Small interference RNA-mediated knockdown of sperm associated antigen 9 having structural homology with c-Jun N-terminal kinase-interacting protein

Recently, we reported a novel testis-specific sperm associated antigen 9 (SPAG9) protein, a new member of the JNK-interacting protein family, having a functional role in sperm-egg fusion [N. Jagadish, R. Rana, R. Selvi, D. Mishra, M. Garg, S. Yadav, J.C. Herr, K. Okumura, A. Hasegawa, K. Koyama, A. Suri, Biochem. J. 389 (2005) 73-82]. NCBI Blast searches revealed SPAG9 nucleotide sequence similarities with ESTs of various cancerous tissues. In the present study, we compared the efficiency of two independent SPAG9 specific small interfering RNA (siRNA) constructs, BS/U6/spag9 and BS/U6/spag9-I, to ablate the SPAG9 expression in mammalian cells. A positive correlation between the ratio of target gene versus siRNA and the suppression of SPAG9 expression was observed. Further, the cotransfection of BS/U6/spag9 with pcDNA-SPAG9 and pFlag-CMV2-JNK-3 resulted in specific suppression of SPAG9 without affecting JNK-3 expression. The present investigation will eventually extend the application of SPAG9 siRNA in in vivo targeting experiments that aim to define the SPAG9 functional genomics in tumor and reproductive biology.     

Failed medical management in ovarian pregnancy despite favorable prognostic factors--a case report

Primary ovarian pregnancy is a rare form of ectopic pregnancy that must be demonstrated with use of 4 Spiegelberg criteria. It is usually diagnosed at laparotomy or laparoscopy, although it may resemble a hemorrhagic corpus luteum. Successful conservative management of ovarian pregnancy with methotrexate has been reported only occasionally. This may be partly because of the rarity of this condition and partly because when medical treatment is successful, the patient does not need to undergo laparotomy or laparoscopy, and an occasional ovarian pregnancy may have been diagnosed as a tubal pregnancy. We present a case of ovarian pregnancy (diagnosed at laparotomy) for which initial medical management with methotrexate failed despite favorable prognostic factors. Whether the unusual location (ovary) could have contributed toward treatment failure is unknown.     

I-Ep-bound self-peptides: identification, characterization, and role in alloreactivity

T cell recognition of peptide/allogeneic MHC complexes is a major cause of transplant rejection. Both the presented self-peptides and the MHC molecules are involved; however, the molecular basis for alloreactivity and the contribution of self-peptides are still poorly defined. The murine 2.102 T cell is specific for hemoglobin(64-76)/I-Ek and is alloreactive to I-Ep. The natural self-peptide/I-Ep complex recognized by 2.102 remains unknown. In this study, we characterized the peptides that are naturally processed and presented by I-Ep and used this information to define the binding motif for the murine I-Ep class II molecule. Interestingly, we found that the P9 anchor residue preferred by I-Ep is quite distinct from the residues preferred by other I-E molecules, although the P1 anchor residue is conserved. A degree of specificity for the alloresponse was shown by the lack of stimulation of 2.102 T cells by 19 different identified self-peptides. The binding motif was used to search the mouse genome for candidate 2.102 reactive allopeptides that contain strong P1 and P9 anchor residues and possess previously identified allowable TCR contact residues. Two potential allopeptides were identified, but only one of these peptides, G protein-coupled receptor 128, was able to stimulate 2.102 T cells. Thus, the G protein-coupled receptor 128 peptide represents a candidate allopeptide that is specifically recognized by 2.102 T cells bound to I-Ep and was identified using bioinformatics. These studies highlight the specific involvement of self-peptides in alloreactivity.     

Synthesis and biological evaluation of arylidene analogues of Meldrum’s acid as a new class of antimalarial and antioxidant agents

A series of arylidene analogues of Meldrum’s acid were synthesized and evaluated for in vitro antimalarial and antioxidant activities for the first time. The influence of various physico-chemical parameters such as dielectric constant (ε), donor number (DN), acceptor number (AN), hydrogen bond donor (HBD), hydrogen bond acceptor (HBA), and solubilizing power of the solvents on Meldrum’s acid anion generation and thus on promoting the Knoevenagel condensation of Meldrum’s acid with aryl aldehydes has been discussed. Five compounds 9l, 9m, 9n, 9r, and 9s were found to be most active against Plasmodium falciparum with IC₅₀ values in the range of 9.68–16.11 μM. Compound 9l exhibited the most potent antimalarial activity (IC₅₀ 9.68 μM). The compounds were also found to possess antioxidant activity when tested against DPPH and ABTS free radicals.     

A rational approach for the design and synthesis of 1-acetyl-3,5-diaryl-4,5-dihydro(1H)pyrazoles as a new class of potential non-purine xanthine oxidase inhibitors

Xanthine oxidase is a complex molybdoflavoprotein that catalyses the hydroxylation of xanthine to uric acid. Fifty three analogues of 1-acetyl-3,5-diaryl-4,5-dihydro(1H)pyrazoles were rationally designed and synthesized and evaluated for in vitro xanthine oxidase inhibitory activity for the first time. Some notions about structure activity relationships are presented. Six compounds 41, 42, 44, 46, 55 and 59 were found to be most active against XO with IC(50) ranging from 5.3 μM to 15.2 μM. The compound 59 emerged as the most potent XO inhibitor (IC(50)=5.3 μM). Some of the important interactions of 59 with the amino acid residues of active site of XO have been figured out by molecular modeling.     

Genome-wide association study for atopy and allergic rhinitis in a Singapore Chinese population

Allergic rhinitis (AR) is an atopic disease which affects about 600 million people worldwide and results from a complex interplay between genetic and environmental factors. However genetic association studies on known candidate genes yielded variable results. The aim of this study is to identify the genetic variants that influence predisposition towards allergic rhinitis in an ethnic Chinese population in Singapore using a genome-wide association study (GWAS) approach. A total of 4461 ethnic Chinese volunteers were recruited in Singapore and classified according to their allergic disease status. The GWAS included a discovery stage comparing 515 atopic cases (including 456 AR cases) and 486 non-allergic non-rhinitis (NANR) controls. The top SNPs were then validated in a replication cohort consisting of a separate 2323 atopic cases (including 676 AR cases) and 511 NANR controls. Two SNPs showed consistent association in both discovery and replication phases; MRPL4 SNP rs8111930 on 19q13.2 (OR = 0.69, P_combined = 4.46×10⁻⁰⁵) and BCAP SNP rs505010 on chromosome 10q24.1 (OR = 0.64, P_combined = 1.10×10⁻⁰⁴). In addition, we also replicated multiple associations within known candidates regions such as HLA-DQ and NPSR1 locus in the discovery phase. Our study suggests that MRPL4 and BCAP, key components of the HIF-1α and PI3K/Akt signaling pathways respectively, are two novel candidate genes for atopy and allergic rhinitis. Further study on these molecules and their signaling pathways would help in understanding of the pathogenesis of allergic rhinitis and identification of targets for new therapeutic intervention.     

Soluble Flt-1 and PlGF: New Markers of Early Pregnancy Loss?

Recent data have indicated a relationship between placental oxygen and angiogenic protein levels in the first trimester of normal pregnancies. Our objective was to investigate if maternal serum levels of angiogenic factors Soluble vascular endothelial growth factor (VEGF) receptor 1 (sFlt-1), soluble Endoglin and placental growth factor (PlGF) are altered in women with symptoms of threatened miscarriage (TM) and if they are predictive of a subsequent miscarriage. Blood samples were collected at 6–10 weeks from women presenting with TM (n = 40), from asymptomatic controls (n = 32) and from non- pregnant women in their luteal phase (n = 14). All samples were assayed for serum level of sFLT-1, PlGF, sEndoglin and HSP70 using commercial ELISAs. Samples were analysed retrospectively on the basis of pregnancy outcome. TM group included 21 women with a normal pregnancy outcome and 19 with subsequent complete miscarriage. The latter subgroup had significantly lower mean maternal serum (MS) sFlt-1 (83%, P<0.001) and PlGF (44%, P<0.001) compared to those with a normal pregnancy outcome. Asymptomatic control pregnant women had similar MS levels of sFlt-1 and PlGF compared to the TM patients with a normal outcome. The mean MS sFlt-1 (>10 fold) and MS PlGF (∼2 fold) levels were significantly (P<0.001) higher in control pregnant women compared to the non-pregnant group in the luteal phase of the menstrual cycle. Soluble Endoglin was not altered in the normal pregnant women compared to non pregnant women, although lower in the TM subgroup with a subsequent miscarriage (∼25%, P<0.001) compared to TM with a live birth. There was no significant difference in the mean MS HSP 70 levels between the different groups. This study shows that sFlt1 and PlGF MS levels are increased by several folds in early pregnancy and that MS sFlt-1 and MS PlGF are markedly decreased in threatened miscarriage patients who subsequently have a miscarriage suggesting these proteins are sensitive predictive markers of subsequent pregnancy loss.     

SuperFine: fast and accurate supertree estimation

Many research groups are estimating trees containing anywhere from a few thousands to hundreds of thousands of species, toward the eventual goal of the estimation of a Tree of Life, containing perhaps as many as several million leaves. These phylogenetic estimations present enormous computational challenges, and current computational methods are likely to fail to run even on data sets in the low end of this range. One approach to estimate a large species tree is to use phylogenetic estimation methods (such as maximum likelihood) on a supermatrix produced by concatenating multiple sequence alignments for a collection of markers; however, the most accurate of these phylogenetic estimation methods are extremely computationally intensive for data sets with more than a few thousand sequences. Supertree methods, which assemble phylogenetic trees from a collection of trees on subsets of the taxa, are important tools for phylogeny estimation where phylogenetic analyses based upon maximum likelihood (ML) are infeasible. In this paper, we introduce SuperFine, a meta-method that utilizes a novel two-step procedure in order to improve the accuracy and scalability of supertree methods. Our study, using both simulated and empirical data, shows that SuperFine-boosted supertree methods produce more accurate trees than standard supertree methods, and run quickly on very large data sets with thousands of sequences. Furthermore, SuperFine-boosted matrix representation with parsimony (MRP, the most well-known supertree method) approaches the accuracy of ML methods on supermatrix data sets under realistic conditions.