Studies on the structure of the bioregulator purified from the rat brain

From the brain tissue of Wistar rats, we purified a bioregulator, which is active at ultralow doses. Using reversed-phase HPLC, we prepared a homogenous polypeptide with a molecular weight of 4749 ± 2 Da, which is responsible for the biological activity of the bioregulator. Using the CD spectroscopy method, we calculated the percentage of canonical elements of the secondary polypeptide structure in a solution. Using the methods of proteomics, we revealed that the structure of the investigated polypeptide was similar to the N-terminal sequence of a fragment of guanine-nucleotide binding G₀-protein subunit α-1.     

Dressing the mind properly for the game

Game theory as a theoretical and empirical approach to interaction has spread from economics to psychology, political science, sociology and biology. Numerous social interactions-foraging, talking, trusting, coordinating, competing-can be formally represented in a game with specific rules and strategies. These same interactions seem to rely on an interweaving of mental selves, but an effective strategy need not depend on explicit strategizing and higher mental capabilities, as less sentient creatures or even lines of software can play similar games. Human players are distinct because we are less consistent and our choices respond to elements of the setting that appear to be strategically insignificant. Recent analyses of this variable response have yielded a number of insights into the mental approach of human players: we often mentalize, but not always; we are endowed with social preferences; we distinguish among various types of opponents; we manifest different personalities; we are often guided by security concerns; and our strategic sophistication is usually modest.     

Working in the real and the imaginary

The science we practice is shaped by our interactions with people; the enthusiastic teachers, the fascinating mentors, the inspiring colleagues, and the inquisitive students. The science we enjoy takes us into areas we couldn''t have anticipated. From time to time, we come back to reality and try to find ways to share our new explorations with our friends and relatives and to convert our insights into collective progress. What could be a better job?     

They fought the law and the law won

The new science geo-engineering doesn't try to alter a few corn plants; it aims to tinker with the entire planet, based on the notion that ultimately we can actively manipulate the planet to have any climate pattern we want. But there is no way we can ever assess all of the likely consequences.     

An assessment of the impacts of molecular oxygen on the evolution of proteomes

Oxygen is not only one of life's essential elements but also a source of protein damage, mutagenesis, and ageing. Many proteome adaptations have been proposed to tackle such stresses and we assessed them using comparative genomics in a phylogenetic context. First, we find that aerobiosis is a trait with important phylogenetic inertia but that oxygen content in proteins is not. Instead, oxygen content is close to the expected values given the nucleotide composition. Accordingly, we find no evidence of oxygen being a scarce resource for protein synthesis even among anaerobes. Second, we searched for counterselection of amino acids more prone to oxidation among aerobes. Only cysteine follows the expected trend, whereas tryptophan follows the inverse one. When analyzing composition in the context of protein structures and residue accessibility, we find that all oxidable residues are avoided at the surface of proteins. Yet, there is no difference between aerobes and anaerobes in this respect, and the effect might be explained by the hydrophobicity of these residues. Third, we revisited the hypothesis that atmospheric enrichment in molecular oxygen led to the development of the communication capabilities of eukaryotes. With a larger data set and adequate controls, we confirm the trend of longer oxygen-rich outer domains in transmembrane proteins of eukaryotes. Yet, we find no significant association between oxygen concentration in the environment and this trait within prokaryotes, suggesting that this difference is clade specific and independent of oxygen availability. We find that genes involved in cellular responses to oxygen are much more frequent among aerobes, and we suggest that they erase most expected differences in terms of proteome composition between organisms facing high and low oxygen concentrations.     

Chaos in a minimal model of the alternative pathway of the complement system

In previous work, we introduced a minimal model of the alternative pathway of the complement. We also limited our analysis to a reduced set of parameter values because, for some parameters, experimentally supported estimates were not found. On the other hand, changes in value of some parameters may be a result of a pathological condition. Therefore, here we extend our analysis and include a wider range of values of five of the physiologically relevant parameters. For all the parameters considered, we observe chaotic oscillations, and we construct bifurcation diagrams using Poincaré sections of local maxima.     

Regulation of the anaphase-promoting complex by the COP9 signalosome

The COP9 complex (signalosome) is a known regulator of the proteasome/ubiquitin pathway. Furthermore it regulates the activity of the cullin-RING ligase (CRL) families of ubiquitin E3-complexes. Besides the CRL family, the anaphase-promoting complex (APC/C) is a major regulator of the cell cycle. To investigate a possible connection between both complexes we assessed interacting partners of COP9 using an in vivo protein-protein interaction assay. Hereby, we were able to show for the first time that CSN2, a subunit of the COP9 signalosome, interacts physically with APC/C. Furthermore, we detected a functional influence of the COP9 complex regarding the stability of several targets of the APC/C. Consistent with these data we showed a genetic instability of cells over-expressing CSN2.     

Characterization of the interactions between the nucleoprotein and the phosphoprotein of Henipavirus

The Henipavirus genome is encapsidated by the nucleoprotein (N) within a helical nucleocapsid that recruits the polymerase complex via the phosphoprotein (P). In a previous study, we reported that in henipaviruses, the N-terminal domain of the phosphoprotein and the C-terminal domain of the nucleoprotein (N(TAIL)) are both intrinsically disordered. Here we show that Henipavirus N(TAIL) domains are also disordered in the context of full-length nucleoproteins. We also report the cloning, purification, and characterization of the C-terminal X domains (P(XD)) of Henipavirus phosphoproteins. Using isothermal titration calorimetry, we show that N(TAIL) and P(XD) form a 1:1 stoichiometric complex that is stable under NaCl concentrations as high as 1 M and has a K(D) in the μM range. Using far-UV circular dichroism and nuclear magnetic resonance, we show that P(XD) triggers an increase in the α-helical content of N(TAIL). Using fluorescence spectroscopy, we show that P(XD) has no impact on the chemical environment of a Trp residue introduced at position 527 of the Henipavirus N(TAIL) domain, thus arguing for the lack of stable contacts between the C termini of N(TAIL) and P(XD). Finally, we present a tentative structural model of the N(TAIL)-P(XD) interaction in which a short, order-prone region of N(TAIL) (α-MoRE; amino acids 473-493) adopts an α-helical conformation and is embedded between helices α2 and α3 of P(XD), leading to a relatively small interface dominated by hydrophobic contacts. The present results provide the first detailed experimental characterization of the N-P interaction in henipaviruses and designate the N(TAIL)-P(XD) interaction as a valuable target for rational antiviral approaches.     

Relaxation of the myocardium of the perfused and ischaemic rabbit heart

The decrease in the rate of relaxation of the myocardium during ischaemic impairment of metabolic processes is accompanied by a decrease in the size of contraction. If we stimulate the ischaemic heart with irregularly distributed pulses we can achieve, by an extrasystolic potentiation mechanism, isolated contractions. If we stimulate the ischaemic heart with irregularly distributed pulses we can achieve, by an extrasystolic potentiation mechanism, isolated contractions of the same size as average contractions in normal perfusion. When comparing the relaxation of such contractions in 15 perfused rabbit hearts, we found a linear correlation between the relaxation rate and the size of the contractions. If we relate to relaxation rate to contraction size, the relaxation rate in early ischaemia (1 min after stopping perfusion) is thus in most cases normal, despite the marked decrease in the size of the contractions. The size of the contractions of the ischaemic mammalian myocardium thus seems to diminish before relaxation (which is likewise energy-dependent) is affected.     

From the trap to the basket: getting to the bottom of the nuclear pore complex

Nuclear pore complexes (NPCs) are large supramolecular assemblies that perforate the double-membraned nuclear envelope and serve as the sole gateways of molecular exchange between the cytoplasm and the nucleus in interphase cells. Combining novel specimen preparation regimes with innovative use of high-resolution scanning electron microscopy, Hans Ris produced in the late eighties stereo images of the NPC with unparalleled clarity and structural detail, thereby setting new standards in the field. Since that time, efforts undertaken to resolve the molecular structure and architecture, and the numerous interactions that occur between NPC proteins (nucleoporins), soluble transport receptors, and the small GTPase Ran, have led to a deeper understanding of the functional role of NPCs in nucleocytoplasmic transport. In spite of these breakthroughs, getting to the bottom of the actual cargo translocation mechanism through the NPC remains elusive and controversial. Here, we review recent insights into NPC function by correlating structural findings with biochemical data. By introducing new experimental and computational results, we reexamine how NPCs can discriminate between receptor-mediated and passive cargo to promote vectorial translocation in a highly regulated manner. Moreover, we comment on the importance and potential benefits of identifying and experimenting with individual key components implicated in the translocation mechanism. We conclude by dwelling on questions that we feel are pertinent to a more rational understanding of the physical aspects governing NPC mechanics. Last but not least, we substantiate these uncertainties by boldly suggesting a new direction in NPC research as a means to verify such novel concepts, for example, a de novo designed ‘minimalist’ NPC. This article is dedicated to the memory of Hans Ris.     

Truncation of the cytoplasmic domain of the simian immunodeficiency virus envelope glycoprotein alters the conformation of the external domain.

We previously reported that truncation of the cytoplasmic domain of the macaque simian immunodeficiency virus SIVmac239 envelope glycoprotein enhanced its ability to induce cell fusion in a variety of cell lines. In the present study, we examined the expression of the full-length and truncated SIVmac239 envelope glycoprotein complex on cell surfaces. Using a membrane-impermeable reagent to biotinylate proteins on cell surfaces followed by immunoprecipitation, we found that under conditions in which the full-length TM protein could not be detected on the surfaces of CD4-positive or CD4-negative cell lines, the truncated TM protein was detected efficiently. In contrast, using a membrane-impermeable iodination reagent to label proteins on cell surfaces, we could detect both the full-length and truncated TM proteins. No difference between the full-length and truncated proteins was observed in the detection of the SU proteins in the biotinylation assay. Additionally, we used an assay in which SIV-specific antibodies are prebound to the native envelope proteins expressed on the cell surface and then the proteins are immunoprecipitated. Using this assay, we could not detect the truncated or full-length TM protein on the cell surface, whereas we could detect the SU subunits of both proteins. We also observed that the truncated TM protein formed more stable sodium dodecyl sulfate-resistant oligomers than the full-length TM protein did. These results indicate that truncation of the cytoplasmic domain of the SIVmac239 envelope glycoprotein affects the conformation of the external domain of the TM protein on the cell surface, even though the two proteins have no differences in the amino acid sequences of their external domains. This altered conformation could play a role in the enhanced fusion activity of the truncated SIV glycoprotein.     

Hypothesis Testing of Inclusion of the Tolerance Interval for the Assessment of Food Safety

In the testing of food quality and safety, we contrast the contents of the newly proposed food (genetically modified food) against those of conventional foods. Because the contents vary largely between crop varieties and production environments, we propose a two-sample test of substantial equivalence that examines the inclusion of the tolerance intervals of the two populations, the population of the contents of the proposed food, which we call the target population, and the population of the contents of the conventional food, which we call the reference population. Rejection of the test hypothesis guarantees that the contents of the proposed foods essentially do not include outliers in the population of the contents of the conventional food. The existing tolerance interval (TI₀) is constructed to have at least a pre-specified level of the coverage probability. Here, we newly introduce the complementary tolerance interval (TI₁) that is guaranteed to have at most a pre-specified level of the coverage probability. By applying TI₀ and TI₁ to the samples from the target population and the reference population respectively, we construct a test statistic for testing inclusion of the two tolerance intervals. To examine the performance of the testing procedure, we conducted a simulation that reflects the effects of gene and environment, and residual from a crop experiment. As a case study, we applied the hypothesis testing to test if the distribution of the protein content of rice in Kyushu area is included in the distribution of the protein content in the other areas in Japan.     

Reflections on the responsible conduct of cancer research

Most cancer researchers regularly practice the responsible conduct of research (RCR) without consciously considering it. As professional scientists, we simply do what we are trained to do. However, as we train a new generation of cancer researchers in our laboratories, we must be vigilant against undue complacency. In an age when misconduct in research is receiving more media attention than ever before, we should periodically take a moment of pause and reflect upon the meaning and practice of responsibly conducting research. Rather than meeting minimum standards in a compliance-driven manner, we should practice forethought and periodically consider how we can improve. We, as leaders in cancer research, must then push our peers to do the same. By embedding RCR into the culture of cancer research through a multilayer approach, including regular assessment at the levels of individual research groups, departmentally, and institutionally, we will become a model discipline in the responsible conduct of research.     

On the epistemology of mental illness.

The most important epistemological problem in psychiatry is the detection of malingering. This is a consequence of the fact that there is no objective way to confirm any psychiatric diagnosis. Psychiatric diagnosis is based on subjective complaints. The discovery of objective markers for psychiatric diagnosis is problematic because it presupposes we can tell valid from faked subjective symptoms. But this is the difficulty. If we use pervasive irrationality as a sign of mental illness, we encounter the problem of identifying pervasive irrationality. To understand someone's behaviour, we have to assume it is largely rational. This precludes us from using behaviour to separate genuine from faked mental illness. There are a number of strategies used to solve any epistemological problem, and the most successful is the hypothetico-deductive method. If we use this, we can solve our epistemological problem. Genuine mental illness can be identified when it is the best explanation of the person's overall behaviour. Consilience of inductions is critical in supporting the validity of such explanations. This implies that it is merely a hypothesis that mental illness exists, and that we might discover that many mental illnesses, perhaps all, do not exist. We must embrace this possibility--only if we take a risk will we gain any knowledge.     

Adaptation in the face of internal conflict: the paradox of the organism revisited

The paradox of the organism refers to the observation that organisms appear to function as coherent purposeful entities, despite the potential for within-organismal components like selfish genetic elements and cancer cells to erode them from within. While it is commonly accepted that organisms may pursue fitness maximisation and can be thought to hold particular agendas, there is a growing recognition that genes and cells do so as well. This can lead to evolutionary conflicts between an organism and the parts that reside within it. Here, we revisit the paradox of the organism. We first outline its conception and relationship to debates about adaptation in evolutionary biology. Second, we review the ways selfish elements may exploit organisms, and the extent to which this threatens organismal integrity. To this end, we introduce a novel classification scheme that distinguishes between selfish elements that seek to distort transmission versus those that seek to distort phenotypic traits. Our classification scheme also highlights how some selfish elements elude a multi-level selection decomposition using the Price equation. Third, we discuss how the organism can retain its status as the primary fitness-maximising agent in the face of selfish elements. The success of selfish elements is often constrained by their strategy and further limited by a combination of fitness alignment and enforcement mechanisms controlled by the organism. Finally, we argue for the need for quantitative measures of both internal conflicts and organismality.     

Removal of the pro-domain does not affect the conformation of the procaspase-3 dimer.

We have investigated the oligomeric properties of procaspase-3 and a mutant that lacks the pro-domain (called pro-less variant). In addition, we have examined the interactions of the 28 amino acid pro-peptide when added in trans to the pro-less variant. By sedimentation equilibrium studies, we have found that procapase-3 is a stable dimer in solution at 25 degrees C and pH 7.2, and we estimate an upper limit for the equilibrium dissociation constant of approximately 50 nM. Considering the expression levels of caspase-3 in Jurkat cells, we predict that procaspase-3 exists as a dimer in vivo. The pro-less variant is also a dimer, with little apparent change in the equilibrium dissociation constant. Thus, in contrast with the long pro-domain caspases, the pro-peptide of caspase-3 does not appear to be involved in dimerization. Results from circular dichroism, fluorescence anisotropy, and FTIR studies demonstrate that the pro-domain interacts weakly with the pro-less variant. The data suggest that the pro-peptide adopts a beta-structure when in contact with the protein, but it is a random coil when free in solution. In addition, when added in trans, the pro-peptide does not inhibit the activity of the mature caspase-3 heterotetramer. On the other hand, the active caspase-3 does not efficiently hydrolyze the pro-domain at the NSVD(9) sequence as occurs when the pro-peptide is in cis to the protease domain. Based on these results, we propose a model for maturation of the procaspase-3 dimer.     

Constitutive expression of the maltoporin LamB in the absence of OmpR damages the cell envelope

Cells experience multiple environmental stimuli simultaneously. To survive, they must respond accordingly. Unfortunately, the proper response to one stress easily could make the cell more susceptible to a second coexistent stress. To deal with such a problem, a cell must possess a mechanism that balances the need to respond simultaneously to both stresses. Our recent studies of ompR malT(Con) double mutants show that elevated expression of LamB, the outer membrane porin responsible for maltose uptake, causes cell death when the osmoregulator OmpR is disabled. To obtain insight into the nature of the death experienced by ompR malT(Con) mutants, we described the death process. On the basis of microscopic and biochemical approaches, we conclude that death results from a loss of membrane integrity. On the basis of an unbiased genome-wide search for suppressor mutations, we conclude that this loss of membrane integrity results from a LamB-induced envelope stress that the cells do not sufficiently perceive and thus do not adequately accommodate. Finally, we conclude that this envelope stress involves an imbalance in the lipopolysaccharide/porin composition of the outer membrane and an increased requirement for inorganic phosphate.