Historical introgression and the role of selective vs. neutral processes in structuring nuclear genetic variation (AFLP) in a circumpolar marine fish, the capelin (Mallotus villosus)

The capelin (Mallotus villosus) is a widespread marine fish species for which previous work has identified geographically distinct mtDNA clades, the frontiers of which are well within adult and larval dispersal capabilities. Here, we use AFLPs to test for the presence of nuclear gene flow among clades. In addition, we evaluate genetic structuring within one clade, the Northwest Atlantic (NWA). We found that each of the mtDNA clades corresponds with a unique nuclear DNA genetic cluster. Within the NWA clade, we detected individuals with small but significant amounts of genetic ancestry from other clades, likely due to historical introgression. Further support for historical introgression comes from analyses of variance in locus-specific differentiation, which support introgression between some clades and divergence without gene flow between others. Within the NWA, we identified two genetic clusters that correspond to sites in geographically adjacent areas. However, these clusters differ primarily at 'outlier' loci, and a genetic subdivision (K=2) was not supported by genetic clustering programs using neutral loci. Significant neutral F(ST) differentiation was found only between sites that otherwise differed at outlier loci. Thus, these populations may be in the initial stages of 'isolation by adaptation'. These results suggest strong between-clade reproductive isolation despite opportunities for gene flow and support the hypothesis that selection can contribute to divergence in otherwise 'open' systems.     

Mining the plant-herbivore interface with a leafmining Drosophila of Arabidopsis

Experimental infections of Arabidopsis thaliana (Arabidopsis) with genomically characterized plant pathogens such as Pseudomonas syringae have facilitated the dissection of canonical eukaryotic defence pathways and parasite virulence factors. Plants are also attacked by herbivorous insects, and the development of an ecologically relevant genetic model herbivore that feeds on Arabidopsis will enable the parallel dissection of host defence and reciprocal resistance pathways such as those involved in xenobiotic metabolism. An ideal candidate is Scaptomyza flava, a drosophilid fly whose leafmining larvae are true herbivores that can be found in nature feeding on Arabidopsis and other crucifers. Here, we describe the life cycle of S.?flava on Arabidopsis and use multiple approaches to characterize the response of Arabidopsis to S.?flava attack. Oviposition choice tests and growth performance assays on different Arabidopsis ecotypes, defence-related mutants, and hormone and chitin-treated plants revealed significant differences in host preference and variation in larval performance across Arabidopsis accessions. The jasmonate and glucosinolate pathways in Arabidopsis are important in mediating quantitative resistance against S.?flava, and priming with jasmonate or chitin resulted in increased resistance. Expression of xenobiotic detoxification genes was reduced in S.?flava larvae reared on Arabidopsis jasmonate signalling mutants and increased in plants pretreated with chitin. These results and future research directions are discussed in the context of developing a genetic model system to analyse insect-plant interactions.     

Merotelic kinetochore attachment: causes and effects

Accurate chromosome segregation depends on the proper attachment of sister kinetochores to microtubules emanating from opposite spindle poles. Merotelic kinetochore orientation is an error in which a single kinetochore is attached to microtubules emanating from both spindle poles. Despite correction mechanisms, merotelically attached kinetochores can persist until anaphase, causing chromatids to lag on the mitotic spindle and hindering their timely segregation. Recent studies showing that merotelic kinetochore attachment represents a major mechanism of aneuploidy in mitotic cells and is the primary mechanism of chromosomal instability in cancer cells have underlined the importance of studying merotely. Here, we highlight recent progress in our understanding of how cells prevent and correct merotelic kinetochore attachments.     

An inhibitor-like binding mode of a carbonic anhydrase activator within the active site of isoform II

The 2,4,6-trimethylpyridinium derivative of histamine is an effective activator of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1). However, unlike other CA activators, which bind at the entrance of the active site cavity, an X-ray crystal structure of hCA II in complex with the 1-[2-(1H-imidazol-4-yl)-ethyl]-2,4,6-trimethylpyridinium salt evidenced a binding mode never observed before either for activators or inhibitors of this enzyme, with the 2,4,6-trimethylpyridinium ring pointing towards the metal ion deep within the enzyme cavity, and several strong hydrophobic interactions stabilizing the adduct. Indeed, incubation of the activator with the enzyme for several days leads to potent inhibitory effects. This is the first example of a CA activator which after a longer contact with the enzyme behaves as an inhibitor.     

Inhibition studies with anions and small molecules of two novel β-carbonic anhydrases from the bacterial pathogen Salmonella enterica serovar Typhimurium

Two new β-carbonic anhydrases (CAs, EC 4.2.1.1) from the bacterial pathogen Salmonella enterica serovar Typhimurium, stCA 1 and stCA 2, were characterized kinetically. The two enzymes possess appreciable activity as catalysts for the hydration of CO₂ to bicarbonate, with k_cat of 0.79 × 10⁶ s⁻¹ and 1.0 × 10⁶ s⁻¹, and k_cat/K_m of 5.2 × 10⁷ M⁻¹ s⁻¹ and of 8.3 × 10⁷ M⁻¹ s⁻¹, respectively. A large number of simple/complex inorganic anions as well as other small molecules (sulfamide, sulfamic acid, phenylboronic acid, phenylarsonic acid, dialkyldithiocarbamates) showed interesting inhibitory properties towards the two new enzymes, with several low micromolar inhibitors discovered. As many strains of S. enterica show extensive resistance to classical antibiotics, inhibition of the β-CAs investigated here may be useful for developing lead compounds for novel types of antibacterials.     

GH overexpression causes muscle hypertrophy independent from local IGF-I in a zebrafish transgenic model

The aim of the present study was to analyse the morphology of white skeletal muscle in males and females from the GH-transgenic zebrafish (Danio rerio) lineage F0104, comparing the expression of genes related to the somatotrophic axis and myogenesis. Histological analysis demonstrated that transgenic fish presented enhanced muscle hypertrophy when compared to non-transgenic fish, with transgenic females being more hypertrophic than transgenic males. The expression of genes related to muscle growth revealed that transgenic hypertrophy is independent from local induction of insulin-like growth factor 1 gene (igf1). In addition, transgenic males exhibited significant induction of myogenin gene (myog) expression, indicating that myog may mediate hypertrophic growth in zebrafish males overexpressing GH. Induction of the α-actin gene (acta1) in males, independently from transgenesis, also was observed. There were no significant differences in total protein content from the muscle. Our results show that muscle hypertrophy is independent from muscle igf1, and is likely to be a direct effect of excess circulating GH and/or IGF1 in this transgenic zebrafish lineage.     

Inhibition of the β-carbonic anhydrase from Streptococcus pneumoniae by inorganic anions and small molecules: Toward innovative drug design of antiinfectives?

The Gram-positive bacterium Streptococcus pneumoniae is a human respiratory tract pathogen that contributes significantly to global mortality and morbidity. It was recently shown that this bacterial pathogen depends on a conserved ??-carbonic anhydrase (CA, EC 4.2.1.1) for in vitro growth in environmental ambient air and during intracellular survival in host cells. Hence, it is to be expected that this pneumococcal carbonic anhydrase (PCA) contributes to transmission and pathogenesis of the bacterium, making it a potential therapeutic target. In this study, purified recombinant PCA has been further characterized kinetically and for inhibition with a series of inorganic anions and small molecules useful as leads. PCA has appreciable activity as catalyst for the hydration of CO₂ to bicarbonate, with a k_cat of 7.4 × 10⁵ s⁻¹ and k_cat/K_m of 6.5 × 10⁷ M⁻¹ s⁻¹ at an optimum pH of 8.4. Inorganic anions such as chloride, bromide, iodide, cyanate, selenocyanate, trithiocarbonate, and cyanide were effective inhibitors of PCA (K_Is of 21-98 ??M). Sulfamide, sulfamic acid, phenylboronic, phenylarsonic acid, and diethyldithiocarbamate showed inhibition constants in the low micromolar/submicromolar range (K_Is of 0.61-6.68 ??M), whereas that of the sulfonamide acetazolamide was in the nanomolar range (K_Is 89 nM). In conclusion, our results show that PCA can effectively be inhibited by a range of molecules that could be interesting leads for obtaining more potent PCA inhibitors. PCA might be a novel target for designing antimicrobial drugs with a new mechanism of action.     

Antiviral activity of seed extract from Citrus bergamia towards human retroviruses

The effects of an extract from Citrus bergamia (BSext) and those of two products purified from the same extract, that is, nomilin and limonin, and reference compounds, towards HTLV-1 have been reported. Moreover, they were also compared with those obtained towards HIV-1. Results showed that the efficacy of both BSext and limonin in inhibiting HTLV-1 as well as HIV-1 expression in infected cells, as evaluated by comparable quantitative assays, was close to that of the effective, reference compounds, respectively. The protective effect of BSext and of the purified products was associated with the inhibition of both HTLV-1 and HIV-1 RT activities in conceptually similar, cell-free assays. The cytotoxicity of the assayed compounds of natural origin was substantially less pronounced than that of the reference compounds, thus showing a favourable selectivity index for the novel BSext product.     

Synthesis and biological profile of new 1,2,3,4-tetrahydroisoquinolines as selective carbonic anhydrase inhibitors

In a previous paper we identified several 1-aryl-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline-2-sulfonamides that displayed inhibitory effects toward selected carbonic anhydrase isozymes at micromolar concentration. In order to deepen the structure-activity relationships (SARs) and identify novel compounds with improved activity, we synthesized a series of monomethoxy analogues of the previously investigated dimethoxy derivatives. The evaluation of biological profile has been focused on in vitro effects against several CA isoforms. The new monomethoxy derivatives showed higher hCA inhibitory effects against several isoforms compared to the dimethoxy analogues. Particularly, some of these compounds (e.g., 1b and 1h) showed low nanomolar K(I) values and excellent selectivity for hCA IX and hCA XIV versus hCA I and II inhibition.     

Modular strategy for the construction of radiometalated antibodies for positron emission tomography based on inverse electron demand Diels-Alder click chemistry

A modular system for the construction of radiometalated antibodies was developed based on the bioorthogonal cycloaddition reaction between 3-(4-benzylamino)-1,2,4,5-tetrazine and the strained dienophile norbornene. The well-characterized, HER2-specific antibody trastuzumab and the positron emitting radioisotopes (64)Cu and (89)Zr were employed as a model system. The antibody was first covalently coupled to norbornene, and this stock of norbornene-modified antibody was then reacted with tetrazines bearing the chelators 1,4,7,10-tetraazacyclo-dodecane-1,4,7,10-tetraacetic acid (DOTA) or desferrioxamine (DFO) and subsequently radiometalated with (64)Cu and (89)Zr, respectively. The modification strategy is simple and robust, and the resultant radiometalated constructs were obtained in high specific activity (2.7-5.3 mCi/mg). For a given initial stoichiometric ratio of norbornene to antibody, the (64)Cu-DOTA- and (89)Zr-DFO-based probes were shown to be nearly identical in terms of stability, the number of chelates per antibody, and immunoreactivity (>93% in all cases). In vivo PET imaging and acute biodistribution experiments revealed significant, specific uptake of the (64)Cu- and (89)Zr-trastuzumab bioconjugates in HER2-positive BT-474 xenografts, with little background uptake in HER2-negative MDA-MB-468 xenografts or other tissues. This modular system-one in which the divergent point is a single covalently modified antibody stock that can be reacted selectively with various chelators-will allow for both greater versatility and more facile cross-comparisons in the development of antibody-based radiopharmaceuticals.