In vitro eradication of abasic site-mediated DNA–peptide/protein cross-links by Escherichia coli long-patch base excision repair

Apurinic/apyrimidinic (AP or abasic) sites are among the most abundant DNA lesions. Numerous proteins within different organisms ranging from bacteria to human have been demonstrated to react with AP sites to form covalent Schiff base DNA–protein cross-links (DPCs). These DPCs are unstable due to their spontaneous hydrolysis, but the half-lives of these cross-links can be as long as several hours. Such long-lived DPCs are extremely toxic due to their large sizes, which physically block DNA replication. Therefore, these adducts must be promptly eradicated to maintain genome integrity. Herein, we used in vitro reconstitution experiments with chemically synthesized, stable, and site-specific Schiff base AP-peptide/protein cross-link analogs to demonstrate for the first time that this type of DPC can be repaired by Escherichia coli (E. coli) long-patch base excision repair. We demonstrated that the repair process requires a minimum of three enzymes and five consecutive steps, including: (1) 5′-DNA strand incision of the DPC by endonuclease IV; (2 to 4) strand-displacement DNA synthesis, removal of the 5′-deoxyribose phosphate-peptide/protein adduct-containing flap, and gap-filling DNA synthesis by DNA polymerase I; and (5) strand ligation by a ligase. We further demonstrated that endonuclease IV plays a major role in incising an AP-peptide cross-link within E. coli cell extracts. We also report that eradicating model AP-protein (11.2–36.1 kDa) DPCs is less efficient than that of an AP-peptide_10mer cross-link, supporting the emerging model that proteolysis is likely required for efficient DPC repair.     

Light Activation of Rhodopsin: Insights from Molecular Dynamics Simulations Guided by Solid-State NMR Distance Restraints

Structural restraints provided by solid-state NMR measurements of the metarhodopsin II intermediate are combined with molecular dynamics simulations to help visualize structural changes in the light activation of rhodopsin. Since the timescale for the formation of the metarhodopsin II intermediate (> 1 ms) is beyond that readily accessible by molecular dynamics, we use NMR distance restraints derived from ¹³C dipolar recoupling measurements to guide the simulations. The simulations yield a working model for how photoisomerization of the 11-cis retinylidene chromophore bound within the interior of rhodopsin is coupled to transmembrane helix motion and receptor activation. The mechanism of activation that emerges is that multiple switches on the extracellular (or intradiscal) side of rhodopsin trigger structural changes that converge to disrupt the ionic lock between helices H3 and H6 on the intracellular side of the receptor.     

Ligneous membranitis in Scottish Terriers is associated with a single nucleotide polymorphism in the plasminogen (PLG) gene

Ligneous membranitis (LM) is a rare chronic inflammatory condition of the mucous membranes associated with plasminogen (encoded by PLG) deficiency in affected humans and dogs. In human, the condition is genetic in nature with numerous mutations and polymorphisms in PLG identified in affected individuals and related family members. The condition is uncommonly reported in dogs and, to date, no genetic studies have been performed. We identified related Scottish Terriers (littermates) with severe LM and unaffected relatives (sire, dam and a sibling from a previous litter). Plasma plasminogen activity was below normal in one affected dog but within normal reference intervals for the other. Sequencing of PLG from the affected dogs revealed a homozygous A>T single nucleotide polymorphism in an intron donor site (c.1256+2T>A). The related, unaffected dogs displayed heterozygous alleles at this position (c.1256+2T/A), whereas no mutation was detected in unaffected, non‐related control dogs. This is the first report to identify gene polymorphisms associated with LM in dogs.     

Geographic and breed distribution of an Msp I PCR-RFLP in the bovine growth hormone (bGH) gene

Information is presented on the frequency of the Msp I (-) allele in the third intron of the bovine growth hormone gene in a large number of cattle breeds. Consideration of the breed frequencies in relation to their geographic origin shows a low frequency for breeds originating in Northern Europe, moderate frequencies for breeds originating in Eastern Europe or the countries surrounding the Mediterranean basin, and very high frequencies for breeds originating in the Indian subcontinent. Consideration of breed frequencies in relation to breed type, shows low to moderate frequencies for the humpless breeds, high frequencies for the humped breeds. Various explanations for this distribution are discussed, among them the possibility that the Msp I (-) allele originated in the Bos indicus breeds of the Indian subcontinent, from which it diffused through the humpless Bos taurus breeds of Eastern Europe, the Mediterranean basin, eventually reaching Western, Northern Europe, Western Africa in low frequencies.     

The Role of the N-Terminus of the Myosin Essential Light Chain in Cardiac Muscle Contraction

To study the regulation of cardiac muscle contraction by the myosin essential light chain (ELC) and the physiological significance of its N-terminal extension, we generated transgenic (Tg) mice by partially replacing the endogenous mouse ventricular ELC with either the human ventricular ELC wild type (Tg-WT) or its 43-amino-acid N-terminal truncation mutant (Tg-Δ43) in the murine hearts. The mutant protein is similar in sequence to the short ELC variant present in skeletal muscle, and the ELC protein distribution in Tg-Δ43 ventricles resembles that of fast skeletal muscle. Cardiac muscle preparations from Tg-Δ43 mice demonstrate reduced force per cross-sectional area of muscle, which is likely caused by a reduced number of force-generating myosin cross-bridges and/or by decreased force per cross-bridge. As the mice grow older, the contractile force per cross-sectional area further decreases in Tg-Δ43 mice and the mutant hearts develop a phenotype of nonpathologic hypertrophy while still maintaining normal cardiac performance. The myocardium of older Tg-Δ43 mice also exhibits reduced myosin content. Our results suggest that the role of the N-terminal ELC extension is to maintain the integrity of myosin and to modulate force generation by decreasing myosin neck region compliance and promoting strong cross-bridge formation and/or by enhancing myosin attachment to actin.     

Mononuclear and dinuclear bromo bridged iridium(I) complexes with N-allyl substituted imidazolin-2-ylidene ligands

The reaction of 1-methyl-3-(2-propenyl)imidazolium bromide (1) or 1,3-bis(2-propenyl)-imidazolium bromide (2) with [Ir(μ-OMe)(cod)]₂ afforded the five coordinated iridium(I) carbene complexes [IrBr(L)(cod)] (3) (L=1-methyl-3-(2-propenyl)imidazolin-2-ylidene) and (4) (L=1,3-bis(2-propenyl)imidazolin-2-ylidene). The reaction proceeds via an in situ deprotonation of the imidazolium salt. Molecular structure determinations on 3 and 4 confirmed the coordination of the carbene ligands via the carbene carbon atom and one allyl group in both complexes. Treatment of complex 3 with an excess of AgBF₄ gave the dinuclear bromo bridged complex [(Ir(μ-Br)(L)(cod)]₂BF₄ (5) (L=1-methyl-3-(2-propenyl)imidazolin-2-ylidene). The reaction of complex 4 with an excess of AgBF₄ led to the mononuclear complex [Ir(L)(cod)]BF₄ (6) (L=1,3-bis(2-propenyl)imidazolin-2-ylidene) where both N-allyl substituents are coordinated to the iridium(I) center.     

Sternal gland structures in males of bean flower thrips,Megalurothrips sjostedti,and Poinsettia thrips,Echinothrips americanus,in comparison with those of western flower thrips,Frankliniella occidentalis (Thysanoptera: Thripidae)

Sternal pores are important features for identification of male thrips, especially within the subfamily Thripinae. They vary in shape, size and distribution even between species of one genus. Their functional role is speculated to be that of sex- and/or aggregation pheromone production. Yet, sexual aggregations are not reported in Echinothrips americanus, known to have sternal pores, while we observed aggregations in Megalurothrips sjostedti, previously reported to lack them.We examined the sternal glands and pores of the thripine species E. americanus and M. sjostedti males, in comparison with those of Frankliniella occidentalis using light microscopy, as well as scanning and transmission electron microscopy. Pore plates of F. occidentalis were ellipsoid and medial on sternites III–VII, while in E. americanus they were distributed as multiple micro pore plates on sternites III–VIII. In M. sjostedti they appeared as an extremely small pore in front of the posterior margin of each of sternites IV–VII. Pore plate and pore plate area were distributed similarly on sternites III–VII in F. occidentalis. However, in E. americanus the total pore plate area increased significantly from sternites III to VIII. Ultrastructure of cells associated with sternal glands showed typical characteristics of gland cells that differ in size, shape and number. The function of sternal glands is further discussed on the basis of morphological comparisons with other thrips species.     

A simple test of the homogeneity of risk difference in sparse data: an application to a multicenter study

In this paper, we focus discussion on testing the homogeneity of risk difference for sparse data, in which we have few patients in each stratum, but a moderate or large number of strata. When the number of patients per treatment within strata is small (2 to 5 patients), none of test procedures proposed previously for testing the homogeneity of risk difference for sparse data can really perform well. On the basis of bootstrap methods, we develop a simple test procedure that can improve the power of the previous test procedures. Using Monte Carlo simulations, we demonstrate that the test procedure developed here can perform reasonable well with respect to Type I error even when the number of patients per stratum for each treatment is as small as two patients. We evaluate and study the power of the proposed test procedure in a variety of situations. We also include a comparison of the performance between the test statistics proposed elsewhere and the test procedure developed here. Finally, we briefly discuss the limitation of using the proposed test procedure. We use the data comparing two chemotherapy treatments in patients with multiple myeloma to illustrate the use of the proposed test procedure.     

Search for the tumor-related proteins of transition cell carcinoma in Taiwan by proteomic analysis

To better understand the carcinogenesis of bladder cancer in Taiwan, we utilized the proteomic approach to search for potential biomarkers of transitional cell carcinoma (TCC). Analysis by 2-DE and MS/MS indicated that seven proteins are down-regulated and three proteins up-regulated in grade III samples as compared with those of grade II. Of these deregulated proteins, fatty acid binding proteins, annexin V, heat-shock protein 27, and lactate dehydrogenase have been shown to be associated with bladder cancer. Our studies also found altered expression of a group of proteins that have not been documented previously in bladder cancer, including annexin I, 15-hydroxyprostaglandin dehydrogenase, galectin-1, lysophospholipase and mitochondrial short-chain enoyl-coenzyme A hydratase 1 precursor. These results illustrate a pattern of differential protein expression between low- and high-grade tumors and it may be utilized as the molecular fingerprinting of a subset of bladder cancers. In addition, the present study provides a valuable resource in the study of pathological mechanisms in cancers of urothelial origin. The immunohistochemical staining of grade II and III TCC samples with antiserum to annexin I protein was utilized to confirm that the annexin I protein is up-regulated in grade III TCC.     

No postcopulatory selection against MHC‐homozygous offspring: Evidence from a pedigreed captive rhesus macaque colony

The heterozygosity status of polymorphic elements of the immune system, such as the major histocompatibility complex (MHC), is known to increase the potential to cope with a wider variety of pathogens. Pre‐ and postcopulatory processes may regulate MHC heterozygosity. In a population where mating occurs among individuals that share identical MHC haplotypes, postcopulatory selection may disfavour homozygous offspring or ones with two MHC haplotypes identical to its mother. We tested these ideas by determining the incidence of MHC‐heterozygous and MHC‐homozygous individuals in a pedigreed, partially consanguineous captive rhesus monkey colony. Bayesian statistics showed that when parents share MHC haplotypes, the distribution of MHC‐heterozygous and MHC‐homozygous individuals significantly fitted the expected Mendelian distribution, both for the complete MHC haplotypes, and for MHC class I or II genes separately. Altogether, we found in this captive colony no evidence for postcopulatory selection against MHC‐homozygous individuals. However, the distribution of paternally and maternally inherited MHC haplotypes tended to differ significantly from expected. Individuals with two MHC haplotypes identical to their mother were underrepresented and offspring with MHC haplotypes identical to their father tended to be overrepresented. This suggests that postcopulatory processes affect MHC haplotype combination in offspring, but do not prevent low MHC heterozygosity.