Simulating the fidelity and the three Mg mechanism of pol η and clarifying the validity of transition state theory in enzyme catalysis

Pol η belongs to the important Y family of DNA polymerases that can catalyze translesion synthesis across sites of damaged DNA. This activity involves the reduced fidelity of Pol η for 8‐oxo‐7,8‐dhyedro‐2′‐deoxoguanosin(8‐oxoG). The fundamental interest in Pol η has grown recently with the demonstration of the importance of a 3rd Mg2+ ion. The current work explores both the fidelity of Pol η and the role of the 3rd metal ion, by using empirical valence bond (EVB) simulations. The simulations reproduce the observed trend in fidelity and shed a new light on the role of the 3rd metal ion. It is found that this ion does not lead to a major catalytic effect, but most probably plays an important role in reducing the product release barrier. Furthermore, it is concluded, in contrast to some implications, that the effect of this metal does not violate transition state theory, and the evaluation of the catalytic effect must conserve the molecular composition upon moving from the reactant to the transition state. Proteins 2017; 85:1446–1453. © 2017 Wiley Periodicals, Inc.     

Transition state theory can be used in studies of enzyme catalysis: lessons from simulations of tunnelling and dynamical effects in lipoxygenase and other systems

The idea that enzyme catalysis involves special factors such as coherent fluctuations, quantum mechanical tunnelling and non-equilibrium solvation (NES) effects has gained popularity in recent years. It has also been suggested that transition state theory (TST) cannot be used in studies of enzyme catalysis. The present work uses reliable state of the art simulation approaches to examine the above ideas. We start by demonstrating that we are able to simulate any of the present catalytic proposals using the empirical valence bond (EVB) potential energy surfaces, the dispersed polaron model and the quantized classical path (QCP) approach, as well as the approximate vibronic method. These approaches do not treat the catalytic effects by phenomenological treatments and thus can be considered as first principles approaches (at least their ability to compare enzymatic reaction to the corresponding solution reactions). This work will consider the lipoxygenase reaction, and to lesser extent other enzymes, for specific demonstration. It will be pointed out that our study of the lipoxygenase reaction reproduces the very large observed isotope effect and the observed rate constant while obtaining no catalytic contribution from nuclear quantum mechanical (NQM) effects. Furthermore, it will be clarified that our studies established that the NQM effect decreases rather than increases when the donor-acceptor distance is compressed. The consequences of these findings in terms of the temperature dependence of the kinetic isotope effect and in terms of different catalytic proposals will be discussed. This paper will also consider briefly the dynamical effects and conclude that such effects do not contribute in a significant way to enzyme catalysis. Furthermore, it will be pointed out that, in contrast to recent suggestions, NES effects are not dynamical effects and should therefore be part of the activation free energy rather than the transmission factor. In view of findings of the present work and our earlier works, it seems that TST provides a quantitative tool for studies of enzyme catalysis and that the key open questions are related to the nature of the factors that lead to transition state stabilization.     

Transgenic organisms expressing genes from Bacillus thuringiensis to combat insect pests

Various subspecies (ssp.) of Bacillus thuringiensis (Bt) are considered the best agents known so far to control insects, being highly specific and safe, easily mass produced and with long shelf life.1 The para-crystalline body that is produced during sporulation in the exosporium includes polypeptides named δ-endotoxins, each killing a specific set of insects. The different entomopathogenic toxins of various Bt ssp. can be manipulated genetically in an educated way to construct more efficient transgenic bacteria or plants that express combinations of toxin genes to control pests.2 Joint research projects in our respective laboratories during the last decade demonstrate what can be done by implementing certain ideas using molecular biology with Bt ssp. israelensis (Bti) as a model system. Here, we describe our progress achieved with Gram-negative bacterial species, including cyanobacteria, and some preliminary experiments to form transgenic plants, mainly to control mosquitoes (Diptera), but also a particular Lepidopteran and Coleopteran pest species. In addition, a system is described by which environment-damaging genes can be removed from the recombinants thus alleviating procedures for obtaining permits to release them in nature.     

Challenges and advances in validating enzyme design proposals: the case of kemp eliminase catalysis

One of the fundamental challenges in biotechnology and biochemistry is the ability to design effective enzymes. Despite recent progress, most of the advances on this front have been made by placing the reacting fragments in the proper places, rather than by optimizing the preorganization of the environment, which is the key factor in enzyme catalysis. Thus, rational improvement of the preorganization would require approaches capable of evaluating reliably the actual catalytic effect. This work considers the catalytic effects in different Kemp eliminases as a benchmark for a computer-aided enzyme design. It is shown that the empirical valence bond provides a powerful screening tool, with significant advantages over current alternative strategies. The insights provided by the empirical valence bond calculations are discussed with an emphasis on the ability to analyze the difference between the linear free energy relationships obtained in solution and those found in the enzymes. We also point out the trade-off between the reliability and speed of the calculations and try to determine what it takes to realize reliable computer-aided screening.     

Propagating Waves of Directionality and Coordination Orchestrate Collective Cell Migration

The ability of cells to coordinately migrate in groups is crucial to enable them to travel long distances during embryonic development, wound healing and tumorigenesis, but the fundamental mechanisms underlying intercellular coordination during collective cell migration remain elusive despite considerable research efforts. A novel analytical framework is introduced here to explicitly detect and quantify cell clusters that move coordinately in a monolayer. The analysis combines and associates vast amount of spatiotemporal data across multiple experiments into transparent quantitative measures to report the emergence of new modes of organized behavior during collective migration of tumor and epithelial cells in wound healing assays. First, we discovered the emergence of a wave of coordinated migration propagating backward from the wound front, which reflects formation of clusters of coordinately migrating cells that are generated further away from the wound edge and disintegrate close to the advancing front. This wave emerges in both normal and tumor cells, and is amplified by Met activation with hepatocyte growth factor/scatter factor. Second, Met activation was found to induce coinciding waves of cellular acceleration and stretching, which in turn trigger the emergence of a backward propagating wave of directional migration with about an hour phase lag. Assessments of the relations between the waves revealed that amplified coordinated migration is associated with the emergence of directional migration. Taken together, our data and simplified modeling-based assessments suggest that increased velocity leads to enhanced coordination: higher motility arises due to acceleration and stretching that seems to increase directionality by temporarily diminishing the velocity components orthogonal to the direction defined by the monolayer geometry. Spatial and temporal accumulation of directionality thus defines coordination. The findings offer new insight and suggest a basic cellular mechanism for long-term cell guidance and intercellular communication during collective cell migration.     

How well can post-traumatic stress disorder be predicted from pre-trauma risk factors? An exploratory study in the WHO World Mental Health Surveys

Post‐traumatic stress disorder (PTSD) should be one of the most preventable mental disorders, since many people exposed to traumatic experiences (TEs) could be targeted in first response settings in the immediate aftermath of exposure for preventive intervention. However, these interventions are costly and the proportion of TE‐exposed people who develop PTSD is small. To be cost‐effective, risk prediction rules are needed to target high‐risk people in the immediate aftermath of a TE. Although a number of studies have been carried out to examine prospective predictors of PTSD among people recently exposed to TEs, most were either small or focused on a narrow sample, making it unclear how well PTSD can be predicted in the total population of people exposed to TEs. The current report investigates this issue in a large sample based on the World Health Organization (WHO)'s World Mental Health Surveys. Retrospective reports were obtained on the predictors of PTSD associated with 47,466 TE exposures in representative community surveys carried out in 24 countries. Machine learning methods (random forests, penalized regression, super learner) were used to develop a model predicting PTSD from information about TE type, socio‐demographics, and prior histories of cumulative TE exposure and DSM‐IV disorders. DSM‐IV PTSD prevalence was 4.0% across the 47,466 TE exposures. 95.6% of these PTSD cases were associated with the 10.0% of exposures (i.e., 4,747) classified by machine learning algorithm as having highest predicted PTSD risk. The 47,466 exposures were divided into 20 ventiles (20 groups of equal size) ranked by predicted PTSD risk. PTSD occurred after 56.3% of the TEs in the highest‐risk ventile, 20.0% of the TEs in the second highest ventile, and 0.0‐1.3% of the TEs in the 18 remaining ventiles. These patterns of differential risk were quite stable across demographic‐geographic sub‐samples. These results demonstrate that a sensitive risk algorithm can be created using data collected in the immediate aftermath of TE exposure to target people at highest risk of PTSD. However, validation of the algorithm is needed in prospective samples, and additional work is warranted to refine the algorithm both in terms of determining a minimum required predictor set and developing a practical administration and scoring protocol that can be used in routine clinical practice.     

Seminal plasma induces the expression of IL-1α in normal and neoplastic cervical cells via EP2/EGFR/PI3K/AKT pathway

Background

Cervical cancer is a chronic inflammatory disease of multifactorial etiology usually presenting in sexually active women. Exposure of neoplastic cervical epithelial cells to seminal plasma (SP) has been shown to promote the growth of cancer cells in vitro and tumors in vivo by inducing the expression of inflammatory mediators including pro-inflammatory cytokines. IL-1α is a pleotropic pro-inflammatory cytokine induced in several human cancers and has been associated with virulent tumor phenotype and poorer prognosis. Here we investigated the expression of IL-1α in cervical cancer, the role of SP in the regulation of IL-1α in neoplastic cervical epithelial cells and the molecular mechanism underlying this regulation.

Methods and results

Real-time quantitative RT-PCR confirmed the elevated expression of IL-1α mRNA in cervical squamous cell carcinoma and adenocarcinoma tissue explants, compared with normal cervix. Using immunohistochemistry, IL-1α was localized to the neoplastically transformed squamous, columnar and glandular epithelium in all cases of squamous cell carcinoma and adenocarcinomas explants studied. We found that SP induced the expression of IL-α in both normal and neoplastic cervical tissue explants. Employing HeLa (adenocarcinoma) cell line as a model system we identified PGE₂ and EGF as possible ligands responsible for SP-mediated induction of IL-1α in these neoplastic cells. In addition, we showed that SP activates EP2/EGFR/PI3kinase-Akt signaling to induce IL-1α mRNA and protein expression. Furthermore, we demonstrate that in normal cervical tissue explants the induction of IL-1α by SP is via the activation of EP2/EGFR/PI3 kinase-Akt signaling.

Conclusion

SP-mediated induction of IL-1α in normal and neoplastic cervical epithelial cells suggests that SP may promote cervical inflammation as well as progression of cervical cancer in sexually active women.

     

Decrease in Vitamin D Status in the Greenlandic Adult Population from 1987–2010

Background

Low vitamin D status may be pronounced in Arctic populations due to limited sun exposure and decreasing intake of traditional food.

Objective

To investigate serum 25(OH)D3 as a measure of vitamin D status among adult Inuit in Greenland, predictors of low serum 25(OH)D3 concentrations and the trend from 1987 to 2005–2010.

Design

A total of 2877 randomly selected Inuit (≥18 years) from the Inuit Health in Transition study were included. A sub-sample (n = 330) donated a blood sample in 1987 which allowed assessment of time trends in vitamin D status.

Results

The geometric mean serum 25(OH)D3 (25[OH]D2 concentrations were negligible and not reported) in 2005–2010 was lowest among the 18–29 year old individuals (30.7 nmol/L; 95% CI: 29.7; 31.7) and increased with age. In all age-groups it decreased from 1987 to 2005–2010 (32%–58%). Low 25(OH)D3 concentrations (<50 nmol/L) were present in 77% of the 18–29 year old and decreased with age. A characteristic seasonal variation in 25(OH)D3 concentrations was observed (range 33.2–57.1 nmol/L, p<0.001), with the highest concentrations in August to October. Age (2.0% per year increase; CI: 1.7, 2.2), female gender (7.1%; CI: 2.0; 12.5), alcohol intake (0.2% per increase in drinks/week; 0.0; 0.4), and traditional diet (10.0% per 100 g/d increase; CI: 7.9; 12.1) were associated with increased serum 25(OH)D3, whereas smoking (−11.6%; CI: −16.2; −6.9), BMI (−0.6%; CI: −1.1; −0.2) and latitude (−0.7% per degree increase; CI: −1.3; −0.2) were associated with decreased concentrations.

Conclusion

We identified a remarkable decrease in vitamin D status from 1987 to 2005–2010 and a presently low vitamin D status among Inuit in Greenland. A change away from a traditional diet may well explain the observed decline. The study argues for the need of increased dietary intake of vitamin D and supplementation might be considered.     

Nuclear accumulation of multiple protein kinases during prolactin-induced proliferation of Nb2 rat lymphoma cells

Intracellular kinases play important roles in signal transduction and are involved in the surface receptor-mediated regulation of cellular functions, including mitogenesis. In the present study, we examined the possible involvement of various protein kinases in the passage of a mitogenic signal from the cell surface to the nucleus of Nb₂ cells, a rat nodal lymphoma cell line in which prolactin is a mitogen. Following a prolactin challenge, various kinase activities were monitored at short intervals in different cellular fractions over a 60 min period. Protein kinase C (PKC) activity in the cytosolic fraction rapidly declined to 50% of its original activity within the first 30 min, while PKC activity in the nuclear fractions increased sharply, reaching its highest level by 30 min following a prolactin challenge. There were also increases in both casein kinase and protein tyrosine kinase (PTK) activities in the nuclear fractions during the first 30 min following a prolactin challenge that paralleled PKC activity. The activities of all three kinases declined thereafter, reaching levels close to their respective basal values by 60 min following initiation of prolactin treatment. These observations suggest the possibility that multiple protein kinases may be involved in mitogenic signal transduction for prolactin in Nb₂ cells. © 1996 Wiley-Liss, Inc.     

The fate of Bacillus thuringiensis var. israelensis in B. thuringiensis var. israelensis-killed pupae of Aedes aegypti

Carcasses of mosquito larvae killed by Bacillus thuringiensis var. israelensis allow its complete growth cycle (germination, vegetative growth, and sporulation), thus becoming toxic themselves to scavenging larvae. In this study, we demonstrate that the bacterium is capable of inducing death of Aedes aegypti pupae and of recycling in the resulting carcasses. B. thuringiensis var. israelensis-killed pupae were obtained by treating 40-hr-old synchronized fourth instar larvae with a low dose of spores (8000/ml). The fraction of dead pupae was reduced by higher or lower spore concentrations as well as by treating younger or older larval populations (both fourth instar): Increased proportions of dead larvae were obtained at higher concentration or by earlier treatment, whereas lower concentrations or later treatment resulted in more living pupae. Multiplication of B. thuringiensis var. israelensis is shown to occur in the carcasses of dead pupae. The number of spores in each pupal carcass followed a similar kinetic as in larval carcasses, but the final yield was about 10-fold higher, apparently reflecting the difference in dry weight between the two mosquito developmental stages (426 micrograms vs 83 micrograms, respectively). The specific larvicidal activity in a homogenized dead pupa was similar to that of B. thuringiensis var. israelensis powder, LC50 of about 600 spores/ml.