Discovery of a new RNA-containing nuclear structure in UVC-induced apoptotic cells by integrated laser electron microscopy

Background information. Treatment of cells with UVC radiation leads to the formation of DNA cross‐links which, if not repaired, can lead to apoptosis. γ‐H2AX and cleaved caspase 3 are proteins formed during UVC‐induced DNA damage and apoptosis respectively. The present study sets out to identify early morphological markers of apoptosis using a new method of correlative microscopy, ILEM (integrated laser electron microscopy). Cleaved caspase 3 and γ‐H2AX were immunofluorescently labelled to mark the cells of interest. These cells were subsequently searched in the fluorescence mode of the ILEM and further analysed at high resolution with TEM (transmission electron microscopy). Results. Following the treatment of HUVECs (human umbilical vein endothelial cells) with UVC radiation, in the majority of the cells γ‐H2AX was formed, whereas only in a subset of cells caspase 3 was activated. In severely damaged cells with high levels of γ‐H2AX a round, electron‐dense nuclear structure was found, which was hitherto not identified in UV‐stressed cells. This structure exists only in nuclei of cells containing cleaved caspase 3 and is present during all stages of the apoptotic process. Energy‐loss imaging showed that the nuclear structure accumulates phosphorus, indicating that it is rich in nucleic acids. Because the nuclear structure did not label for DNA and was not affected by regressive EDTA treatment, it is suggested that the UV‐induced nuclear structure contains a high amount of RNA. Conclusions. Because the UV‐induced nuclear structure was only found in cells labelled for cleaved caspase 3 it is proposed as an electron microscopic marker for all stages of apoptosis. Such a marker will especially facilitate the screening for early apoptotic cells, which lack the well‐known hallmarks of apoptosis within a cell population. It also raises new questions on the mechanisms involved in the UV‐induced apoptotic pathway.     

Organotin meclofenamic complexes: Synthesis, crystal structures and antiproliferative activity of the first complexes of meclofenamic acid - Novel anti-tuberculosis agents

The complexes [Me₂(Meclo)SnOSn(Meclo)Me₂]₂ (2) and [Ph₃Sn(Meclo)] (3) where HMeclo is meclofenamic acid, N-(2,6-dichloro-m-tolylanthranilic acid)], have been prepared and structurally characterized by means of vibrational, ¹H and ¹³C NMR spectroscopies. The crystal structure of complexes (2) and (3) have been determined by X-ray crystallography. Three distannoxane rings are present to the dimeric tetraorganodistannoxane of planar ladder arrangement of (2). The structure is centro symmetric and features a central rhombus Sn₂O₂ unit two additional tin atoms linked at the oxygen atoms. Five- and six-coordinated tin centers are present in the dimer distannoxane. X-ray analysis of (3) revealed a penta-coordinated structure containing Ph₃Sn coordinated to the chelated carboxylato group. The polar imino hydrogen atom participates in intra-molecular hydrogen bonds. Complexes (2) and (3) are self-assembled via π → π, C-H-π, stacking interactions and intra-molecular hydrogen bonds. Meclofenamic acid and [Ph₃Sn(Meclo)] have been evaluated for antiproliferative activity in vitro against three human cancer cell lines: MCF-7 (human breast cancer cell line), T24 (bladder cancer cell line), A-549 (non-small cell lung carcinoma) and a mouse L-929 (a fibroblast-like cell line cloned from strain L). The [Ph₃Sn(Meclo)] complex exhibited high cytotoxic activity against all the cancer cell lines. Meclofenamic and [Ph₃Sn(Meclo)] were tested for anti-mycobacterial activity against Mycobacterium tuberculosis H37Rv. The [Ph₃Sn(Meclo)] complex was found to be a promising anti-mycobacterial lead compound, displaying high activity against M. tuberculosis H37Rv.     

Complex dynamics of microbial competition in the gradostat

We examine the conditions necessary for the emergence of complex dynamic behavior in systems of microbial competition. In particular, we study the effect of spatial heterogeneity and substrate-inhibition on the dynamics of such a system. This is accomplished through the study of a mathematical model of two microbial populations competing for a single nutrient in a configuration of two interconnected chemostats. Microbial growth is assumed to follow substrate-inhibited kinetics for both species. Such a system with sterile feed has been shown in a previous work to exhibit stable periodic states. In the present work we study the system for the case of non-sterile feed, i.e., when the two species are present in the feed of the chemostats. The analysis is done by numerical bifurcation theory methods. We demonstrate that, in addition to periodic states, the system possesses stable quasi-periodic states resulting from Neimark-Sacker bifurcations of limit cycles. Also, periodic states may undergo successive period doublings leading to periodic states of increasing period and indicating that chaotic states might be possible. Multistability is also observed, consisting in the coexistence of several stable steady states and possibly stable periodic or quasi-periodic states for given operating conditions. It appears that substrate-inhibition, spatial heterogeneity and presence of microorganisms in the inflow are all necessary conditions for complex dynamics to arise in a microbial system of pure and simple competition.     

Does begging call convergence increase feeding rates to nestling tree swallows Tachycineta bicolor?

Some studies suggest that offspring might coordinate their begging displays to send a more effective brood signal, which in turn, could increase parental feeding rates. In tree swallows Tachycineta bicolor , when nestlings call together, their calls are more similar in structure than when they call alone. Here, we tested the hypothesis that call convergence enhances the overall brood signal, thus increasing parental provisioning rates. We played back similar and dissimilar calls (as measured by cross-correlation) to parents during a one-h playback period, and filmed the response of parents and nestlings. Contrary to our hypothesis, parental feeding rates did not differ in relation to call similarity. Based on these results, call similarity does not appear to function as a coordinated brood signal in tree swallows.     

Origin and seasonality of subfossil caprine dung from the discovery site of the Iceman (Eastern Alps)

The discovery of a Neolithic glacier mummy (dated to 3300–3100 cal b.c.) on a remote site of an Alpine pass at 3,200 m in the Ötztal Mountains is still puzzling. In the initial phase of the Iceman research, four hypotheses were suggested to explain the find in its entirety. The speculations vary from a hunter or warrior to a shaman, a miner or a shepherd. None of these proposals is accepted or corroborated by archaeological findings, but on the basis of palynological investigations conducted in the vicinity of the discovery site the assumption that the Iceman was involved in an early form of transhumance has now gained general acceptance. Concerning this assumption we present in this paper a recent study conducted on about a hundred caprine (sheep/goat or ibex/chamois) dung pellets recovered from the find spot of the Iceman and which were dated from 5400 to 2000 cal b.c. The approach was to determine through plant remains from these faeces whether they were droppings derived from animals grazing in anthropogenic habitats at low altitudes or in alpine grasslands. The former case would suggest they were livestock, the latter game. The results showed that all droppings derive from animals grazing at high altitudes.     

The genome and variation of Bacillus anthracis

The Bacillus anthracis genome reflects its close genetic ties to Bacillus cereus and Bacillus thuringiensis but has been shaped by its own unique biology and evolutionary forces. The genome is comprised of a chromosome and two large virulence plasmids, pXO1 and pXO2. The chromosome is mostly co-linear among B. anthracis strains and even with the closest near neighbor strains. An exception to this pattern has been observed in a large inversion in an attenuated strain suggesting that chromosome co-linearity is important to the natural biology of this pathogen. In general, there are few polymorphic nucleotides among B. anthracis strains reflecting the short evolutionary time since its derivation from a B. cereus-like ancestor. The exceptions to this lack of diversity are the variable number tandem repeat (VNTR) loci that exist in genic and non genic regions of the chromosome and both plasmids. Their variation is associated with high mutability that is driven by rapid insertion and deletion of the repeats within an array. A notable example is found in the vrrC locus which is homologous to known DNA translocase genes from other bacteria.     

Induction of Therapeutic Antibodies by Vaccination against External Loops of Tumor-Associated Viral Latent Membrane Protein

Some human herpesviruses (HHV) are etiological contributors to a wide range of malignant diseases. These HHV express latent membrane proteins (LMPs), which are type III membrane proteins consistently exposed at the cell surface in these malignancies. These LMPs have relatively large cytoplasmic domains but only short extracellular loops connecting transmembrane segments that are accessible at the surface of infected cells, but they do not elicit antibodies in the course of natural infection and tumorigenesis. We report here that conformational peptides mimicking two adjacent loops of the Epstein-Barr virus (EBV) LMP1 (2LS peptides) induce high-affinity antibodies with remarkable antitumor activities in mice. In active immunization experiments, LMP1-targeting 2LS vaccine conferred tumor protection in BALB/c mice. Moreover, this tumor protection is dependent upon a humoral anti-2LS immune response as demonstrated in DO11.10 (TCR-OVA) mice challenged with LMP1-expressing tumor and in SCID mice xenografted with human EBV-positive lymphoma cells. These data provide a proof of concept for 2LS immunization against short external loops of viral LMPs. This approach might possibly be extended to other infectious agents expressing type III membrane proteins.After the primary infection, some viruses, especially human herpesviruses (HHV) such as Epstein-Barr virus (EBV), cytomegalovirus, Kaposi''s sarcoma herpesvirus (HHV8), varicella-zoster virus, and herpes simplex virus, persist lifelong in all infected individuals, most often in an asymptomatic latent form. However, in the long term, some HHV can be involved in the emergence of malignant diseases in a small subset of infected individuals. EBV-associated lymphomas and carcinomas (22, 37), HHV8-associated Kaposi''s sarcomas (30), and human cytomegalovirus-associated glioblastomas (24) are examples of beta- and gammaherpesvirus-related human malignancies. All these malignancy-associated viruses encode type III membrane proteins which are expressed during the latent state of infection and thus can be called latent membrane proteins (LMPs). These viral LMPs (vLMPs), or “multipass” membrane proteins, appeared to be necessary for virus-driven host cell survival and/or transforming activity (1, 3, 28, 31). They are regarded by some authors as evolutionary mimics of cellular chemokine/cytokine receptors, and, like cellular receptors, they recruit numerous cytoplasmic adaptors. The several transmembrane domains of these vLMPs seem to mimic activated cellular chemokine/cytokine receptor structures and to function with versatile signaling devices, reprogramming cellular signaling networks to modulate cellular function after infection. They contribute prominently to virus survival in latently infected individuals and to virus-related human pathologies, including cancer (8, 14, 19, 34, 36). Despite expressing vLMP antigens at their membrane surface, these latently infected cells are very poor in initiating effective immune responses in infected individuals, thus facilitating viral persistence in humans (2, 17, 38). One reason for this poor immunogenicity may be the constitutive cell signaling property reported for these vLMPs in latently infected cells (3, 16, 35, 38). Consequently, unnecessary overexpression and large extracellular domains for ligand binding may facilitate vLMP immune escape (3, 35, 38). Thus, a major therapeutic approach involved the discovery of naturally active compounds or pharmacological agents that specifically block viral receptor functioning (12, 35). Compounds emerged from high-throughput screening of synthetic chemical libraries, but we still lack specific agents for vLMPs, as they cross-react with cellular chemokine/cytokine receptors and cellular signaling pathways (35). Functional antibodies (Abs) recognizing membrane proteins for anticancer therapies have recently emerged, but there are very few of these and they resulted mostly from serendipity rather than from a systematic design strategy (5). To date, LMPs as a target for a virus-specific immunotherapeutic Ab strategy have not been explored extensively. Some studies have been conducted with purified full-length LMPs from EBV, a gammaherpesvirus, but these studies failed to produce or detect Abs recognizing LMP extracellular domains (10, 20, 29). One reason for this poor immunogenicity could be the too-short extracellular structure of these LMPs, which could explain the failure of latently infected individuals to produce cytolytic Abs (21). To test this hypothesis, we used as an LMP model the EBV-encoded oncoprotein LMP1 which mimics a constitutively active tumor necrosis factor receptor-like molecule and is expressed during EBV latent infection (16). This LMP1 expression was observed in most EBV-carrying malignancies (16, 22, 37), therefore causing EBV to be classified as a class I human carcinogenic agent (11). Here, we report an original humoral approach, because Abs have unlimited diversity and are often exquisitely specific and readily produced. Indeed, to overcome the too-short extracellular size of LMP, we hypothesized that synthesis of a peptide mimicking several extracellular loops of LMP would be a successful general strategy for the development of Abs with the high-pressure liquid chromatography affinity necessary for neutralizing and cytolytic effectiveness, as described previously (4, 33a). We argue here that this new process (D. Tranchand Bunel, 28 January 2003, French patent application FR0300943; D. Tranchand Bunel, 28 July 2005, U.S. Patent Office, US069140) was rewarding, as by vaccinating mice with peptides that covered two adjacent extracellular loops of LMP1 (2LS peptides), we obtained the production of neutralizing and cytolytic high-affinity Abs. Moreover, these Abs induced by 2LS peptide vaccination appeared to confer protection of mice against the development of tumors expressing LMP1.     

A preliminary phylogeny of the 'didymocarpoid Gesneriaceae' based on three molecular data sets: Incongruence with available tribal classifications

The 'didymocarpoid Gesneriaceae' (traditional subfam. Cyrtandroideae excluding Epithemateae) are the largest group of Old World Gesneriaceae, comprising 85 genera and 1800 species. We attempt to resolve their hitherto poorly understood generic relationships using three molecular markers on 145 species, of which 128 belong to didymocarpoid Gesneriaceae. Our analyses demonstrate that consistent topological relationships can be retrieved from data sets with missing data using subsamples and different combinations of gene sequences. We show that all available classifications in Old World Gesneriaceae are artificial and do not reflect natural relationships. At the base of the didymocarpoids are grades of clades comprising isolated genera and small groups from Asia and Europe. These are followed by a clade comprising the African and Madagascan genera. The remaining clades represent the advanced Asiatic and Malesian genera. They include a major group with mostly twisted capsules. The much larger group of remaining genera comprises exclusively genera with straight capsules and the huge genus Cyrtandra with indehiscent fruits. Several genera such as Briggsia, Henckelia, and Chirita are not monophyletic; Chirita is even distributed throughout five clades. This degree of incongruence between molecular phylogenies, traditional classifications, and generic delimitations indicates the problems with classifications based on, sometimes a single, morphological characters.