DNA damage response in peripheral mouse blood leukocytes in vivo after variable,low-dose rate exposure

Environmental contamination and ingestion of the radionuclide Cesium-137 (137Cs) is a large concern in fallout from a nuclear reactor accident or improvised nuclear device, and highlights the need to develop biological assays for low-dose rate, internal emitter radiation. To mimic low-dose rates attributable to fallout, we have developed a VAriable Dose-rate External 137Cs irradiatoR (VADER), which can provide arbitrarily varying and progressive low-dose rate irradiations in the range of 0.1–1.2 Gy/day, while circumventing the complexities of dealing with radioactively contaminated biomaterials. We investigated the kinetics of mouse peripheral leukocytes DNA damage response in vivo after variable, low-dose rate 137Cs exposure. C57BL/6 mice were placed in the VADER over 7 days with total accumulated dose up to 2.7 Gy. Peripheral blood response including the leukocyte depletion, apoptosis as well as its signal protein p53 and DNA repair biomarker γ-H2AX was measured. The results illustrated that blood leukocyte numbers had significantly dropped by day 7. P53 levels peaked at day 2 (total dose = 0.91 Gy) and then declined; whereas, γ-H2AX fluorescence intensity (MFI) and foci number generally increased with accumulated dose and peaked at day 5 (total dose = 2.08 Gy). ROC curve analysis for γ-H2AX provided a good discrimination of accumulated dose < 2 Gy and ≥ 2 Gy, highlighting the potential of γ-H2AX MFI as a biomarker for dosimetry in a protracted, environmental exposure scenario.     

On the Importance of Amino Acid Sequence and Spatial Proximity of Interacting Residues for Protein Folding

Abstract

Membrane proteins can be classified among the following five types: (1) type I membrane protein. (2) type II membrane protein. (3) multipass transmembrane proteins. (4) lipid chain- anchored membrane proteins, and (5) GPI-anchored membrane proteins. T. Kohonen's self-organization model which is a typical neural network is applied for predicting the type of a given membrane protein based on its amino acid composition. As a result, the high rates of self-consistency (94.80%) and cross-validation (77.76%), and stronger fault-tolerant ability were obtained.     

146 Amyloid fibril polymorphism probed by advanced vibrational spectroscopy

Amyloid fibrils are associated with many neurodegenerative diseases. All known amyloids including pathogenic and nonpathogenic forms display functional and structural heterogeneity (polymorphism) which determines the level of their toxicity. Despite a significant biological and biomedical importance, the nature of the amyloid fibril polymorphism remains elusive. We utilized for the first time three most advanced vibrational techniques to probe the core, the surface, and supramolecular chirality of fibril polymorphs. A new type of folding, aggregation phenomenon, spontaneous refolding from one polymorph to another, was discovered (Kurouski, Lauro et al., 2010). Hydrogen–deuterium exchange deep UV resonance Raman spectroscopy (Oladepo, Xiong et al., 2012) combined with advanced statistical analysis (Shashilov & Lednev, 2010) allowed for structural characterization of the highly ordered cross-β core of amyloid fibrils. We reported several examples showing significant variations in the core structure for fibril polymorphs. Amyloid fibrils are generally composed of several protofibrils and may adopt variable morphologies, such as twisted ribbons or flat-like sheets. We discovered the existence of another level of amyloid polymorphism, namely, that associated with fibril supramolecular chirality. Two chiral polymorphs of insulin, which can be controllably grown by means of small pH variations, exhibit opposite signs of vibrational circular dichroism (VCD) spectra (Kurouski, Dukor et al. 2012). VCD supramolecular chirality is correlated not only by the apparent fibril handedness but also by the sense of supramolecular chirality from a deeper level of chiral organization at the protofilament level of fibril structure. A small pH change initiates spontaneous transformation of insulin fibrils from one polymorph to another. As a result, fibril supramolecular chirality overturns both accompanying morphological and structural changes (Kurouski, Dukor et al. 2012). No conventional methods could probe the fibril surface despite its significant role in the biological activity. We utilized tip-enhanced Raman spectroscopy (TERS) to characterize the surface structure of an individual fibril due to a high depth and lateral spatial resolution of the method in the nanometer range (Kurouski, Deckert-Gaudig et al. 2012). It was found that the surface is strongly heterogeneous and consists of clusters with various protein conformations and amino acid composition.     

From spring sources to springbrook: Changes in environmental characteristics and benthic fauna

Spatial patterns in macroinvertebrate communities and some abiotic factors were examined in three rheocrene springs and their springbrooks (Kraków-Cz?stochowa Upland, southern Poland). The mean discharge of particular springs ranged from 5 to 11 L s^?1, and its annual fluctuations were small. Water temperature was very stable at all sampling sites. In the eucrenon the number of benthic taxa was the smallest (9–14 determined to the family level), but the densities were the highest (approx. 14000 ind. m^?2). The biggest changes in macroinvertebrate composition were observed in the modified hypocrenon, which is an artificial pond. The lowest number of taxa were found in a natural, short springbrook with a nondiversified bottom substrate. The density of crenophilic taxa (Drusus trifidus, Dugesia gonocephala, Elmidae) diminished along the springbrooks, while the opposite trend was observed for ubiquitous taxa (some Oligochaeta, Asellus aquaticus and Chironomidae). Even in a very short natural springbrook (30 m), Drusus trifidus, the only species of Trichoptera found in the springs discussed here, goes through the entire development cycle. The strongest influence of a big river was observed at the outflow of one of the natural springbrooks, where the highest number of riverine oligochaete species were found. The benthic fauna of the springs studied here differed from that found in other springs in this area — the absence of the typical crenophilic species Bithynella austriaca (Gastropoda) and the presence of Gianus aquedulcis (Oligochaeta) may indicate the autonomy of the spring fauna in the Mstów area, possibly resulting from the postglacial geomorphological formation of this region or differences in habitat conditions.     

Development of a pharmacophore model for the catecholamine release-inhibitory peptide catestatin: Virtual screening and functional testing identify novel small molecule therapeutics of hypertension

The endogenous catecholamine release-inhibitory peptide catestatin (CST) regulates events leading to hypertension and cardiovascular disease. Earlier we studied the structure of CST by NMR, molecular modeling, and amino acid scanning mutagenesis. That structure has now been exploited for elucidation of interface pharmacophores that mediate binding of CST to its target, with consequent secretory inhibition. Designed pharmacophore models allowed screening of 3D structural domains. Selected compounds were tested on both cultured catecholaminergic cells and an in vivo model of hypertension; in each case, the candidates showed substantial mimicry of native CST actions, with preserved or enhanced potency and specificity. The approach and compounds have thus enabled rational design of novel drug candidates for treatment of hypertension or autonomic dysfunction.     

The central role of selenium in the biochemistry and ecology of the harmful pelagophyte,Aureococcus anophagefferens

The trace element selenium (Se) is required for the biosynthesis of selenocysteine (Sec), the 21st amino acid in the genetic code, but its role in the ecology of harmful algal blooms (HABs) is unknown. Here, we examined the role of Se in the biology and ecology of the harmful pelagophyte, Aureococcus anophagefferens, through cell culture, genomic analyses, and ecosystem studies. This organism has the largest and the most diverse selenoproteome identified to date that consists of at least 59 selenoproteins, including known eukaryotic selenoproteins, selenoproteins previously only detected in bacteria, and novel selenoproteins. The A. anophagefferens selenoproteome was dominated by the thioredoxin fold proteins and oxidoreductase functions were assigned to the majority of detected selenoproteins. Insertion of Sec in these proteins was supported by a unique Sec insertion sequence. Se was required for the growth of A. anophagefferens as cultures grew maximally at nanomolar Se concentrations. In a coastal ecosystem, dissolved Se concentrations were elevated before and after A. anophagefferens blooms, but were reduced by >95% during the peak of blooms to 0.05 nℳ. Consistent with this pattern, enrichment of seawater with selenite before and after a bloom did not affect the growth of A. anophagefferens, but enrichment during the peak of the bloom significantly increased population growth rates. These findings demonstrate that Se inventories, which can be anthropogenically enriched, can support proliferation of HABs, such as A. anophagefferens through its synthesis of a large arsenal of Se-dependent oxidoreductases that fine-tune cellular redox homeostasis.     

MOR Is Not Enough: Identification of Novel mu-Opioid Receptor Interacting Proteins Using Traditional and Modified Membrane Yeast Two-Hybrid Screens

The mu-opioid receptor (MOR) is the G-protein coupled receptor primarily responsible for mediating the analgesic and rewarding properties of opioid agonist drugs such as morphine, fentanyl, and heroin. We have utilized a combination of traditional and modified membrane yeast two-hybrid screening methods to identify a cohort of novel MOR interacting proteins (MORIPs). The interaction between the MOR and a subset of MORIPs was validated in pulldown, co-immunoprecipitation, and co-localization studies using HEK293 cells stably expressing the MOR as well as rodent brain. Additionally, a subset of MORIPs was found capable of interaction with the delta and kappa opioid receptors, suggesting that they may represent general opioid receptor interacting proteins (ORIPS). Expression of several MORIPs was altered in specific mouse brain regions after chronic treatment with morphine, suggesting that these proteins may play a role in response to opioid agonist drugs. Based on the known function of these newly identified MORIPs, the interactions forming the MOR signalplex are hypothesized to be important for MOR signaling and intracellular trafficking. Understanding the molecular complexity of MOR/MORIP interactions provides a conceptual framework for defining the cellular mechanisms of MOR signaling in brain and may be critical for determining the physiological basis of opioid tolerance and addiction.     

Shift work,circadian gene variants and risk of breast cancer

Circadian (clock) genes have been linked with several functions relevant to cancer, and epidemiologic research has suggested relationships with breast cancer risk for variants in NPAS2, CLOCK, CRY2 and TIMELESS. Increased breast cancer risk has also been observed among shift workers, suggesting potential interactions in relationships of circadian genes with breast cancer. Relationships with breast cancer of 100 SNPs in 14 clock-related genes, as well as potential interactions with shift work history, were investigated in a case–control study (1042 cases, 1051 controls). Odds ratios in an additive genetic model for European-ancestry participants (645 cases, 806 controls) were calculated, using a two-step correction for multiple testing: within each gene through permutation testing (10,000 permutations), and correcting for the false discovery rate across genes. Interactions of genotypes with ethnicity and shift work (<2 years vs ≥2 years) were evaluated individually. Following permutation analysis, two SNPs (rs3816360 in ARNTL and rs11113179 in CRY1) displayed significant associations with breast cancer and one SNP (rs3027188 in PER1) was marginally significant; however, none were significant following adjustment for the false discovery rate. No significant interaction with shift work history was detected. If shift work causes circadian disruption, this was not reflected in associations between clock gene variants and breast cancer risk in this study. Larger studies are needed to assess interactions with longer durations (>30 years) of shift work that have been associated with breast cancer.