Role of anoctamins in cancer and apoptosis

Anoctamin 1 (TMEM16A, Ano1) is a recently identified Ca²⁺-activated chloride channel and a member of a large protein family comprising 10 paralogues. Before Ano1 was identified as a chloride channel protein, it was known as the cancer marker DOG1. DOG1/Ano1 is expressed in gastrointestinal stromal tumours (GIST) and particularly in head and neck squamous cell carcinoma, at very high levels never detected in other tissues. It is now emerging that Ano1 is part of the 11q13 locus, amplified in several types of tumour, where it is thought to augment cell proliferation, cell migration and metastasis. Notably, Ano1 is upregulated through histone deacetylase (HDAC), corresponding to the known role of HDAC in HNSCC. As Ano1 does not enhance proliferation in every cell type, its function is perhaps modulated by cell-specific factors, or by the abundance of other anoctamins. Thus Ano6, by regulating Ca²⁺-induced membrane phospholipid scrambling and annexin V binding, supports cellular apoptosis rather than proliferation. Current findings implicate other cellular functions of anoctamins, apart from their role as Ca²⁺-activated Cl⁻ channels.     

New approach of monitoring changes in chlorophyll a fluorescence of single guard cells and protoplasts in response to physiological stimuli

A new type of microfluorometer was applied to assess photosynthesis at the single-cell level by chlorophyll fluorescence using the saturation pulse method. A microscopy–pulse amplitude modulation (PAM) chlorophyll fluorometer was combined with a Zeiss Axiovert 25 inverted epifluorescence microscope for high-resolution measurements on single mesophyll and guard cells and the respective protoplasts. Available information includes effective quantum yield of photosystem II, relative electron transport rate and energization of the thylakoid membrane due to the transthylakoidal proton gradient. Dark–light induction curves of guard cell (GCPs) and mesophyll cell protoplasts (MCPs) displayed very similar characteristics, indicating similar functional organization of thylakoid membranes in both types of chloroplasts. Light response curves, however, revealed much earlier saturation of photosynthetic electron flow in GCPs than in MCPs. Under anaerobiosis, photosynthetic electron flow and membrane energization were severely suppressed. A similar effect was observed in guard cells when epidermal peels were incubated with the fungal toxin fusicoccin which activates the plasma membrane H⁺-ATPase and causes irreversible opening of stomata. The drop in electron transport rate was prevented by blocking ATP consumption of the H⁺ pump or by glucose addition. These results show that chlorophyll fluorescence quenching analysis allows profound insights into stomatal physiology.     

Continuous ECS-indicated recording of the proton-motive charge flux in leaves

Technical features and examples of application of a special emitter–detector module for highly sensitive measurements of the electrochromic pigment absorbance shift (ECS) via dual-wavelength (550–520 nm) transmittance changes (P515) are described. This device, which has been introduced as an accessory of the standard, commercially available Dual-PAM-100 measuring system, not only allows steady-state assessment of the proton motive force (pmf) and its partitioning into ΔpH and ΔΨ components, but also continuous recording of the overall charge flux driven by photosynthetic light reactions. The new approach employs a double-modulation technique to derive a continuous signal from the light/dark modulation amplitude of the P515 signal. This new, continuously measured signal primarily reflects the rate of proton efflux via the ATP synthase, which under quasi-stationary conditions corresponds to the overall rate of proton influx driven by coupled electron transport. Simultaneous measurements of charge flux and CO₂ uptake as a function of light intensity indicated a close to linear relationship in the light-limited range. A linear relationship between these two signals was also found for different internal CO₂ concentrations, except for very low CO₂, where the rate of charge flux distinctly exceeded the rate of CO₂ uptake. Parallel oscillations in CO₂ uptake and charge flux were induced by high CO₂ and O₂. The new device may contribute to the elucidation of complex regulatory mechanisms in intact leaves.     

Tm studies of a tertiary structure from the human hepatitis delta agent which functions in vitro as a ribozyme control element.

Viroids and other circular subviral RNA pathogens, such as the hepatitis delta agent, use a rolling circle replication cycle requiring an intact circular RNA. However, many infectious RNAs have the potential to form self-cleavage structures, whose formation must be controlled in order to preserve the circular replication template. The native structure of delta RNA contains a highly conserved element of local tertiary structure which is composed of sequences partially overlapping those needed to form the self-cleavage motif. A bimolecular complex containing the tertiary structure can be made. We show that when it is part of this bimolecular complex the potential cleavage site is protected and is not cleaved by the delta ribozyme, demonstrating that the element of local tertiary structure can function as a ribozyme control element in vitro. Physical studies of the complex containing this element were carried out. The complex binds magnesium ions and is not readily dissociated by EDTA under the conditions tested; > 50% of the complexes remain following incubation in 1 mM EDTA at 60 degrees C for 81 min. The thermal stability of the complex is reduced in the presence of sodium ions. A DNA complex and a perfect RNA duplex studied in parallel showed a similar effect, but of lesser magnitude. The RNA complex melts at temperatures approximately 10 degrees C lower in buffers containing 0.5 mM MgCl2 and 100 mM NaCl than in buffers containing 0.5 mM MgCl2 with no NaCl (78.1 compared with 87.7 degrees C). The element of local tertiary structure in delta genomic RNA appears to be a molecular clamp whose stability is highly sensitive to ion concentration in the physiological range.     

Development and Validation of a Novel Leishmania donovani Screening Cascade for High-Throughput Screening Using a Novel Axenic Assay with High Predictivity of Leishmanicidal Intracellular Activity

Visceral leishmaniasis is an important parasitic disease of the developing world with a limited arsenal of drugs available for treatment. The existing drugs have significant deficiencies so there is an urgent need for new and improved drugs. In the human host, Leishmania are obligate intracellular parasites which poses particular challenges in terms of drug discovery. To achieve sufficient throughput and robustness, free-living parasites are often used in primary screening assays as a surrogate for the more complex intracellular assays. We and others have found that such axenic assays have a high false positive rate relative to the intracellular assays, and that this limits their usefulness as a primary platform for screening of large compound collections. While many different reasons could lie behind the poor translation from axenic parasite to intracellular parasite, we show here that a key factor is the identification of growth slowing and cytostatic compounds by axenic assays in addition to the more desirable cytocidal compounds. We present a screening cascade based on a novel cytocidal-only axenic amastigote assay, developed by increasing starting density of cells and lowering the limit of detection, and show that it has a much improved translation to the intracellular assay. We propose that this assay is an improved primary platform in a new Leishmania screening cascade designed for the screening of large compound collections. This cascade was employed to screen a diversity-oriented-synthesis library, and yielded two novel antileishmanial chemotypes. The approach we have taken may have broad relevance to anti-infective and anti-parasitic drug discovery.     

Poly(ADP-ribose) polymerases in double-strand break repair: Focus on PARP1, PARP2 and PARP3

Poly(ADP-ribosyl)ation (PARylation) is a post-translational modification of proteins catalysed by Poly(ADP-ribose) polymerases (PARP). A wealth of recent advances in the biochemical and functional characterization of the DNA-dependent PARP family members have highlighted their key contribution in the DNA damage response network, the best characterized being the role of PARP1 and PARP2 in the resolution of single-strand breaks as part of the BER/SSBR process. How PARylation contributes to the repair of double-strand breaks is less well defined but has become recently the subject of significant research in the field. The aim of this review is to provide an overview of the current knowledge concerning the role of the DNA-activated PARP1, PARP2 and PARP3 in cellular response to double-strand breaks (DSB). In addition, we outline the biological significance of these properties in response to programmed DNA lesions formed during physiological processes such as antibody repertoire assembly and diversification.     

Nucleolar retention of a translational C/EBPα isoform stimulates rDNA transcription and cell size

The messenger RNA of the intronless CEBPA gene is translated into distinct protein isoforms through the usage of consecutive translation initiation sites. These translational isoforms have distinct functions in the regulation of differentiation and proliferation due to the presence of different N‐terminal sequences. Here, we describe the function of an N‐terminally extended protein isoform of CCAAT enhancer‐binding protein α (C/EBPα) that is translated from an alternative non‐AUG initiation codon. We show that a basic amino‐acid motif within its N‐terminus is required for nucleolar retention and for interaction with nucleophosmin (NPM). In the nucleoli, extended‐C/EBPα occupies the ribosomal DNA (rDNA) promoter and associates with the Pol I‐specific factors u pstream‐b inding f actor 1 (UBF‐1) and SL1 to stimulate rRNA synthesis. Furthermore, during differentiation of HL‐60 cells, endogenous expression of extended‐C/EBPα is lost concomitantly with nucleolar C/EBPα immunostaining probably reflecting the reduced requirement for ribosome biogenesis in differentiated cells. Finally, overexpression of extended‐C/EBPα induces an increase in cell size. Altogether, our results suggest that control of rRNA synthesis is a novel function of C/EBPα adding to its role as key regulator of cell growth and proliferation.     

G‐fibres in storage roots of Trifolium pratense (Fabaceae): tensile stress generators for contraction

Root contraction has been described for many species within the plant kingdom for over a century, and many suggestions have been made for mechanisms behind these contractions. To move the foliage buds deeper into the soil, the proximal part of the storage root of Trifolium pratense contracts by up to 30%. Anatomical studies have shown undeformed fibres next to strongly deformed tissues. Raman imaging revealed that these fibres are chemically and structurally very similar to poplar (Populus) tension wood fibres, which are known to generate high tensile stresses and bend leaning stems or branches upright. Analogously, an almost pure cellulosic layer is laid down in the lumen of certain root fibres, on a thin lignified secondary cell wall layer. To reveal its stress generation capacities, the thick cellulosic layer, reminiscent of a gelatinous layer (G‐layer) in tension wood, was selectively removed by enzymatic treatment. A substantial change in the dimensions of the isolated wood fibre bundles was observed. This high stress relaxation indicates the presence of high tensile stress for root contraction. These findings indicate a mechanism of root contraction in T. pratense (red clover) actuated via tension wood fibres, which follows the same principle known for poplar tension wood.