ATP-competitive inhibitors of the mitotic kinesin KSP that function via an allosteric mechanism

The mitotic kinesin KSP (kinesin spindle protein, or Eg5) has an essential role in centrosome separation and formation of the bipolar mitotic spindle. Its exclusive involvement in the mitotic spindle of proliferating cells presents an opportunity for developing new anticancer agents with reduced side effects relative to antimitotics that target tubulin. Ispinesib is an allosteric small-molecule KSP inhibitor in phase 2 clinical trials. Mutations that attenuate ispinesib binding to KSP have been identified, which highlights the need for inhibitors that target different binding sites. We describe a new class of selective KSP inhibitors that are active against ispinesib-resistant forms of KSP. These ATP-competitive KSP inhibitors do not bind in the nucleotide binding pocket. Cumulative data from generation of resistant cells, site-directed mutagenesis and photo-affinity labeling suggest that they compete with ATP binding via a novel allosteric mechanism.     

Parallels,nonparallels, and plasticity in population differentiation of threespine stickleback within a lake

The frequent occurrence of parallel phenotypic divergence in similar habitats is often evoked when emphasizing the role of ecology in adaptive radiation and speciation. However, because phenotypic plasticity can contribute to the observed pattern of divergence, confirmation of divergence at loci underlying phenotypic traits is important for confirming adaptive divergence. In the present study, we examine parallel morphological, neutral, and potentially adaptive genetic divergence of threespine stickleback inhabiting different habitats within a lake. Three genetic clusters best explained the neutral genetic structure within the lake; however, morphological differences were only weakly connected to genetic clusters and there was considerable phenotypic variation within clusters. Among the factors that could contribute to the observed pattern of morphological and genetic divergence are phenotypic plasticity, selective mortality of hybrids, and habitat choice based on morphology. Several loci are identified as outliers indicating divergent selection between the morphs and some parallels in morphological and adaptive genetic divergence are found in stickleback spawning at two lava sites. However, neutral genetic structure indicates considerable genetic connectivity among the two lava sites, and the parallels in morphology may therefore represent selective distribution of phenotypes rather than parallel divergence. © 2009 The Linnean Society of London, Biological Journal of the Linnean Society, 2009, 98 , 803–813.     

Genetic structure of Anogeissus dhofarica (Combretaceae) populations endemic to the monsoonal fog oases of the southern Arabian Peninsula

Anogeissus dhofarica (Combretaceae) is an endemic tree of the monsoon affected coastal mountains of the southern Arabian Peninsula, being the character species of the Hybantho durae–Anogeissetum dhofaricae association, a drought deciduous, monsoon forest community found only in the Dhofar region of southern Oman and the eastern Al‐Mahra region of south‐east Yemen. Due to the steep precipitation gradient from the centre to the edges in this monsoon affected area, A. dhofarica is found in two different habitat types: in continuous woodland belts of the Hawf and Dhofar mountains, and in isolated, scattered woodland patches, as found especially in the Fartak Mts (south‐east Yemen). Fifteen populations (212 individuals) from across the whole distribution area of the species were analysed using amplified fragment length polymorphism fingerprinting to: (1) evaluate the consequences of population fragmentation on the genetic diversity harboured in isolated patches versus cohering stands of the species and (2) to reconstruct the phylogeographical pattern of A. dhofarica as a consequence of oscillations in the monsoon activity during the Pleistocene and Holocene. The analysis of among‐population genetic differentiation and within‐population genetic diversity in A. dhofarica populations resulted in a lack of genetic pauperization and genetic differentiation of populations of the distinctly isolated patches of the Fartak Mts compared to the more luxurious forests of the Hawf and Dhofar regions. This is considered to be due to the high buffer capacity against the loss of genetic diversity caused by the long‐lived life‐form of the species combined with the capability to propagate clonally and the relatively recent fragmentation of Anogeissus forests into the described patches rather than due to high values of gene flow among remnant populations caused by bee pollination and anemochorical and hydrochorical diaspore dispersal. The phylogeographical pattern of the species argues for a quite recent fragmentation of a once continuous forest belt of A. dhofarica that is rather connected with climate changes in the Holocene than triggered by aridity–humidity oscillations reported for the Pleistocene. © 2009 The Linnean Society of London, Biological Journal of the Linnean Society, 2009, 97 , 40–51.     

Xiphograptus and the evolution of virgella‐bearing graptoloids

Abstract: The virgellar spine is one of the most consistent features of the graptolite sicula. It is present in a large number of graptoloid groups, but evolved separately and independently in these as it is seen from the presence of the spine in either ventral (Axonophora) or dorsal (Phyllograptus, Xiphograptus) position. The evolution of the virgellar spine in the Pan‐Bireclinata in the Upper Dapingian to Lower Darriwilian time interval is known to follow four main steps, from a simple rutellum, through a lamelliform rutellum and a lanceolate virgella to the true virgellar spine. For the xiphograptids and in Phyllograptus, the origin and early development is less well documented, but appears to follow a similar path. However, the individual stages are condensed, and a true virgellar spine emerges already in the Floian time interval. A virgellar spine was found in Didymograptellus bifidus, necessitating a revision of the diagnosis of the genus Didymograptellus. A number of species of the virgellate genera Xiphograptus, Yutagraptus and Didymograptellus are described from isolated material for the first time. The species are useful for the biostratigraphic correlation of endemic mid‐continent North American faunas with the Pacific Type faunal realm. Xiphograptus artus sp. nov., Didymograptellus primus sp. nov. and Didymograptellus cowheadensis sp. nov. from the Cow Head Group of western Newfoundland are described as new.     

Crosstalk between desmoglein-2/desmocollin-2/Src kinase and coxsackie and adenovirus receptor/ZO-1 protein complexes,regulates blood-testis barrier dynamics

Morphological studies in the testis reported the presence of ‘desmosome-like’ junctions between Sertoli cells at the blood-testis barrier, whose function is also constituted by tight junctions and basal ectoplasmic specializations. Unfortunately, little is known about the role of desmosomes in blood-testis barrier dynamics. This study aims to fill this gap with the functional investigation of two desmosomal cadherins, desmoglein-2 and desmocollin-2, by their specific knockdown in Sertoli cells cultured in vitro. Reminiscent of the blood-testis barrier in vivo, desmosome-like structures were visible by electron microscopy when Sertoli cells were cultured at high density, thereby forming a polarized epithelium with functional cell junctions. At this point, we opted to focus our efforts on desmoglein-2 and desmocollin-2 based on results which illustrated desmosomal mRNAs to be expressed by Sertoli and germ cells, as well as on results which illustrated desmoglein-2 to co-immunoprecipitate with plakoglobin, c-Src and desmocollin-2. Simultaneous knockdown of desmoglein-2 and desmocollin-2 not only led to a reduction in and mislocalization of zonula occludens-1, but also perturbed the localization of c-Src and coxsackie and adenovirus receptor at the cell–cell interface, resulting in disruption of tight junction permeability barrier. We hereby propose a novel regulatory protein complex composed of desmoglein-2, desmocollin-2, c-Src, coxsackie and adenovirus receptor and zonula occludens-1 at the blood-testis barrier.     

IRAK1 and IRAK4 Promote Phosphorylation,Ubiquitination, and Degradation of MyD88 Adaptor-like (Mal)

Signal transduction by Toll-like receptor 2 (TLR2) and TLR4 requires the adaptors MyD88 and Mal (MyD88 adaptor-like) and serine/threonine kinases, interleukin-1 receptor-associated kinases IRAK1 and IRAK4. We have found that both IRAK1 and IRAK4 can directly phosphorylate Mal. In addition, co-expression of Mal with either IRAK resulted in depletion of Mal from cell lysates. This is likely to be due to Mal phosphorylation by the IRAKs because kinase-inactive forms of either IRAK had no effect. Furthermore, lipopolysaccharide stimulation resulted in ubiquitination and degradation of Mal, which was inhibited using an IRAK1/4 inhibitor or by knocking down expression of IRAK1 and IRAK4. MyD88 is not a substrate for either IRAK and did not undergo degradation. We therefore conclude that Mal is a substrate for IRAK1 and IRAK4 with phosphorylation promoting ubiquitination and degradation of Mal. This process may serve to negatively regulate signaling by TLR2 and TLR4.     

An Artemis polymorphic variant reduces Artemis activity and confers cellular radiosensitivity

Artemis is required for V(D)J recombination and the repair of a subset of radiation-induced DNA double strand breaks (DSBs). Artemis-null patients display radiosensitivity (RS) and severe combined immunodeficiency (SCID), classified as RS-SCID. Strongly impacting hypomorphic Artemis mutations confer marked infant immunodeficiency and a predisposition for EBV-associated lymphomas. Here, we provide evidence that a polymorphic Artemis variant (c.512C > G: p.171P > R), which has a world-wide prevalence of 15%, is functionally impacting. The c.512C > G mutation causes an ～3-fold decrease in Artemis endonuclease activity in vitro. Cells derived from a patient who expressed a single Artemis allele with the polymorphic mutational change, showed radiosensitivity and a DSB repair defect in G2 phase, with Artemis cDNA expression rescuing both phenotypes. The c.512C > G change has an additive impact on Artemis function when combined with a novel C-terminal truncating mutation (p.436C > X), which also partially inactivates Artemis activity. Collectively, our findings provide strong evidence that monoallelic expression of the c.512C > G variant impairs Artemis function causing significant radiosensitivity and a G2 phase DSB repair defect. The patient exhibiting monoallelic c.512C > G-Artemis expression showed immunodeficiency only in adulthood, developed bilateral carcinoma of the nipple and myelodysplasia raising the possibility that modestly decreased Artemis function can impact clinically.