Sub-Angstrom resolution enzyme X-ray structures: is seeing believing?

Recent technical advances in crystallographic analysis, particularly highly focused and high brilliance synchrotron beam lines, have significantly improved the resolutions that are attainable for many macromolecular crystal structures. The Protein Data Bank (http://www.rcsb.org/pdb/) contains an increasing number of atomic resolution structures, which are providing a wealth of structural information that was not previously visible in lower resolution electron density maps. Here, we review the importance of visualizing hydrogen atoms and multiple sidechain conformations or anisotropy, as well as substrate strain, at sub-Angstrom resolution. The additional structural features that are visible in the electron density maps as a result of atomic resolution data provide a better understanding of the catalytic mechanisms of cholesterol oxidase, ribonuclease A, beta-lactamase, serine proteases, triosephosphate isomerase and endoglucanase.     

Phosphorylation during mitosis: How many kinases are out there?

Comment on: Rizkallah R, et al. Cell Cycle 2011; 10: 3327-36.     

Proline-rich domain in dynamin-2 has a low microtubule-binding activity: how is this activity controlled during mitosis in HeLa cells?

The large GTPase dynamin is strongly accumulated in the constricted area including midzonal microtubules of dividing cells. The proline-rich domain (PRD) of dynamin has been considered as a microtubule-binding domain. However, it remains unclear how PRD controls dynamin-microtubule interaction in mitotic cells. Here, we found that the microtubule-binding activity of PRD is low in dynamin-2. One of the mitosis-specific kinase activities to PRD in HeLa cells was identified as cyclin B-Cdc2 kinase. The kinase phosphorylated PRD at Ser(764) and/or Thr(766) and reduced the microtubule-binding activity of PRD. These results suggest that phosphorylation of PRD by cyclin B-Cdc2 kinase plays an important role to control dynamin-2-microtubule interaction in mitotic HeLa cells.     

Seeing is believing? A beginners' guide to practical pitfalls in image acquisition

Imaging can be thought of as the most direct of experiments. You see something; you report what you see. If only things were truly this simple. Modern imaging technology has brought about a revolution in the kinds of questions we can approach, but this comes at the price of increasingly complex equipment. Moreover, in an attempt to market competing systems, the microscopes have often been inappropriately described as easy to use and suitable for near-beginners. Insufficient understanding of the experimental manipulations and equipment set-up leads to the introduction of errors during image acquisition. In this feature, I review some of the most common practical pitfalls faced by researchers during image acquisition, and how they can affect the interpretation of the experimental data.This article is targeted neither to the microscopy gurus who push forward the frontiers of imaging technology nor to my imaging specialist colleagues who may wince at the overly simplistic comments and lack of detail. Instead, this is for beginners who gulp with alarm when they hear the word "confocal pinhole" or sigh as they watch their cells fade and die in front of their very eyes time and time again at the microscope. Take heart, beginners, if microscopes were actually so simple then many people (including myself) would suddenly be out of a job!     

NO-sensitive guanylyl cyclase β1 subunit is peripherally associated to chromosomes during mitosis. Novel role in chromatin condensation and cell cycle progression

NO-sensitive guanylyl cyclase (GCNO), the major NO target, is involved in important regulatory functions in the cardiovascular, gastrointestinal and central nervous systems. GC_NO exists as heterodimers of α(1/2) and β1 subunits. Deletion of the obligate β1 dimerizing partner abrogates NO/cGMP signaling and shortens the life span of KO mice. Localization studies in the CNS have shown that β1 is more widespread than α subunits and in some areas is the only GC_NO subunit expressed, suggesting that β1 may have GC_NO-independent functions. GC_NO is predominantly cytosolic, but association to membranes and other intracellular structures has been described. Here, we show localization of β1 in cytoplasm and nucleus of cells expressing α subunits and GC_NO activity (astrocytes, C6 cells), as well as in cells devoid of α subunits and GC_NO activity (microglia). In both cell types β1 associates peripherally to chromosomes in all phases of mitosis. Immunodepletion of β1 in C6 cells enhances chromatin condensation in an in vitro assay. Moreover, silencing β1 by siRNA induces cell cycle re-entry as determined by flow cytometry, and increases proliferation rate in a MTT-assay, whereas infection with β1-containing adenovirus has the opposite effect. These actions are independent of cGMP formation. We postulate that β1 is a multifunctional protein that regulates chromatin condensation and cell cycle progression, in addition to being an obligate monomer in functional GC_NO heterodimers.     

Adenosine and cardioprotection during reperfusion – an overview

Ischemic heart disease includes a number of entities that have been grouped in accordance with physiopathology and evolutive criteria. In recent years new ischemic syndromes have been described. Within the new ischemic syndromes, ventricular post-ischemic dysfunction – also known as stunned myocardium – is worth mentioning. In this route, several studies have suggested that reperfusion per se could cause cellular injury (reperfusion injury). In previous years, a protective effect on the injury caused by ischemia and reperfusion in the heart has been attributed to adenosine. These effects have been documented in different experimental in vivo and in vitro models. Thus, the administration of exogenous adenosine, or agonists of adenosine receptors prior to ischemia reduces the size of the infarction, improves the recovery of the ventricular function during reperfusion (attenuating stunning) and prolongs the time period to the ischemic contracture. However, focusing on a potential therapeutic application, it is of the utmost importance to find this protection and learn the mechanisms involved when procedures are applied during early reperfusion.We showed that adenosine, administered from the beginning of reperfusion, attenuated systolic and diastolic (myocardial stiffness) alterations of the stunned myocardium. This protective effect was mediated by the activation of A₁ adenosine receptors, and without modification on infarct size. According to some authors, adenosine can decrease the release of endothelin, during early reperfusion, and reduce an overload of Ca²⁺ that could cause a cellular lesion. Finally, ischemic preconditioning involves a series of intracellular events that are initiated with the activation of the A1 receptor, and end at the sensitive K⁺ ATP channels of the mitochondria. The phosphorylation and opening of these channels would cause the protective effect. Activation of this specific mechanism during reperfusion has not been studied extensively.     

BimEL is phosphorylated at mitosis by Aurora A and targeted for degradation by βTrCP1

Bcl-2-interacting mediator of cell death (Bim) is a pro-apoptotic B-cell lymphoma 2 family member implicated in numerous apoptotic stimuli. In particular, Bim is required for cell death mediated by antimitotic agents, however, mitotic regulation of Bim remains poorly understood. Here, we show that the major splice variant of Bim, BimEL, is regulated during mitosis by the Aurora A kinase and protein phosphatase 2A (PP2A). We observed that BimEL is phosphorylated by Aurora A early in mitosis and reversed by PP2A after mitotic exit. Aurora A phosphorylation stimulated binding of BimEL to the F-box protein beta-transducin repeat containing E3 ubiquitin protein ligase and promoted ubiquitination and degradation of BimEL. These findings describe a novel mechanism by which the oncogenic kinase Aurora A promotes cell survival during mitosis by downregulating proapoptotic signals. Notably, we observed that knockdown of Bim significantly increased resistance of cells to the Aurora A inhibitor MLN8054. Inhibitors of Aurora A are currently under investigation as cancer chemotherapeutics and our findings suggest that efficacy of this class of drugs may function in part by enhancing apoptotic activity of BimEL.     

Perturbing mitosis for anti‐cancer therapy: is cell death the only answer?

Interfering with mitosis for cancer treatment is an old concept that has proven highly successful in the clinics. Microtubule poisons are used to treat patients with different types of blood or solid cancer since more than 20 years, but how these drugs achieve clinical response is still unclear. Arresting cells in mitosis can promote their demise, at least in a petri dish. Yet, at the molecular level, this type of cell death is poorly defined and cancer cells often find ways to escape. The signaling pathways activated can lead to mitotic slippage, cell death, or senescence. Therefore, any attempt to unravel the mechanistic action of microtubule poisons will have to investigate aspects of cell cycle control, cell death initiation in mitosis and after slippage, at single‐cell resolution. Here, we discuss possible mechanisms and signaling pathways controlling cell death in mitosis or after escape from mitotic arrest, as well as secondary consequences of mitotic errors, particularly sterile inflammation, and finally address the question how clinical efficacy of anti‐mitotic drugs may come about and could be improved.     

The nutrient games – Plasmodium metabolism during hepatic development

Malaria is a febrile illness caused by species of the protozoan parasite Plasmodium and is characterized by recursive infections of erythrocytes, leading to clinical symptoms and pathology. In mammals, Plasmodium parasites undergo a compulsory intrahepatic development stage before infecting erythrocytes. Liver-stage parasites have a metabolic configuration to facilitate the replication of several thousand daughter parasites. Their metabolism is of interest to identify cellular pathways essential for liver infection, to kill the parasite before onset of the disease. In this review, we summarize the current knowledge on nutrient acquisition and biosynthesis by liver-stage parasites mostly generated in murine malaria models, gaps in knowledge, and challenges to create a holistic view of the development and deficiencies in this field.     

Selenium – an antioxidative protectant in soybean during senescence

Selenium (Se) is regarded as an antioxidant in animals and plants, even though considered as non-essential element in plants. To test its ability to counteract senescence related oxidative stress in soybean a pot culture experiment was conducted. The soybean plant was sprayed with sodium selenate (50ppm) at 78days after sowing (DAS). Soybean leaves were harvested at 80 and 90 DAS for analysis of oxidant production and antioxidative enzymes activity. Se positively promoted growth and acted as antioxidant by inhibiting lipid peroxidation and per cent injury of cell membrane. The antioxidative effect was associated with an increase in superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) enzymes activity. Significant increase in antioxidant enzyme activity was positively related to Se content. The decrease in antioxidative enzymes at 90 DAS was much faster in control plants than Se-sprayed plants. The reduction in SOD and GSH-Px may be associated with senescence-induced oxidative burst.     

What is truth – in science and beyond

Truth is threatened in our societies and one might wish that scientists should stand up for truth, but in order to do so, one needs to know what is truth and how it can be recognized. The oldest and most widely accepted concept of truth is the Correspondence Theory requesting a fit of propositions and reality. In the Coherence Theory truth is a consistent property of a whole system of propositions. In the Pragmatic Theory truth works in practical terms. Scientists have defined criteria to verify true statements by experiments and by the simplicity of theories. Aristotle proposed parsimony claiming the superiority of theories which derive from fewer hypotheses. David Hume suggested probability arguments to assess the force of evidence. Nicolai Hartmann elaborated a model based on the congruence of a priori logical arguments with a posteriori empirical observations. Karl Popper introduced the falsification of testable theories as a way to better theories. The analysis shows that scientific and medical research uses classical philosophical criteria of truth in their daily work. Humanities use different, hermeneutic criteria of truth. Finally, societies need for their coherence a dialectic approach to truth based on honest discussion of opposing views.     

Bioprospecting – why is it so unrewarding?

Some economic analyses have placed high values on the chemicaldiversity residing in threatened habitats[, Conservation Biology 6:128–130; , InBiodiversity and its Importance to Human Health, Columbia University Press, NewYork; , Journal of PoliticalEconomy 108: 173–206]. Consequently, bioprospecting (searching for newbiologically active chemicals in organisms) is considered by some to be a way offunding the preservation of biodiversity, especially in the less developedcountries. However, the large multinational pharmaceutical andagrochemical companies spend very little of their research effort onbioprospecting [, Phytochemistry55: 463–480]. Why is this? The answer lies in the fact that any chemical(whether a synthetic or a natural product) has a very low probability ofpossessing useful biological activity. The common belief that every naturalproduct has been selected by its producer such that only biologically activenatural products are made is not correct. Given that random collections ofsynthetic or natural products have a similar chance of containing a chemicalwith specific activity against any one target, and given that syntheticchemicals are nearly always much easier to synthesise on an industrial scale, itis predictable that major agrochemical and pharmaceutical companies will devoteonly a limited amount of their R &; D budget to bioprospecting. Although argued that scientificadvances will make bioprospecting more cost-effective in future, an alternativescenario is presented where current biotechnological developments will furthererode the value of bioprospecting. It is concluded that there should be noreliance on large-income streams being available from bioprospecting agreementsto help fund the preservation of biodiversity.     

Loading of the centromeric histone H3 variant during meiosis–how does it differ from mitosis?

In eukaryotic phyla studied so far, the essential centromeric histone H3 variant (CENH3) is loaded to centromeric nucleosomes after S-phase (except for yeast) but before mitotic segregation (except for metazoan). While the C-terminal part of CENH3 seems to be sufficient for mitotic centromere function in plants, meiotic centromeres neither load nor tolerate impaired CENH3 molecules. However, details about CENH3 deposition in meiocytes are unknown (except for Drosophila). Therefore, we quantified fluorescence signals after the immunostaining of CENH3 along meiotic and mitotic nuclear division cycles of rye, a monocotyledonous plant. One peak of fluorescence intensity appeared in the early meiotic prophase of pollen mother cells and a second one during interkinesis, both followed by a decrease of CENH3. Then, the next loading occurred in the male gametophyte before its first mitotic division. These data indicate that CENH3 loading differs between mitotic and meiotic nuclei. Contrary to the situation in mitotic cycles, CENH3 deposition is biphasic during meiosis and apparently linked with a quality check, a removal of impaired CENH3 molecules, and a general loss of CENH3 after each loading phase. These steps ensure an endowment of centromeres with a sufficient amount of correct CENH3 molecules as a prerequisite for centromere maintenance during mitotic cycles of the microgametophyte and the progeny. From a comparison with data available for Drosophila, we hypothesise that the post-divisional mitotic CENH3 loading in metazoans is evolutionarily derived from the post-divisional meiotic loading phase, while the pre-divisional first meiotic loading has been conserved among eukaryotes.