Modulation of drug sensitivity in yeast cells by the ATP-binding domain of human DNA topoisomerase IIalpha

Epipodophyllotoxins are effective antitumour drugs that trap eukaryotic DNA topoisomerase II in a covalent complex with DNA. Based on DNA cleavage assays, the mode of interaction of these drugs was proposed to involve amino acid residues of the catalytic site. An in vitro binding study, however, revealed two potential binding sites for etoposide within human DNA topoisomerase IIα (htopoIIα), one in the catalytic core of the enzyme and one in the ATP-binding N-terminal domain. Here we have tested how N-terminal mutations that reduce the affinity of the site for etoposide or ATP affect the sensitivity of yeast cells to etoposide. Surprisingly, when introduced into full-length enzymes, mutations that lower the drug binding capacity of the N-terminal domain in vitro render yeast more sensitive to epipodophyllotoxins. Consistently, when the htopoIIα N-terminal domain alone is overexpressed in the presence of yeast topoII, cells become more resistant to etoposide. Point mutations that weaken etoposide binding eliminate this resistance phenotype. We argue that the N-terminal ATP-binding pocket competes with the active site of the holoenzyme for binding etoposide both in cis and in trans with different outcomes, suggesting that each topoisomerase II monomer has two non-equivalent drug-binding sites.     

The DNA sequence of chromosome I of an African trypanosome: gene content,chromosome organisation,recombination and polymorphism

The African trypanosome, Trypanosoma brucei, causes sleeping sickness in humans in sub-Saharan Africa. Here we report the sequence and analysis of the 1.1 Mb chromosome I, which encodes approximately 400 predicted genes organised into directional clusters, of which more than 100 are located in the largest cluster of 250 kb. A 160-kb region consists primarily of three gene families of unknown function, one of which contains a hotspot for retroelement insertion. We also identify five novel gene families. Indeed, almost 20% of predicted genes are members of families. In some cases, tandemly arrayed genes are 99–100% identical, suggesting an active process of amplification and gene conversion. One end of the chromosome consists of a putative bloodstream-form variant surface glycoprotein (VSG) gene expression site that appears truncated and degenerate. The other chromosome end carries VSG and expression site-associated genes and pseudogenes over 50 kb of subtelomeric sequence where, unusually, the telomere-proximal VSG gene is oriented away from the telomere. Our analysis includes the cataloguing of minor genetic variations between the chromosome I homologues and an estimate of crossing-over frequency during genetic exchange. Genetic polymorphisms are exceptionally rare in sequences located within and around the strand-switches between several gene clusters.     

Structure-activity studies of a series of dipyrazolo[3,4-b:3',4'-d]pyridin-3-ones binding to the immune regulatory protein B7.1

The interaction of co-stimulatory molecules on T cells with B7 molecules on antigen presenting cells plays an important role in the activation of naive T cells. Consequently, agents that disrupt these interactions should have applications in treatment of transplant rejection as well as autoimmune diseases. To this end, specific small molecule inhibitors of human B7.1 were identified and characterized. Herein, we report the identification of potent small molecule inhibitors of the B7.1-CD28 interaction. In a high-throughput screen we identified several leads that prevented the interaction of B7.1 with CD28 with activities in the nanomolar to low micromolar range. One of these, the dihydrodipyrazolopyridinone 1, was subsequently shown to bind the V-like domain of human B7.1 at equimolar stoichiometry. With this as a starting point, we report here the synthesis and initial in vitro structure-activity relationships of a series of these compounds.     

Functional and structural basis of carnitine palmitoyltransferase 1A deficiency

Carnitine palmitoyltransferase 1A (CPT1A) is the key regulatory enzyme of hepatic long-chain fatty acid beta-oxidation. Human CPT1A deficiency is characterized by recurrent attacks of hypoketotic hypoglycemia. We presently analyzed at both the functional and structural levels five missense mutations identified in three CPT1A-deficient patients, namely A275T, A414V, Y498C, G709E, and G710E. Heterologous expression in Saccharomyces cerevisiae permitted to validate them as disease-causing mutations. To gain further insights into their deleterious effects, we localized these mutated residues into a three-dimensional structure model of the human CPT1A created from the crystal structure of the mouse carnitine acetyltransferase. This study demonstrated for the first time that disease-causing CPT1A mutations can be divided into two categories depending on whether they affect directly (functional determinant) or indirectly the active site of the enzyme (structural determinant). Mutations A275T, A414V, and Y498C, which exhibit decreased catalytic efficiency, clearly belong to the second class. They are located more than 20 A away from the active site and mostly affect the stability of the protein itself and/or of the enzyme-substrate complex. By contrast, mutations G709E and G710E, which abolish CPT1A activity, belong to the first category. They affect Gly residues that are essential not only for the structure of the hydrophobic core in the catalytic site, but also for the chain-length specificity of CPT isoforms. This study provides novel insights into the functionality of CPT1A that may contribute to the design of drugs for the treatment of lipid disorders.     

Incorporation of thioether building blocks into an alphavbeta3-specific RGD peptide: synthesis and biological activity

We report the design, synthesis, and binding affinities of a family of thioether analogues of the alpha(v)beta(3)-specific compound c[(Mpa)RGDD(tBuG)C]-NH(2). The synthesis of the thioether building blocks is scalable and produced the desired products in good yields. The linear peptides were synthesized on solid supports, followed by cyclization in solution. Our analogues demonstrate interesting binding data to the isolated receptors. In particular, the peptide c[NH-Arg-Gly-Asp-Asp-(tBuG)-Cys(S-CH(2)-CO)]NH(2) (1) exhibits differences in binding when compared to the parent compound and demonstrates potent affinity to the alpha(v)beta(3) and alpha(5)beta(1) receptors while having reduced binding to the alpha(IIb)beta(3) receptor. This result combined with the replacement of the disulfide with a thioether makes this compound interesting for further development.     

High yield regioselective monobenzyloxycarbonylation of secondary alcohols in glycopyranoside series

The regioselective monobenzyloxycarbonylation of secondary alcohols in methyl 6-O-(4-methoxytrityl)-alpha-D-manno-, gluco- and galactopyranoside has been achieved in high yields (74-85%) by using benzyl chloroformate in the presence of 4-dimethylaminopyridine and/or 1,4-diazabicyclo[2.2.2]octane.     

A comparison of adductor pollicis fatigue in older men and women

Sex differences in fatigue resistance of the adductor pollicis (AP) muscle were studied in 24 older adults who were divided into three groups: 12 older men (69.8 +/- 4.60 years), 6 older women not on hormone replacement therapy (HRT) (70.2 +/- 4.02 years), and 6 older women on HRT (68.7 +/- 6.47 years). Fatigue in the AP muscle was induced using an intermittent (5 s contraction, 5 s rest) submaximal voluntary contraction (50% of maximal voluntary contraction (MVC)) protocol, which was continued until exhaustion (i.e., when subjects could either no longer maintain a 5-s contraction at 50% MVC or when the MVC was deemed to be lower than the target force). There was no effect of HRT on MVC or time to fatigue (TTF); therefore, the older women were pooled as one subject group. At baseline, men were stronger than women for MVC (75.9 +/- 18.8 N in men vs. 56.8 +/- 10.0 N in women; P < 0.05) and evoked twitch force (7.3 +/- 1.7 N in men vs. 5.2 +/- 0.8 N in women; P < 0.05). There was no difference in TTF between men and women (14.77 +/- 7.06 min in men vs. 11.53 +/- 4.91 min in women; P > 0.20), nor was there a significant relationship between baseline muscle force and TTF (r = 0.14). There was also no difference in the pattern of fatigue and recovery between the men and women. These results suggest that there is no difference in endurance or fatigue characteristics of the AP muscle in men and women over the age of 65 years, and that baseline muscle force does not predict fatigue resistance in this muscle.     

Zygotic Wnt/beta-catenin signaling preferentially regulates the expression of Myf5 gene in the mesoderm of Xenopus

Zygotic Wnt signaling has been shown to be involved in dorsoventral mesodermal patterning in Xenopus embryos, but how it regulates different myogenic gene expression in the lateral mesodermal domains is not clear. Here, we use transient exposure of embryos or explants to lithium, which mimics Wnt/beta-catenin signaling, as a tool to regulate the activation of this pathway at different times and places during early development. We show that activation of Wnt/beta-catenin signaling at the early gastrula stage rapidly induces ectopic expression of XMyf5 in both the dorsal and ventral mesoderm. In situ hybridization analysis reveals that the induction of ectopic XMyf5 expression in the dorsal mesoderm occurs within 45 min and is not blocked by the protein synthesis inhibitor cycloheximide. By contrast, the induction of XMyoD is observed after 2 h of lithium treatment and the normal expression pattern of XMyoD is blocked by cycloheximide. Analysis by RT-PCR of ectodermal explants isolated soon after midblastula transition indicates that lithium also specifically induces XMyf5 expression, which takes place 30 min following lithium treatment and is not blocked by cycloheximide, arguing strongly for an immediate-early response. In the early gastrula, inhibition of Wnt/beta-catenin signaling blocks the expression of XMyf5 and XMyoD, but not of Xbra. We further show that zygotic Wnt/beta-catenin signaling interacts specifically with bFGF and eFGF to promote XMyf5 expression in ectodermal cells. These results suggest that Wnt/beta-catenin pathway is required for regulating myogenic gene expression in the presumptive mesoderm. In particular, it may directly activate the expression of the XMyf5 gene in the muscle precursor cells.     

X-ray structure and ligand binding study of a moth chemosensory protein

Chemosensory proteins (CSPs) are believed to be involved in chemical communication and perception. Such proteins, of M(r) 13,000, have been isolated from several sensory organs of a wide range of insect species. Several CSPs have been identified in the antennae and proboscis of the moth Mamestra brassicae. One of them, CSPMbraA6, a 112-amino acid antennal protein, has been expressed in large quantities and is soluble in the Escherichia coli periplasm. X-ray structure determination has been performed in parallel with ligand binding assays using tryptophan fluorescence quenching. The protein has overall dimensions of 25 x 30 x 32 A and exhibits a novel type of alpha-helical fold with six helices connected by alpha-alpha loops. A narrow channel extends within the protein hydrophobic core. Fluorescence quenching with brominated alkyl alcohols or fatty acids and modeling studies indicates that CSPMbraA6 is able to bind such compounds with C12-18 alkyl chains. These ubiquitous proteins might have the role of extracting hydrophobic linear compounds (pheromones, odors, or fatty acids) dispersed in the phospholipid membrane and transporting them to their receptor.