome in males since they result in functional null alleles. So far, microdeletions at Xq24 reported in males always have been associated with a syndromic form of ID due to the loss of UBE2A. Here, we report on overlapping microdeletions at Xq24 that do not include UBE2A or affect its expression, in patients with non-syndromic ID plus some additional features from three unrelated families. The smallest region of overlap, confirmed by junction sequencing, harbors two members of the mitochondrial solute carrier family 25, SLC25A5 and SLC25A43. However, identification of an intragenic microdeletion including SLC25A43 but not SLC25A5 in a healthy boy excluded a role for SLC25A43 in cognition. Therefore, our findings point to SLC25A5 as a novel gene for non-syndromic ID. This highly conserved gene is expressed ubiquitously with high levels in cortex and hippocampus, and a presumed role in mitochondrial exchange of ADP/ATP. Our data indicate that SLC25A5 is involved in memory formation or establishment, which could add mitochondrial processes to the wide array of pathways that regulate normal cognitive functions.     

Cis-Amide isosteric replacement in thienobenzoxepin inhibitors of PI3-kinase

Substructural class effects surrounding replacement of a ‘cis’ N-methyl aniline amide within potent and selective thienobenzoxepin PI3-kinase inhibitors are disclosed. While a simple aryl to alkyl switch was not tolerated due to differences in preferred amide conformation, heterocyclic amide isosteres with maintained aryl substitution improved potency and metabolic stability at the cost of physical properties. These gains in potency allowed lipophilic deconstruction of the arene to simple branched alkyl substituents. As such, overall lipophilicity-neutral, MW decreases were realized relative to the aniline amide series. The improved properties for lead compound 21 resulted in high permeability, solubility and bioavailability.     

Identification of GNE-293, a potent and selective PI3Kδ inhibitor: Navigating in vitro genotoxicity while improving potency and selectivity

In an effort to identify potent and isoform selective inhibitors of PI3Kδ, GNE-293 (34) was identified. Inhibitor 2 was found to induce micronuclei formation in both the MNT and HCA in vitro assays. Compounds testing negative for genotoxicity were successfully identified through modifications of the 2-benzimidazole substituent and the methylene moiety to disrupt planarity. A variety of heteroatom linkers were explored to examine their effect on potency and isoform selectivity by restricting torsional angles to favor ligand interactions with PI3Kδ’s Trp760. These modifications also resulted in an improved in vivo pharmacokinetic profile.     

Method for milk oligosaccharide profiling by 2-aminobenzamide labeling and hydrophilic interaction chromatography

Although the properties of milk oligosaccharides have been of scientific interest for many years, their structural diversity presents a challenging analytical task. In the quest for a simple and robust technology to characterize the different oligosaccharides present in milk, we developed an analytical scheme based on their fluorescent labeling, pre-fractionation by weak anionic exchange chromatography and separation by hydrophilic interaction liquid chromatography (HILIC)-high performance liquid chromatography (HPLC). HILIC relies on the hydrophilic potential of the molecule, which accounts for differences in properties such as molecular volume, lipophilic surface area, charge, composition, structure, linkage and oligosaccharide branching. The robustness of the methodology has been demonstrated using bovine colostrum oligosaccharides as a case study. Structural assignments for 37 free glycans, including 20 sialylated species, were obtained by a combination of HILIC-HPLC, exoglycosidase digestion and offline negative-ion mode mass spectrometry (MS)/MS. Parameters obtained for each glycan, including linkages, enzymatic digestion products and glucose unit values, will be added to GlycoBase, a public access database (http://glycobase.nibrt.ie/glycobase.html). This approach provides a basis for the analysis of free milk oligosaccharides in a fast and sensitive manner and could be adapted for an automated technology platform amenable to diverse environments. Indeed, our approach, in conjunction with bacterial-binding assays, can provide a better understanding of the structural elements required for biological activity of free milk oligosaccharides and could serve as a scientific basis for the selection of such bioactives from various food sources.     

Regulation of PKC alpha activity by C1-C2 domain interactions

In this study, the role of interdomain interactions involving the C1 and C2 domains in the mechanism of activation of PKC was investigated. Using an in vitro assay containing only purified recombinant proteins and the phorbol ester, 4 beta-12-O-tetradecanoylphorbol-13-acetate (TPA), but lacking lipids, it was found that PKC alpha bound specifically, and with high affinity, to a alpha C1A-C1B fusion protein of the same isozyme. The alpha C1A-C1B domain also potently activated the isozyme in a phorbol ester- and diacylglycerol-dependent manner. The level of this activity was comparable with that resulting from membrane association induced under maximally activating conditions. Furthermore, it was found that alpha C1A-C1B bound to a peptide containing the C2 domain of PKC alpha. The alpha C1A-C1B domain also activated conventional PKC beta I, -beta II, and -gamma isoforms, but not novel PKC delta or -epsilon. PKC delta and -epsilon were each activated by their own C1 domains, whereas PKC alpha, -beta I, -beta II, or -gamma activities were unaffected by the C1 domain of PKC delta and only slightly activated by that of PKC epsilon. PKC zeta activity was unaffected by its own C1 domain and those of the other PKC isozymes. Based on these findings, it is proposed that the activating conformational change in PKC alpha results from the dissociation of intra-molecular interactions between the alpha C1A-C1B domain and the C2 domain. Furthermore, it is shown that PKC alpha forms dimers via inter-molecular interactions between the C1 and C2 domains of two neighboring molecules. These mechanisms may also apply for the activation of the other conventional and novel PKC isozymes.     

Cancer/testis antigens expression and autologous serological response in a set of Brazilian non-Hodgkin’s lymphoma patients

Background

Based on their tumor-associated expression pattern, cancer/testis antigens (CTAs) are considered potential targets for cancer immunotherapy. We aim to evaluate the expression of CTAs in non-Hodgkin??s lymphoma (NHL) samples and the ability of these patients to elicit spontaneous humoral immune response against CTAs.

Methods

Expression of MAGE-A family, CT7/MAGE-C1, CT10/MAGE-C2, GAGE and NY-ESO-1 was analyzed by immunohistochemistry in a tissue microarray generated from 106 NHL archival cases. The humoral response against 19 CTAs was tested in 97 untreated NHL serum samples using ELISA technique.

Results

11.3?% of NHL tumor samples expressed at least 1 CTA. MAGE-A family (6.6?%), GAGE (5.7?%) and NY-ESO-1(4.7?%) were the most frequently expressed antigens. We found no statistically significant correlation between CTA positivity and clinical parameters such as NHL histological subtype, Ann Arbor stage, international prognostic index score, response to treatment and overall survival. Humoral response against at least 1 CTA was observed in 16.5?% of NHL serum samples. However, overall seroreactivity was low, and strong titers (>1:1000) were observed in only two diffuse large B-cell lymphomas patients against CT45.

Conclusion

Our findings are in agreement with most of published studies in this field to date and suggest an overall low expression of CTAs in NHL patients. However, as many new CTAs have been described recently and some of them are found to be highly expressed in NHL cell lines and tumor samples, further studies exploring the expression of different panels of CTAs are needed to evaluate their role as candidates for immunotherapy in NHL patients.     

Genetics of symbiosis in Lotus japonicus: recombinant inbred lines, comparative genetic maps, and map position of 35 symbiotic loci

Development of molecular tools for the analysis of the plant genetic contribution to rhizobial and mycorrhizal symbiosis has provided major advances in our understanding of plant-microbe interactions, and several key symbiotic genes have been identified and characterized. In order to increase the efficiency of genetic analysis in the model legume Lotus japonicus, we present here a selection of improved genetic tools. The two genetic linkage maps previously developed from an interspecific cross between L. japonicus Gifu and L. filicaulis, and an intraspecific cross between the two ecotypes L. japonicus Gifu and L. japonicus MG-20, were aligned through a set of anchor markers. Regions of linkage groups, where genetic resolution is obtained preferentially using one or the other parental combination, are highlighted. Additional genetic resolution and stabilized mapping populations were obtained in recombinant inbred lines derived by a single seed descent from the two populations. For faster mapping of new loci, a selection of reliable markers spread over the chromosome arms provides a common framework for more efficient identification of new alleles and new symbiotic loci among uncharacterized mutant lines. Combining resources from the Lotus community, map positions of a large collection of symbiotic loci are provided together with alleles and closely linked molecular markers. Altogether, this establishes a common genetic resource for Lotus spp. A web-based version will enable this resource to be curated and updated regularly.     

KIR2DL4 (CD158d) genotype influences expression and function in NK cells

The expression and function of the NK cell receptor KIR2DL4 are controversial. Two common alleles of the transmembrane domain of KIR2DL4 exist. The 10A allele with 10 adenines at the end of the transmembrane exon encodes a full length receptor, whereas the 9A allele has only 9 adenines resulting in a frame shift which in turn generates a stop codon early in the first cytoplasmic exon. The possibility that the 10A and 9A alleles might result in differences in expression and function of KIR2DL4 was explored using mAbs to KIR2DL4. Transfection experiments with cDNA from the 10A and 9A alleles revealed significant membrane expression only with the protein encoded by the 10A allele. Analysis of peripheral blood NK cells demonstrated that only in subjects with at least one 10A allele was cell surface expression of KIR2DL4 detectable, and then only on the minor CD56(bright) NK cell subset. The major CD56(dim) NK cell subset did not cell surface express KIR2DL4 but, interestingly, did so after in vitro culture. Functional analysis using cultured NK cells in redirected lysis assays demonstrated that KIR2DL4 is an activating receptor for NK cells with at least one 10A allele. No significant activity was detected for NK cells generated from subjects homozygous for the 9A allele. These data show that genotype influences cell surface expression and function of KIR2DL4 which may account for reported differences in KIR2DL4 expression and function.     

Delayed Cerebroventricular Metabolism of [125I]Angiotensins in the Spontaneously Hypertensive Rat

This study was designed to evaluate the hypothesis that impaired brain angiotensin signal termination contributes to the sustained blood pressure elevations noted in the genetically hypertensive rat model of human essential hypertension. A technique that combined the intracerebroventricular injection of [125I]angiotensins, followed by focused microwave fixation to stop all peptidase activity and subsequent HPLC analyses, was used for determining half-lives of [125I]angiotensin II and [125I]angiotensin III in the ventricular space. The results indicate that the spontaneously hypertensive rat evidenced significantly longer half-lives for intracerebroventricularly injected [125I]angiotensin II over those measured for the Wistar-Kyoto and Sprague-Dawley normotensive rat strains: 45.0, 27.2, and 25.0 s, respectively. This was also true for intracerebroventricularly administered [125I]angiotensin III: 19.5, 11.4, and 9.0 s, respectively. These results support the notion that a dysfunction in central aminopeptidase activity in the spontaneously hypertensive rat may result in prolonged half-lives of endogenously synthesized angiotensins II and III, which are known to serve as ligands at central angiotensin receptors responsible for the control of cardiovascular function. The extended half-lives of these ligands may contribute to the sustained elevations in blood pressure observed in this animal model.