On the origin of the stereoselective affinity of Nutlin-3 geometrical isomers for the MDM2 protein

The stereoselective affinity of small-molecule binding to proteins is typically broadly explained in terms of the thermodynamics of the final bound complex. Using Brownian dynamics simulations, we show that the preferential binding of the MDM2 protein to the geometrical isomers of Nutlin-3, an effective anticancer lead that works by inhibiting the interaction between the proteins p53 and MDM2, can be explained by kinetic arguments related to the formation of the MDM2:Nutlin-3 encounter complex. This is a diffusively bound state that forms prior to the final bound complex. We find that the MDM2 protein stereoselectivity for the Nutlin-3a enantiomer stems largely from the destabilization of the encounter complex of its mirror image enantiomer Nutlin-3b, by the K70 residue that is located away from the binding site. On the other hand, the trans-Nutlin-3a diastereoisomer exhibits a shorter residence time in the vicinity of MDM2 compared with Nutlin-3a due to destabilization of its encounter complex by the collective interaction of pairs of charged residues on either side of the binding site: Glu25 and Lys51 on one side, and Lys94 and Arg97 on the other side. This destabilization is largely due to the electrostatic potential of the trans-Nutlin-3a isomer being largely positive over extended continuous regions around its structure, which are otherwise well-identified into positive and negative regions in the case of the Nutlin-3a isomer. Such rich insight into the binding processes underlying biological selectivity complements the static view derived from the traditional thermodynamic analysis of the final bound complex. This approach, based on an explicit consideration of the dynamics of molecular association, suggests new avenues for kinetics-based anticancer drug development and discovery.     

Effect of boundary type and season on predatory arthropods associated with field margins on New Zealand farmland

Pitfall traps were used to monitor predatory arthropod numbers along two types of field boundary, a post and wire fence line and a Cupressus macrocarpa hedge, along the same paddock margin in Canterbury, New Zealand, over 24 months. The seven most abundant predator groups recorded were: Araneae > Phalangiidae > Staphylinidae > Coccinellidae > Chilopoda > Hemerobiidae > Carabidae. Araneae, Phalangiidae, Staphylinidae, Chilopoda and Hemerobiidae were found in larger numbers at the wire fence than at the hedge site, whereas the numbers of Carabidae and Coccinellidae adults exhibited no field margin preference. However, more species of Araneae and Staphylinidae were caught at the hedge site, whereas species richness of carabid beetles was greatest at the wire fence. Principal component analysis clearly separated the samples collected from the two habitats based on the assemblages of Araneae, Staphylinidae and Carabidae, and certain species in each of these taxonomic groups appeared to be particularly associated with one boundary type or the other. All the main taxonomic groups exhibited clear seasonal patterns, with distinct peaks in abundance occurring at certain times of the year. The results of the study reinforce the idea that management of field boundaries can be used to manipulate the type and abundance of particular groups of predatory arthropods, and that seasonal patterns should be taken into account in schemes of integrated pest management so that any adverse effects of biocide application on these beneficial species may be minimised.     

Isolation of two populations of sperm cells and microelectrophoresis of pairs of sperm cells from pollen tubes of tobacco (Nicotiana tabacum)

Prior research has indicated that the two sperm cells of Nicotiana tabacum are dimorphic, suggesting that they may participate in preferential fertilization during in vivo fusion with the egg and central cells. To probe the mechanism of potential preferential fertilization in this plant, it will be necessary to use modern sensitive molecular techniques. For this purpose, two individual populations of two sperm cells, constituting the Svn (associated with the vegetative nucleus) and Sua (unassociated with the vegetative nucleus), were isolated in the thousands from tobacco pollen tubes with a micromanipulator as a preliminary step toward research on gametic recognition using molecular techniques. Microelectrophoresis of paired sperm cells from a single pollen tube was conducted at different developmental stages. Sperm cells isolated from 1-, 2-, 3- and 4-cm stylar lengths migrated to the negative pole, with the Sua displaying significantly greater electrophoretic mobility than the Svn, reflecting a more positively charged cell surface on the Sua. The sperm cells isolated from 1-cm style are very sensitive to electron potential in an electrophoretic field, presumably reflecting that they are still in a young state. Differences in cell surface charge between the Sua and Svn may be related with cell fate during fertilization. Supported by National Natural Science Foundation of CHINA (30170060)     

Prevalence and Molecular Genetic Typing of Nonsyndromic Sensorineural Deafness in Chuvash Republic

Summarized genetic epidemiological characteristics of nonsyndromic sensorineural deafness in six raions of Chuvash Republic (Cheboksary, Kanash, Morgaushi, Tsivil'sk, Mariinski Posad, and Alatyr') are presented. A total of 264419 individuals were examined. Forty-five families (60 affected individuals) with autosomal recessive (AR) and 8 families (18 affected individuals) with autosomal dominant (AD) nonsyndromic sensorineural deafness (NSSD) were identified. The load of AD and AR NSSD in the raions examined was estimated. A correlation between the distribution of AR NSSD and genetic drift was demonstrated. Furthermore, the load of AR NSSD was substantially higher in the regions with higher differentiation level. The Spearman's correlation coefficient value was 0.87. Typing of the 35delG mutation in the gene for connexion 26 was carried out in 34 patients from 26 families with AR NSSD. Comparative estimates of the NSSD prevalence in a number of Russian populations were performed.     

Selective structure of the gene pool. IV. Estimation from the selection intensity index Rs

A new approach for investigating the selective structure of the gene pool reflecting the type and intensity of selection is proposed. Selection pressure is estimated on the basis of interpopulation gene diversity with the use of the selection intensity index: RS(i) = NeS(i) = 1/4(1/FST(i)-1/Fe). Distributions of RS(i) in gene pools of indigenous populations from all continents and five subregions of the northeastern Eurasia were examined. It was shown that, of all theoretical distributions, only beta-distributions provide a good approximation of RS(i) estimates. Based on the confidence intervals of RS obtained from beta-distributions, genes can be grouped into the three following classes according to their selective structure: LOWER DIFF, NEUTRAL, and SUPER DIFF. These classes, respectively, include genes subjected mainly to stabilizing selection (RS(i) > 0; LOWER DIFF), genes subjected mainly to differentiating selection (RS(i) < 0; SUPER DIFF), and arbitrarily selectively neutral genes (RS(i) approximately 0; NEUTRAL). Simulation of gene pool sampling (10(6) samples from 50 markers for each gene pool) allowed us to characterize the selective structure by determining markers that fall into the same selective class irrespective of the variant for the sampling process. The selective structure of gene pools from six continents (Europe, Asia, Africa, Australia, America, and southeastern Eurasia) and five subregions of northeastern Eurasia was characterized. It was shown that approximately one-third of genes is subjected to selection irrespective of the hierarchical level of the region. In gene pools of Europe, northeastern Eurasia, and European and Ural subregions, the proportion of genes under stabilizing selection was higher, the proportion of selectively neutral genes, lower. Debatable issues of tests for selective neutrality based on heterogeneity of interpopulation gene diversity are considered. These issues include the effect on FST of the hierarchical population structure, sample size, number of subpopulations, and other factors that shift estimates of gene selective values.     

"Synthetic" maps of the Mari gene pool (from immunobiochemical polymorphism data)]

Models of geographic distribution of 33 alleles of 10 loci (AB0, TF, GC, PI, HP, AHS, F13B, ACP1, PGM1, GLO1) in the indigenous population of five raions (districts) of Marii El Republic were analyzed by cartographic statistical methods. Based on 33 maps for individual alleles, synthetic maps were constructed; they reflected the general characteristics of the spatial variability of the Mari gene pool. A map of reliability of the synthetic maps was also obtained. This study was the first to use estimates of the reliability of the gene-geographic prognosis for constructing and interpreting the maps of principal components. Synthetic maps of principal components reveal the geography of the main factors that determine the genetic diversity of the Maris. In the map of the first principal component (accounting for 25.5% of the total variation of the Mari gene pool), isolines clearly ran in the latitudinal direction; i.e., the variability exhibited a north-south gradient. The direction of changes reflects the characteristic features of the microevolution of the Mari gene pool, because it differs from the direction of the principal components of in the total Ural gene pool. The second principal component (24.3% of variation) also exhibited a latitudinal gradient in the western part of Marii El. In the eastern part of the republic, isolines drastically change their direction and display a marked west-east gradient. This longitudinal orientation of principal components is characteristic of the Maris in the synthetic maps of the Ural region. Contributions of individual genes in the variation of principal components were analyzed. In proceeding from the geographic space to the space of principal components, it was found that Highland Maris are separated from Meadow Maris not only geographically, but also genetically.     

Pulse Decomposition Analysis of the digital arterial pulse during hemorrhage simulation

Background

Markers of temporal changes in central blood volume are required to non-invasively detect hemorrhage and the onset of hemorrhagic shock. Recent work suggests that pulse pressure may be such a marker. A new approach to tracking blood pressure, and pulse pressure specifically is presented that is based on a new form of pulse pressure wave analysis called Pulse Decomposition Analysis (PDA). The premise of the PDA model is that the peripheral arterial pressure pulse is a superposition of five individual component pressure pulses, the first of which is due to the left ventricular ejection from the heart while the remaining component pressure pulses are reflections and re-reflections that originate from only two reflection sites within the central arteries. The hypothesis examined here is that the PDA parameter T13, the timing delay between the first and third component pulses, correlates with pulse pressure. T13 was monitored along with blood pressure, as determined by an automatic cuff and another continuous blood pressure monitor, during the course of lower body negative pressure (LBNP) sessions involving four stages, -15 mmHg, -30 mmHg, -45 mmHg, and -60 mmHg, in fifteen subjects (average age: 24.4 years, SD: 3.0 years; average height: 168.6 cm, SD: 8.0 cm; average weight: 64.0 kg, SD: 9.1 kg).

Results

Statistically significant correlations between T13 and pulse pressure as well as the ability of T13 to resolve the effects of different LBNP stages were established. Experimental T13 values were compared with predictions of the PDA model. These interventions resulted in pulse pressure changes of up to 7.8 mmHg (SE = 3.49 mmHg) as determined by the automatic cuff. Corresponding changes in T13 were a shortening by -72 milliseconds (SE = 4.17 milliseconds). In contrast to the other two methodologies, T13 was able to resolve the effects of the two least negative pressure stages with significance set at p < 0.01.

Conclusions

The agreement of observations and measurements provides a preliminary validation of the PDA model regarding the origin of the arterial pressure pulse reflections. The proposed physical picture of the PDA model is attractive because it identifies the contributions of distinct reflecting arterial tree components to the peripheral pressure pulse envelope. Since the importance of arterial pressure reflections to cardiovascular health is well known, the PDA pulse analysis could provide, beyond the tracking of blood pressure, an assessment tool of those reflections as well as the health of the sites that give rise to them.     

Mutation C3256T of Mitochondrial Genome in White Blood Cells: Novel Genetic Marker of Atherosclerosis and Coronary Heart Disease

This study was undertaken to examine the association between the level of heteroplasmy for the mutation C3256T in human white blood cells and the extent of carotid atherosclerosis, as well as the presence of coronary heart disease (CHD), the major clinical manifestation of atherosclerosis. Totally, 191 participants (84 men, 107 women) aged 65.0 years (SD 9.4) were recruited in the study; 45 (24%) of them had CHD. High-resolution B-mode ultrasonography of carotids was used to estimate the extent of carotid atherosclerosis by measuring of the carotid intima-media thickness (cIMT). DNA samples were obtained from whole venous blood, and then PCR and pyrosequencing were carried out. On the basis of pyrosequencing data, the levels of C3256T heteroplasmy in DNA samples were calculated. The presence of the mutant allele was detected in all study participants; the level of C3256T heteroplasmy in white blood cells ranged from 5% to 74%. The highly significant relationship between C3256T heteroplasmy level and predisposition to atherosclerosis was revealed. In individuals with low predisposition to atherosclerosis the mean level of C3256T heteroplasmy was 16.8%, as compared to 23.8% in moderately predisposed subjects, and further to 25.2% and 28.3% in significantly and highly predisposed subjects, respectively. The level of C3256T heteroplasmy of mitochondrial genome in human white blood cells is a biomarker of mitochondrial dysfunction and risk factor for atherosclerosis; therefore, it can be used as an informative marker of genetic susceptibility to atherosclerosis, coronary heart disease and myocardial infarction.     

MADS about MOSS

Classic MIKC-type MADS-box genes (MIKC^c) play diverse and crucial roles in angiosperm development, the most studied and best understood of which is the specification of floral organ identities. To shed light on how the flower evolved, phylogenetic and functional analyses of genes involved in its ontogeny, such as the MIKC^c genes, must be undertaken in as broad a selection as possible of plants with disparate ancestries. Since little is known about the functions of these genes in non-seed plants, we investigated the developmental roles of a subset of the MIKC^c genes present in the moss, Physcomitrella patens, which is positioned informatively near the base of the land plant evolutionary tree. We observed that transgenic lines possessing an antisense copy of a MIKC^c gene characteristically displayed knocked-down expression of the corresponding native MIKC^c gene as well as multiple diverse phenotypic alterations to the haploid gametophytic and diploid sporophytic generations of the life cycle.¹ In this addendum, we re-examine our findings in the light of recent pertinent literature and provide additional data concerning the effects of simultaneously knocking out multiple MIKC^c genes in this moss.Key words: Physcomitrella, moss, MADS-box gene functions, gene knock-down, gene knockout, gene expression, evo-devoThe moss, Physcomitrella patens, is the only non-seed plant that is amenable to an investigation of MADS-box gene function comparable to that achieved in angiosperms. P. patens possesses six MIKC^c genes which cluster into two distinct phylogenetic clades.² We recently reported a functional genetic analysis of the three genes (PPM1, PPM2 and PpMADS1) within the PPM2-like clade as an initial contribution towards gaining an understanding of the role(s) of MIKC^c genes in this moss.¹By fusing the respective MIKC^c promoters to a GUS reporter gene, we found that both PPM1 and PpMADS1 exhibited fairly ubiquitous expression patterns in both gametophytic and sporophytic tissues. The levels of PPM1 expression were generally higher than those of PpMADS1, and PpMADS1 was not expressed in antheridia, suggesting subtle differences in the functions of these genes. The observed patterns of widespread expression resemble those characterising the majority of vascular, non-seed plant MIKC^c genes^3–7 and accord with RT-PCR results of Quodt and coworkers.² Our in situ GUS expression and RT-PCR results¹ showed that PPM2 was not expressed or was expressed at levels too low to be detected by these methods. Conversely, the original isolation of PPM2 cDNA⁸ and data from more recent expression studies² indicated that PPM2 is expressed (albeit inconsistently and weakly) ubiquitously with elevated levels of expression sometimes observed in gametangia, sporophytic feet and basal portions of sporophytic setae.² The contradictory expression data for PPM2 may derive from differences between the PPM2-reporter gene constructs used by the respective research groups^1,2 or perhaps from variations in moss culture conditions.We also employed an antisense approach designed to knock down expression of PPM1, and perhaps closely related MIKC^c genes, in order to discern MADS-box gene function in P. patens. Knocked-down strains displayed a complex mutant phenotype comprising delayed gametangia formation and sporophyte production, diminished sporophyte yields, and morphological abnormalities in both leaves and sporophytes, findings that are generally consistent with the ubiquitous expression pattern of PPM1¹ and PPM2''s expression as described by Quodt et al.²The phenotypes of strains with single gene knockouts of PPM1, PPM2 or PpMADS1 appeared to be perfectly normal, not displaying any of the phenotypic alterations observed in PPM1 gene knock-down mutants. While it is possible that subtle, transient or conditional phenotypic changes went unnoticed, it seems more probable that genetic redundancy is responsible for these results since the PPM2-like genes exhibit a very high level of sequence similarity. In an effort to circumvent the problem of functional redundancy, we generated all double knockout combinations for PPM1, PPM2 and PpMADS1. However, the double mutants were also phenotypically unchanged. Finally we attempted to produce triple mutants by co-transforming single PPM2 knockout lines with PPM1 and PpMADS1 linear knockout constructs. Of the 31 stable transformants from two transformation experiments, 55% were shown to be double mutants in which the original PPM2 knockout was accompanied by a second gene knockout in either PPM1 or PpMADS1. However, no triple knockouts were obtained. Given the knockout frequencies generally observed in batch transformation experiments in our laboratory and those of others,⁹ between two and five of the transformants had been expected to be triple mutants. These preliminary data, albeit involving a relatively small sample of transformants, suggest that PPM1, PPM2 and PpMADS1 triple knockouts may be lethal.We have related compelling evidence that functionally redundant PPM2-like MIKC^c genes are involved in several aspects of the moss developmental program. It has been argued that broad expression patterns like theirs represent the ancestral state of MADS-box genes in land plants, and that the sporophytic- and organ-specific expression patterns that characterise many MIKC^c genes in extant spermatophytes, including those that specify floral organ identity, correspond to a derived condition that evolved in the spermatophyte lineage following its separation from lineages that led to bryophytes and ferns and fern allies.¹⁰ Nevertheless, it is the apparent participation of PPM2-like genes in the formation of gametangia (the differentiation of reproductive organs from non-reproductive tissues at the gametophore apex) that is particularly interesting and assumes a special significance because of its analogy to the proposed role for ancestors of seed plant C-function MADS-box genes (identifying those regions of the vegetative SAM that will become reproductive organs).11 Furthermore, expression studies of MIKC^c genes in two charophycean algae, the presumed progenitors of all terrestrial plants,^12–14 suggest that they too are involved in haploid reproductive cell differentiation.¹⁵ While these functional similarities do not infer orthology and may be coincidental, we should not discount yet the admittedly controversial hypothesis that some MIKC^c genes in non-seed plants, for example PPM2-like genes of Physcomitrella, are homologous to spermatophyte class C genes and that the ancient role proposed for ancestral class C genes¹¹ has been conserved, in some form, in all major terrestrial plant taxa.     

Quantitative analysis of repetitive sequences in human genomic DNA and detection of an elevated ribosomal repeat copy number in patients with schizophrenia (the results of molecular and cytogenetic analysis)

A modified version of quantitating repetitive sequences in genomic DNA was developed to allow comparisons for numerous individual genomes and simultaneous analysis of several sequences in each DNA specimen. The relative genomic content of ribosomal repeats (rDNA) was estimated for 75 individuals, including 33 healthy donors (HD) and 42 schizophrenic patients (SP). The rDNA copy number in HD was 427 +/- 18 (mean SE) per diploid nucleus, ranging 250-600. In SP, the rDNA copy number was 494 +/- 15 and ranged 280-670, being significantly higher than in HD. The two samples did not differ in contents of sequences hybridizing with probes directed to a subfraction of human satellite III or to the histone genes. Cytogenetic analysis (silver staining of metaphase chromosomes) showed that the content of active rRNA genes in nucleolus organizer regions is higher in SP compared with HD. The possible causes of the elevated rRNA gene dosage in SP were considered. The method employed was proposed for studying the polymorphism for genomic content of various repeats in higher organisms, including humans.