Stability of 3' double nucleotide overhangs that model the 3' ends of siRNA

Thermodynamic parameters are reported for duplex formation in 1 M NaCl for 16 RNA sequences, each containing a core tetramer duplex, GGCC, and a 3' overhang consisting of two bases. The results indicate additional double-helical stability is conferred by the double 3' terminal overhang relative to the single 3' terminal overhang. A nearest-neighbor analysis of the data indicates that the free energy contribution at 37 degrees C of the second base in the double 3' terminal overhang varies from 0 to 0.7 kcal/mol. The second base in the 3' double overhang can contribute nearly the same stability to a duplex as a base pair or a 3' dangling overhang. Stability contribution of a dangling base, two nucleotides removed from the 3' end of a duplex, is dependent upon both the identity of the base as well as that of the dangling base that it neighbors. A second dangling base only increases the stability of the duplex when it is neighboring a 3' purine dangling nucleotide. Furthermore, a second dangling pyrimidine provides a greater contribution to duplex stability than a purine. A nearest-neighbor model was developed to predict the influence of 3' double overhang on the stability of duplex formation. The model improves the prediction of free energy and melting temperature when tested against six sequences with different core duplexes.     

Boro-norleucine as a P1 residue for the design of selective and potent DPP7 inhibitors

Dipeptide-based inhibitors with C-substituted (alkyl or aminoalkyl) alpha-amino acids in the P2 position and boro-norleucine (boro-Nle) in the P1 position were synthesized. Relative to boro-proline, boro-Nle as a P1 residue was shown able to significantly dial out DPP4, FAP, DPP8, and DPP9 activity. Dab-boro-Nle (4g) proved to be the most selective and potent DPP7 inhibitor with a DPP7 IC50 value of 480 pM.     

Novel triazole based inhibitors of Ras farnesyl transferase

A novel series of potent inhibitors of Ras farnesyl transferase possessing a 1,2,4-triazole pharmacophore is described. These inhibitors were discovered from a parallel synthesis effort and were subsequently optimized to in vitro IC₅₀ value of less than 1 nM.     

Validation of an enzyme-linked immunosorbent assay kit using herpesvirus papio 2 (HVP2) antigen for detection of herpesvirus simiae (B virus) infection in rhesus monkeys

Serologic testing for antibody to monkey B virus (BV) in macaque sera is problematic due to the biohazardous nature of BV antigens. Herpesvirus papio 2 (HVP2), a herpesvirus of baboons, is nonpathogenic to humans and is genetically and antigenically more closely related to BV than is human herpes simplex virus 1. This paper describes the results of our in-house laboratory that compared a BV antigen-based enzyme-linked immunosorbent assay (ELISA) by commercial testing laboratory and an HVP2-based ELISA in our laboratory by using 447 sera from 290 rhesus monkeys. The HVP2-based ELISA identified as positive 99.11% of the sera identified as BV-positive by the BV ELISA. The BV antigen-based ELISA identified as positive 98.21% of the sera identified as BV-positive by the HVP2-based ELISA. The HVP2 ELISA also identified two BV-negative and six BV-equivocal sera as positive. Both ELISAs identified the same 85 negative and three equivocal samples as negative and equivocal, respectively. The high degree of correlation (weighted kappa coefficient, 0.94) between the two tests indicates that the HVP2 ELISA is a sensitive and reliable assay for in-house testing of the BV status of rhesus monkeys.     

Receptor for retrograde transport in the apicomplexan parasite Toxoplasma gondii

Toxoplasma gondii and its apicomplexan relatives (such as Plasmodium falciparum, which causes malaria) are obligate intracellular parasites that rely on sequential protein release from specialized secretory organelles for invasion and multiplication within host cells. Because of the importance of these unusual membrane trafficking pathways for drug development and comparative cell biology, characterizing them is essential. In particular, it is unclear what role retrieval mechanisms play in parasite membrane trafficking or where they operate. Previously, we showed that T. gondii's beta-COP (TgBetaCOP; a subunit of coatomer protein complex I, COPI) and retrieval reporters localize exclusively to the zone between the parasite endoplasmic reticulum (ER) and Golgi apparatus. This suggested the existence of an HDEL receptor in T. gondii. We have now identified, cloned, and sequenced this receptor, TgERD2. TgERD2 localizes in a Golgi or ER pattern suggestive of the HDEL retrieval reporter (K. M. Hager, B. Striepen, L. G. Tilney, and D. S. Roos, J. Cell Sci. 112:2631-2638, 1999). A functional assay reveals that TgERD2 is able to complement the Saccharomyces cerevisiae ERD2 null mutant. Retrieval studies reveal that stable expression of a fluorescent exogenous retrieval ligand results in a dispersal of betaCOP signal throughout the cytoplasm and, surprisingly, results in betaCOP staining of the vacuolar space of the parasite. In contrast, stable expression of TgERD2GFP does not appear to disturb betaCOP staining. In addition to TgERD2, Toxoplasma contains two more divergent ERD2 relatives. Phylogenetic analysis reveals that these proteins belong to a previously unrecognized ERD2 subfamily common to plants and alveolate organisms and as such could represent mediators of parasite-specific retrieval functions. No evidence of class 2 ERD2 proteins was found in metazoan organisms or fungi.     

Determination of 8-oxoguanine and 8-hydroxy-2'-deoxyguanosine in the rat cerebral cortex using microdialysis sampling and capillary electrophoresis with electrochemical detection

A rapid and sensitive method to determine 8-oxoguanine (8oxoG) and 8-hydroxydeoxyguanosine (8OHdG), biomarkers for oxidative DNA damage, in cerebral cortex microdialysate samples using capillary electrophoresis (CE) with electrochemical detection (CEEC) was developed. Samples were concentrated on-column using pH-mediated stacking for anions. On-column anodic detection was performed with a carbon fiber working electrode and laser-etched decoupler. The method is linear over the expected extracellular concentration range for 8oxoG and 8-OHdG during induced ischemia-reperfusion, with R.S.D. values 相似文献   

Genomewide significant linkage to stuttering on chromosome 12

Stuttering is a common and sometimes severe communication disorder, of unknown primary etiology, that exists in populations worldwide. Many types of evidence suggest a genetic contribution to stuttering; however, the complex inheritance of this disorder has hindered identification of these factors. We have employed highly inbred families to increase the power of linkage analysis of this disorder. Forty-four Pakistani families with documented or probable consanguinity, from the city of Lahore and surrounding areas, were included. Each family contained multiple cases of stuttering, which were diagnosed using the Stuttering Severity Instrument. Using the Marshfield Weber 9 marker panel, we performed a genomewide linkage scan focused on affected individuals and their parents. The analysis included 199 genotyped individuals, 144 affected and 55 unaffected. The Pedigree Relationship Statistical Test (PREST) was used to identify pedigrees that required additional specification of inbreeding. Initial nonparametric analysis gave evidence of linkage on chromosomes 1, 5, 7, and 12. Additional genotyping was performed on chromosome 12 to a 5-cM level of resolution, and 16 additional individuals were then included, bringing the number of families to 46. Analysis of the enlarged data set provided consistent evidence of linkage on chromosome 12: the S(homoz) scoring function gave a nonparametric LOD score of 4.61, and a LOD score of 3.51 was obtained using the S(all) scoring function. These results suggest that a locus on chromosome 12q may contain a gene with a large effect in this sample.