The role of control groups in mutagenicity studies: matching biological and statistical relevance

The statistical test of the conventional hypothesis of "no treatment effect" is commonly used in the evaluation of mutagenicity experiments. Failing to reject the hypothesis often leads to the conclusion in favour of safety. The major drawback of this indirect approach is that what is controlled by a prespecified level alpha is the probability of erroneously concluding hazard (producer risk). However, the primary concern of safety assessment is the control of the consumer risk, i.e. limiting the probability of erroneously concluding that a product is safe. In order to restrict this risk, safety has to be formulated as the alternative, and hazard, i.e. the opposite, has to be formulated as the hypothesis. The direct safety approach is examined for the case when the corresponding threshold value is expressed either as a fraction of the population mean for the negative control, or as a fraction of the difference between the positive and negative controls.     

On the distribution of the epoch of the first contact of immunocompetent cell with antigen

A hypothesis on the dynamics of the immune reaction at a cellular level is considered, as was presented by Šterzl (1967). According to this hypothesis, the onset of the immune response depends on the first contact of a so-called virgin immunocompetent cell with the antigen. In the present paper a conditional probability distribution of the epoch of this first contact is derived, assuming that such a contact occurs at all (probability of non-occurrence of the contact being, in the general case, positive); the density of this distribution (7) is then specified (8) for the case that decrease of the quantity of an active antigen follows an exponential curve, and moments of this distribution are calculated (12).     

Reactions between half- and full-FLP recombination target sites. A model system for analyzing early steps in FLP protein-mediated site-specific recombination.

The FLP recombination target (FRT) can be cut in half so that only one FLP protein binding site is present (a "half site"). FLP protein binds the half sites and joins them into dimeric, asymmetric head-to-head complexes held together chiefly by strong noncovalent interactions. These complexes react with full (normal) FRT sites to generate a variety of products. Analysis of these DNA species reveals that the reaction follows a well-defined reaction pathway that generally parallels the normal reaction pathway. The system is useful in analyzing early steps in recombination, since the identity of the products in a given recombination event unambiguously pinpoints the order in which the cleavage and strand exchange reactions occur. Two conclusions are derived from the present study: (i) Formation of the dimeric head-to-head complex of half sites is a prerequisite to further steps in recombination. (ii) The identity of the base pairs at positions 6 and -6 within the FRT site has a subtle effect in directing the first strand exchange event in the reaction to predominantly one of two possible cleavage sites. In addition, results are presented that suggest that a DNA-DNA pairing intermediate involving only two base pairs of the core sequence is formed prior to the first cleavage and strand exchange. DNA-DNA interactions may therefore not be limited to the isomerization step that follows the first strand exchange.     

Monte Carlo simulation of the enzymatic lysis of yeast

The overall reaction in the enzymatic lysis of yeast takes place in three major steps: (i) the two-layer wall is digested, (ii) the cell bursts under the osmotic pressure difference to release its intracellular material, and (iii) the intracellular material is digested by the enzymes still present in the solution. The first and third steps are continuous processes, adequately described by Michaelis-Menten kinetic models. The second step is a discrete event, statistical in nature. A model of engineering value should effectively bridge the gap between the two continuous processes (first and third steps). In this work, Monte Carlo simulations are used to identify a suitable function that captures the statistical nature of cell rupture and represents the rate of release of intracellular material. It is shown that the two-parameter beta distribution function serves this purpose most effectively. Comparisons with experimental results indicate that the cell rupture ratio is a widely distributed statistical function. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 290-295, 1997.     

Theoretical insights into the protonation states of active site cysteine and citrullination mechanism of Porphyromonas gingivalis peptidylarginine deiminase

Porphyromonas gingivalis peptidylarginine deiminase (PPAD) catalyzes the citrullination of peptidylarginine, which plays a critical role in the rheumatoid arthritis (RA) and gene regulation. For a better understanding of citrullination mechanism of PPAD, it is required to establish the protonation states of active site cysteine, which is still a controversial issue for the members of guanidino‐group‐modifying enzyme superfamily. In this work, we first explored the transformation between the two states: State N (both C351 and H236 are neutral) and State I (both residues exist as a thiolate–imidazolium ion pair), and then investigated the citrullination reaction of peptidylarginine, using a combined QM/MM approach. State N is calculated to be more stable than State I by 8.46 kcal/mol, and State N can transform to State I via two steps of substrate‐assisted proton transfer. Citrullination of the peptidylarginine contains deamination and hydrolysis. Starting from State N, the deamination reaction corresponds to an energy barrier of 18.82 kcal/mol. The deprotonated C351 initiates the nucleophilic attack to the substrate, which is the key step for deamination reaction. The hydrolysis reaction contains two chemical steps. Both the deprotonated D238 and H236 can act as the bases to activate the hydrolytic water, which correspond to similar energy barriers (～17 kcal/mol). On the basis of our calculations, C351, D238, and H236 constitute a catalytic triad, and their protonation states are critical for both the deamination and hydrolysis processes. In view of the sequence similarity, these findings may be shared with human PAD1–PAD4 and other guanidino‐group‐modifying enzymes. Proteins 2017; 85:1518–1528. © 2017 Wiley Periodicals, Inc.     

Coupling of conformational folding and disulfide-bond reactions in oxidative folding of proteins

The oxidative folding of proteins consists of conformational folding and disulfide-bond reactions. These two processes are coupled significantly in folding-coupled regeneration steps, in which a single chemical reaction (the "forward" reaction) converts a conformationally unstable precursor species into a conformationally stable, disulfide-protected successor species. Two limiting-case mechanisms for folding-coupled regeneration steps are described. In the folded-precursor mechanism, the precursor species is preferentially folded at the moment of the forward reaction. The (transient) native structure increases the effective concentrations of the reactive thiol and disulfide groups, thus favoring the forward reaction. By contrast, in the quasi-stochastic mechanism, the forward reaction occurs quasi-stochastically in an unfolded precursor; i.e., reactive groups encounter each other with a probability determined primarily by loop entropy, albeit modified by conformational biases in the unfolded state. The resulting successor species is initially unfolded, and its folding competes with backward chemical reactions to the unfolded precursors. The folded-precursor and quasi-stochastic mechanisms may be distinguished experimentally by the dependence of their kinetics on factors affecting the rates of thiol--disulfide exchange and conformational (un)folding. Experimental data and structural and biochemical arguments suggest that the quasi-stochastic mechanism is more plausible than the folded-precursor mechanism for most proteins.     

A knowledge-based three dimensional model of the Photosystem II reaction center of Chlamydomonas reinhardtii

In this Minireview, a comparison of the binding niches of the PS II cofactors from several existing models of the PS II reaction center is provided. In particular, it discusses a three dimensional model of the Photosystem II (PS II) reaction center including D1, D2 and cytochrome b559 proteins from the green alga Chlamydomonas reinhardtii that was specifically generated for this Minireview. This model is the most complete to date and includes accessory chlorophyll_zs, a manganese cluster, two molecules of -carotene and cytochrome b559, all of which are essential components of the PS II reaction center. The modeling of the D1 and D2 proteins was primarily based on homology with the L and M subunits of the anoxygenic purple bacterial photosynthetic reaction centers. The non-homologous loop regions were built using a sequence specific approach by searching for the best-matched protein segments in the Protein Data Bank, and by imposing the matching conformations on the corresponding D1 and D2 regions. Cytochrome b559 which is in close proximity to D1 and D2 was tentatively modeled in / conformation and docked on the Q_B side of the PS II reaction center according to experimental suggestions. An alternate docking on the Q_A side is also shown for comparison. The cofactors in the PS II reaction center were modeled either by adopting the structures from the bacterial counterparts, when available, with modifications based on existing experimental data or by de novo modeling and docking in the most probable positions in the reaction center complex. The specific features of this model are the inclusion of the tetramanganese cluster (with calcium and chloride ions) in a open, C-shaped structure modeled within the D1/D2/cytochrome b559 complex with D1-D170, D1-E189, D1-D342 and D1-A344 as putative ligands; and the modeling of two cis -carotenes and two accessory chlorophyll_zs liganded by D1-H118 and D2-H117. We also analyzed residues in the model which may be involved in the D1 and D2 inter-protein interactions, as well as residues which may be involved in putative bicarbonate and water binding and transport.     

Huntington disease in Finland: linkage disequilibrium of chromosome 4 RFLP haplotypes and exclusion of a tight linkage between the disease and D4S43 locus.

The question about heterogeneity of Huntington disease (HD) at the DNA level can be approached by analyzing the RFLP haplotypes formed by several RFLP loci of the diseased chromosome in different populations. In genetically isolated populations such as Finland, it is further possible to use this approach to test the hypothesis of a single mutation enriched in this population demonstrating an exceptionally low prevalence of HD. In this study covering 70% of all diagnosed HD cases in Finland, linkage disequilibrium of RFLP haplotypes of D4S10 and D4S43 loci polymorphisms was found. This phenomenon, not so far reported in any other population, could support the hypothesis of one ancestor HD mutation in the Finnish population. Despite the lower heterozygosity obtained with some RFLP markers, the proportion of individuals receiving informative DNA test results did not significantly differ from that reported in more mixed populations. In one HD family we established a recombination event between HD and the D4S43 locus, an event which can be highly useful in the more precise mapping of the HD gene.     

The design and analysis of microarray experiments: applications in parasitology

Microarray experiments can generate enormous amounts of data, but large datasets are usually inherently complex, and the relevant information they contain can be difficult to extract. For the practicing biologist, we provide an overview of what we believe to be the most important issues that need to be addressed when dealing with microarray data. In a microarray experiment we are simply trying to identify which genes are the most "interesting" in terms of our experimental question, and these will usually be those that are either overexpressed or underexpressed (upregulated or downregulated) under the experimental conditions. Analysis of the data to find these genes involves first preprocessing of the raw data for quality control, including filtering of the data (e.g., detection of outlying values) followed by standardization of the data (i.e., making the data uniformly comparable throughout the dataset). This is followed by the formal quantitative analysis of the data, which will involve either statistical hypothesis testing or multivariate pattern recognition. Statistical hypothesis testing is the usual approach to "class comparison," where several experimental groups are being directly compared. The best approach to this problem is to use analysis of variance, although issues related to multiple hypothesis testing and probability estimation still need to be evaluated. Pattern recognition can involve "class prediction," for which a range of supervised multivariate techniques are available, or "class discovery," for which an even broader range of unsupervised multivariate techniques have been developed. Each technique has its own limitations, which need to be kept in mind when making a choice from among them. To put these ideas in context, we provide a detailed examination of two specific examples of the analysis of microarray data, both from parasitology, covering many of the most important points raised.     

A null allele of esterase D is a marker for genetic events in retinoblastoma formation

Summary The development of homozygosity or hemizygosity in the 13q14 region by deletion, mitotic recombination, or chromosomal loss has been interpreted as a primary event in retinoblastoma. This finding is consistent with the hypothesis that inactivation of both alleles of a gene located at 13q14.11 is required for tumorigenesis. Observations reported by Benedict and colleagues in one case of bilateral retinoblastoma, LA-RB 69, provided early evidence in favor of this hypothesis. By examining levels of esterase D, an enzyme also mapping to 13q14.11, it was previously inferred that one chromosome 13 in this patient's somatic cells contained a submicroscopic deletion of the Rb and esterase D loci and that this chromosome was retained in her tumor while the normal chromosome 13 was lost. Using a rabbit anti-esterase D antibody and the esterase D cDNA probe, we have found that (1) low but detectable quantities of esterase D protein and enzymatic activity are present in tumor cells from LA-RB 69; (2) fibroblast from this patient contain two copies of the esterase D gene, indicated by heterozygosity at an ApaI polymorphic site within this gene; and (3) tumor cells from the same patient are homozygous at this site, indicating loss and reduplication of the esterase D locus. These results demonstrate that one of the two esterase D alleles in this patient acted as a null or silent allele — that is, was present in the genome with markedly decreased protein expression. This mutant allele acted as a marker for tumor-associated loss of chromosome 13 heterozygosity, in concordance with previous proposals.     

A simple mathematical model based on the cancer stem cell hypothesis suggests kinetic commonalities in solid tumor growth

Background

The Cancer Stem Cell (CSC) hypothesis has gained credibility within the cancer research community. According to this hypothesis, a small subpopulation of cells within cancerous tissues exhibits stem-cell-like characteristics and is responsible for the maintenance and proliferation of cancer.

Methodologies/Principal Findings

We present a simple compartmental pseudo-chemical mathematical model for tumor growth, based on the CSC hypothesis, and derived using a “chemical reaction” approach. We defined three cell subpopulations: CSCs, transit progenitor cells, and differentiated cells. Each event related to cell division, differentiation, or death is then modeled as a chemical reaction. The resulting set of ordinary differential equations was numerically integrated to describe the time evolution of each cell subpopulation and the overall tumor growth. The parameter space was explored to identify combinations of parameter values that produce biologically feasible and consistent scenarios.

Conclusions/Significance

Certain kinetic relationships apparently must be satisfied to sustain solid tumor growth and to maintain an approximate constant fraction of CSCs in the tumor lower than 0.01 (as experimentally observed): (a) the rate of symmetrical and asymmetrical CSC renewal must be in the same order of magnitude; (b) the intrinsic rate of renewal and differentiation of progenitor cells must be half an order of magnitude higher than the corresponding intrinsic rates for cancer stem cells; (c) the rates of apoptosis of the CSC, transit amplifying progenitor (P) cells, and terminally differentiated (D) cells must be progressively higher by approximately one order of magnitude. Simulation results were consistent with reports that have suggested that encouraging CSC differentiation could be an effective therapeutic strategy for fighting cancer in addition to selective killing or inhibition of symmetric division of CSCs.     

Carbon-13 and deuterium isotope effects on the catalytic reactions of biotin carboxylase

13C and 2H kinetic isotope effects have been used to investigate the mechanism of enzymic biotin carboxylation. D(V/K) is 0.50 in 80% D2O at pD 8.0 for the forward reaction and 0.57 at pD 8.5 for the phosphorylation of ADP by carbamoyl phosphate. These values approach the theoretical maximum limit for a reaction in which a proton is transferred from a sulfhydryl to a nitrogen or oxygen base. Therefore, it appears that this portion of the reaction is at or near equilibrium. 13(V/K) at pH 8 is 1.007; the small magnitude of this number suggests that the reaction is almost fully committed by the time the carbon-sensitive steps are reached. There does not appear to be a reverse commitment to the reaction under the conditions in which 13(V/K) was determined. A large forward commitment is consistent with the failure to observe positional isotope exchange from the beta gamma-bridge position to the beta-nonbridge position in [18O4]ATP or washout of 18O from the gamma-nonbridge positions. Transfer of 18O from bicarbonate to inorganic phosphate in the forward reaction was clearly observed, however. These observations suggest that biotin carboxylase exists in two distinct forms which differ in the protonation states of the two active-site bases, one of which is a sulfhydryl. Only when the sulfhydryl is ionized and the second base protonated can catalysis take place. Carboxylation of biotin is postulated to occur via a pathway in which carboxyphosphate in formed by nucleophilic attack of bicarbonate on ATP.(ABSTRACT TRUNCATED AT 250 WORDS)     

DNA cleavage in trans by the active site tyrosine during Flp recombination: switching protein partners before exchanging strands.

Each recombination event mediated by the Flp recombinase is the sum of four strand breakage and reunion reactions executed in two steps of two-strand exchanges. The reaction requires four Flp monomers. The key catalytic residue in Flp is Tyr-343. Arg-191, His-305, and Arg-308 appear to facilitate the cleavage and exchange steps of recombination. These four residues constitute the invariant tetrad of the Int family site-specific recombinases. Complementation tests between "step-arrest" mutants of Flp suggest that each Flp protomer harbors a "fractional active site." Hybrid "half site-recombinase" complexes reveal that efficient catalysis occurs when the Arg-His-Arg triad is present on one Flp monomer and the active site Tyr on a second monomer. Strand cleavage by an Flp monomer occurs virtually exclusively on the half site to which its partner protein is bound (cleavage in trans), and almost never on the half site to which it is bound (cleavage in cis). Trans-cleavage by Flp can provide a means for functionally exchanging Flp monomers between two DNA partners. Such a mechanism would be germane to recombination, since cleavage and rejoining in cis can only restore the parental substrate configuration and cannot yield recombinants.     

Folding mechanism of indole-3-glycerol phosphate synthase from Sulfolobus solfataricus: a test of the conservation of folding mechanisms hypothesis in (beta(alpha))(8) barrels

As a test of the hypothesis that folding mechanisms are better conserved than sequences in TIM barrels, the equilibrium and kinetic folding mechanisms of indole-3-glycerol phosphate synthase (sIGPS) from the thermoacidophilic archaebacterium Sulfolobus solfataricus were compared to the well-characterized models of the alpha subunit of tryptophan synthase (alphaTS) from Escherichia coli. A multifaceted approach combining urea denaturation and far-UV circular dichroism, tyrosine fluorescence total intensity, and tyrosine fluorescence anisotropy was employed. Despite a sequence identity of only 13%, a stable intermediate (I) in sIGPS was found to be similar to a stable intermediate in alphaTS in terms of its thermodynamic properties and secondary structure. Kinetic experiments revealed that the fastest detectable folding event for sIGPS involves a burst-phase (<5ms) reaction that leads directly to the stable intermediate. The slower of two subsequent phases reflects the formation/disruption of an off-pathway dimeric form of I. The faster phase reflects the conversion of I to the native state and is limited by folding under marginally stable conditions and by isomerization or rearrangement under strongly folding conditions. By contrast, alphaTS is thought to fold via an off-pathway burst-phase intermediate whose unfolding controls access to a set of four on-pathway intermediates that comprise the stable equilibrium intermediate. At least three proline isomerization reactions are known to limit their interconversions and lead to a parallel channel mechanism. The simple sequential mechanism deduced for sIGPS reflects the dominance of the on-pathway burst-phase intermediate and the absence of prolyl residues that partition the stable intermediate into kinetically distinguishable species. Comparison of the results for sIGPS and alphaTS demonstrates that the thermodynamic properties and the final steps of the folding reaction are better conserved than the early events. The initial events in folding appear to be more sensitive to the sequence differences between the two TIM barrel proteins.     

Evolutionary history and biogeography of the drongos (Dicruridae), a tropical Old World clade of corvoid passerines

We address the phylogenetic relationships of the drongos (Dicruridae) at the species-level using sequences from two nuclear (myoglobin intron-2 and c-mos) and two mitochondrial (ND2 and cytochrome b) loci. The resulting phylogenetic tree shows that the most basal species is D. aeneus, followed in the tree by a trichotomy including (1) the Asian D. remifer, (2) a clade of all African and Indian Ocean islands species as well as two Asian species (D. macrocercus and D. leucophaeus) and (3) a clade that includes all other Asian species as well as two Australasian species (D. megarhynchus and D. bracteatus). Our phylogenetic hypotheses are compared to [Mayr, E., Vaurie, C., 1948. Evolution of the family Dicruridae (Birds). Evolution 2, 238-265.] hypothetical family "tree" based on traditional phenotypic analysis and biogeography. We point out a general discrepancy between the so-called "primitive" or "unspecialized" species and their position in the phylogenetic tree, although our results for other species are congruent with previous hypotheses. We conduct dating analyses using a relaxed-clock method, and propose a chronology of clades formation. A particular attention is given to the drongo radiation in Indian Ocean islands and to the extinction-invasion processes involved. The first large diversification of the family took place both in Asia and Africa at 11.9 and 13.3Myr, respectively, followed by a dispersal event from Africa to Asia at ca 10.6Myr; dispersal over Wallace line occurred later at ca 6Myr. At 5Myr, Principe and Indian Ocean Islands have been colonized from an African ancestor; the most recent colonization event concerned Anjouan by an immigrating population from Madagascar.     

Differences in the catalytic mechanisms of mesophilic and thermophilic indole-3-glycerol phosphate synthase enzymes at their adaptive temperatures

Thermophilic enzymes tend to be less catalytically-active at lower temperatures relative to their mesophilic counterparts, despite having very similar crystal structures. An often cited hypothesis for this general observation is that thermostable enzymes have evolved a more rigid tertiary structure in order to cope with their more extreme, natural environment, but they are also less flexible at lower temperatures, leading to their lower catalytic activity under mesophilic conditions. An alternative hypothesis, however, is that complementary thermophilic-mesophilic enzyme pairs simply operate through different evolutionary-optimized catalytic mechanisms. In this communication, we present evidence that while the steps of the catalytic mechanisms for mesophilic and thermophilic indole-3-glycerol phosphate synthase (IGPS) enzymes are fundamentally similar, the identity of the rate-determining step changes as a function of temperature. Our findings indicate that while product release is rate-determining at 25°C for thermophilic IGPS, near its adaptive temperature (75°C), a proton transfer event, involving a general acid, becomes rate-determining. The rate-determining steps for thermophilic and mesophilic IGPS enzymes are also different at their respective, adaptive temperatures with the mesophilic IGPS-catalyzed reaction being rate-limited before irreversible CO2 release, and the thermophilic IGPS-catalyzed reaction being rate limited afterwards.     

Testing for clonal propagation

The conceptual basis for testing clonal propagation is reconsidered with the result that two steps need to be distinguished clearly: (1) specification of the characteristics of multilocus genotype frequencies that result from sexual reproduction together with the kinds of deviations from these characteristics that are produced by clonal propagation, and (2) a statistical method for detecting these deviations in random samples. It is pointed out that a meaningful characterization of sexual reproduction reflects the association of genes in (multilocus) genotypes within the bounds set by the underlying gene frequencies. An appropriate measure of relative gene association is developed which is equivalent to a multilocus generalization of the standardized gametic disequilibrium (linkage disequilibrium). Its application to the characterization of sexually produced multilocus genotypes is demonstrated. The resulting hypothesis on the frequency of a sexually produced genotype is tested with the help of the (significance) probability of obtaining at least two copies of the genotype in question in a random sample of a given size. If at least two copies of the genotype are observed in a sample, and if the probability is significant, then the hypothesis of sexual reproduction is rejected in favor of the assumption that all copies of the genotype belong to the same clone. Common testing approaches rest on the hypothesis of completely independent association of genes in genotypes and on the (significance) probability of obtaining at least as many copies of a genotype as observed in a sample. The validity of these approaches is discussed in relation to the above considerations and recommendations are set out for conducting appropriate tests.