HIM1, a new yeast Saccharomyces cerevisiae gene playing a role in control of spontaneous and induced mutagenesis

We have identified a new Saccharomyces cerevisiae gene, HIM1, mapped on the right arm of the chromosome IV (ORF YDR317w), mutations in which led to an increase in spontaneous mutation rate and elevated the frequencies of mutations, induced by UV-light, nitrous acid, ethylmethane sulfonate and methylmethane sulfonate. At the same time, him1 mutation did not result in the increase of the sensitivity to the lethal action of these DNA-damaging agents. We tested the induced mutagenesis in double mutants carrying him1 mutation and mutations in other repair genes: apn1, blocking base excision repair; rad2, rev3, and rad54, blocking three principal DNA repair pathways; pms1, blocking mismatch repair; hsm2 and hsm3 mutations, which lead to a mutator effect. Epistatic analysis showed a synergistic interaction of him1 with pms1, apn1, and rad2 mutations, and epistasis with the rev3, the rad54, the hsm2, and the hsm3. To elucidate the role of the HIM1 in control of spontaneous mutagenesis, we checked the repair of DNA mispaired bases in the him1 mutant and discovered that it was not altered in comparison to the wild-type strain. In our opinion, our results suggest that HIM1 gene participates in the control of processing of mutational intermediates appearing during error-prone bypass of DNA damage.     

Syntheses and crystal structures of cyano-bridged cluster-metal layered coordination polymers [Cu(dien)]3[W4Te4(CN)12] · 9H2O and [Ni(en)(NH3)]3[W4Se4(CN)12] · 7.5H2O

Two new tetrahedral tungsten cyanide cluster compounds, [Cu(dien)]₃[W₄Te₄(CN)₁₂] · 9H₂O (1) (dien=diethylenetriamine) and [Ni(en)(NH₃)]₃[W₄Se₄(CN)₁₂] · 7.5H₂O (2) (en=ethylenediamine), were synthesized by treating aqueous solutions of the saltlike cluster compound K₆[W₄Te₄(CN)₁₂] · 5H₂O/K₆[W₄Se₄(CN)₁₂] · 6H₂O with copper(II)/nickel(II) chloride in aqueous ammonia containing dien/en. The cyano-bridged layered coordination polymeric compounds were characterized by single-crystal X-ray diffraction analysis: monoclinic, space group P2₁ for 1; trigonal, space group for 2. Structures of 1 and 2 consist of infinite neutral layers of cluster components {W₄Te₄(CN)₁₂}/{W₄Se₄(CN)₁₂} connected, one another by {Cu(dien)} or {Ni(en)(NH₃)} fragments, respectively.     

Discrimination against deoxyribonucleotide substrates by bacterial RNA polymerase

Nucleic acid polymerases have evolved elaborate mechanisms that prevent incorporation of the non-cognate substrates, which are distinguished by both the base and the sugar moieties. While the mechanisms of substrate selection have been studied in single-subunit DNA and RNA polymerases (DNAPs and RNAPs, respectively), the determinants of substrate binding in the multisubunit RNAPs are not yet known. Molecular modeling of Thermus thermophilus RNAP-substrate NTP complex identified a conserved beta' subunit Asn(737) residue in the active site that could play an essential role in selection of the substrate ribose. We utilized the Escherichia coli RNAP model system to assess this prediction. Functional in vitro analysis demonstrates that the substitutions of the corresponding beta' Asn(458) residue lead to the loss of discrimination between ribo- and deoxyribonucleotide substrates as well as to defects in RNA chain extension. Thus, in contrast to the mechanism utilized by the single-subunit T7 RNAP where substrate selection commences in the inactive pre-insertion site prior to its delivery to the catalytic center, the bacterial RNAPs likely recognize the sugar moiety in the active (insertion) site.     

gamma-synuclein has a dynamic intracellular localization

gamma-Synuclein is a member of the synuclein family consisting of three proteins. Within the last several years increasing attention has focused on these proteins because of their role in human diseases. alpha-Synuclein relevance to Parkinson's disease is based on mutations found in familial cases of the disease and its presence in filaments and inclusion bodies in sporadic cases. gamma-Synuclein is implicated in some forms of cancer and ocular diseases, while beta-synuclein may antagonize their pathological functions. In this paper we present data on the localization and properties of gamma-synuclein in several neuronal and nonneuronal cell cultures. We show that contrary to the current opinion, gamma-synuclein is not an exclusively cytoplasmic protein, but has a dynamic localization and can associate with subcellular structures. It is present in the perinuclear area and may be associated to centrosomes. On late steps of mitosis gamma-synuclein is not found in the centrosomes, and redistributes to the midbody in telophase. Under stress conditions a translocation of gamma-synuclein from the perinuclear area to the nucleus occurs exhibiting nucleocytoplasmic shuttling. gamma-Synuclein overexpression reduces neurite outgrowth in a greater extent then alpha-synuclein overexpression. These data support the view that gamma-synuclein may change its intracellular localization and associate with subcellular structures in response to intracellular signaling or stress.     

RPA repair recognition of DNA containing pyrimidines bearing bulky adducts

Recognition of new DNA nucleotide excision repair (NER) substrate analogs, 48-mer ddsDNA (damaged double-stranded DNA), by human replication protein A (hRPA) has been analyzed using fluorescence spectroscopy and photoaffinity modification. The aim of the present work was to find quantitative characteristics of RPA-ddsDNA interaction and RPA subunits role in this process. The designed DNA structures bear bulky substituted pyrimidine nitrogen bases at the inner positions of duplex forming DNA chains. The photoreactive 4-azido-2,5-difluoro-3- pyridin-6-yl (FAP) and fluorescent antracenyl, pyrenyl (Antr, Pyr) groups were introduced via different linker fragments into exo-4N of deoxycytidine or 5C of deoxyuridine. J-dU-containing DNA was used as a photoactive model of undamaged DNA strands. The reporter group was a fluorescein residue, introduced into the 5'-phosphate end of one duplex-forming DNA strand. RPA-dsDNA association constants and the molar RPA/dsDNA ratio have been calculated based on fluorescence anisotropy measurements under conditions of a 1:1 RPA/dsDNA molar ratio in complexes. The evident preference for RPA binding to ddsDNA over undamaged dsDNA distinctly depends on the adduct type and varies in the following way: undamaged dsDNA < Antr-dC-ddsDNA < mmdsDNA < FAPdU-, Pyr-dU-ddsDNA < FAP-dC-ddsDNA (K(D) = 68 +/- 1; 25 +/- 6; 13 +/- 1; 8 +/- 2, and 3.5 +/- 0.5 nM correspondingly) but weakly depends on the chain integrity. Interestingly the bulkier lesions not in all cases have a greater effect on RPA affinity to ddsDNA. The experiments on photoaffinity modification demonstrated only p70 of compactly arranged RPA directly interacting with dsDNA. The formation of RPA-ddsDNA covalent adducts was drastically reduced when both strands of DNA duplex contained virtually opposite located FAP-dC and Antr-dC. Thus RPA requires undamaged DNA strand presence for the effective interaction with dsDNA bearing bulky damages and demonstrates the early NER factors characteristic features underlying strand discrimination capacity and poor activity of the NER system toward double damaged DNA.     

Taurine replacement attenuates hyperalgesia and abnormal calcium signaling in sensory neurons of STZ-D rats

The etiology of painful diabetic neuropathy is poorly understood, but may result from neuronal hyperexcitability secondary to alterations of Ca2+ signaling in sensory neurons. The naturally occurring amino acid taurine functions as an osmolyte, antioxidant, Ca2+ modulator, inhibitory neurotransmitter, and analgesic such that its depletion in diabetes may predispose one to neuronal hyperexcitability and pain. This study reports the effects of taurine replacement on hyperalgesia and sensory neuron Ca2+ homeostasis in streptozotocin-diabetic (STZ-D) rats. Nondiabetic and STZ-D rats were treated with a 2% taurine-supplemented diet for 6-12 wk. Thermal hyperalgesia and mechanical allodynia were determined by measuring hindpaw withdrawal latency to radiant heat and the withdrawal threshold to the von Frey anesthesiometer. Intracellular Ca2+ signaling was explored in neurons from L4-L6 dorsal root ganglia (DRG), using fura 2 fluorescence. Taurine replacement of diabetic rats attenuated deficits of nerve conduction and prevented reductions of mechanical and thermal withdrawal threshold and latency, respectively. In small DRG sensory neurons from diabetic rats, recovery of intracellular Ca2+ concentration ([Ca2+]i) in response to KCl was slowed and 73% corrected by taurine. The amplitudes of caffeine and ATP-induced [Ca2+]i transients were decreased by 47 and 27% (P < 0.05), respectively, in diabetic rat DRG sensory neurons and corrected by 74 and 93% (P < 0.05), respectively, by taurine replacement. These data indicate that taurine is important in the regulation of neuronal Ca2+ signaling and that taurine deficiency may predispose one to nerve hyperexcitability and pain, complicating diabetes.     

Switching of membrane organelles between cytoskeletal transport systems is determined by regulation of the microtubule-based transport

Intracellular transport of membrane organelles occurs along microtubules (MTs) and actin filaments (AFs). Although transport along each type of the cytoskeletal tracks is well characterized, the switching between the two types of transport is poorly understood because it cannot be observed directly in living cells. To gain insight into the regulation of the switching of membrane organelles between the two major transport systems, we developed a novel approach that combines live cell imaging with computational modeling. Using this approach, we measured the parameters that determine how fast membrane organelles switch back and forth between MTs and AFs (the switching rate constants) and compared these parameters during different signaling states. We show that regulation involves a major change in a single parameter: the transferring rate from AFs onto MTs. This result suggests that MT transport is the defining factor whose regulation determines the choice of the cytoskeletal tracks during the transport of membrane organelles.     

Effects of Zeolite-Containing Material and Ammonium Nitrate on Biodegradation of n-Tridecane in Heavy Clay-Loam Leached Chernozem with High Organic Matter Content

Biostimulation based on usage of soil amendments is growing due to their efficiency in removing different petroleum hydrocarbons (PHC) from contaminated sand or loam-sand soils. However, the research on clay-rich soils with higher organic carbon content, in which PHC biodegradation may proceed differently and which are more difficult to clean up, has been less extensive. In a pot experiment, we studied and compared the effects of two soil amendments, natural zeolite-containing material (ZCM, 50 g kg^?1) as a bulking agent and ammonium nitrate (0.3 g N kg^?1) as a nitrogen fertilizer, on biodegradation of n-tridecane (1 wt.%) in a weakly acidic heavy clay loam leached chernozem with fairly high organic carbon content (3.71%). After 48 days, the nitrogen-amended contaminated soil showed enhancement of both respiratory activity (basal and substrate-induced respiration rates) and the number of n-tridecane- degraders. As a consequence, the extent of n-tridecane biodegradation (86.5%) was essentially higher in the presence of added nitrogen than that in the non-amended soil (73.7%). In contrast, due to the partial retention of n-tridecane molecules in its pores, ZCM retarded biodegradation to 56.0%, showed no significant effect on the number of n-tridecane-degraders and, moreover, enhanced the decomposition of the soil intrinsic organic matter. The obtained data indicate that more precautions should be considered when using porous sorbents such as ZCM for remedial arrangements in PHC-contaminated soils.     

Tranexamic acid suppresses the release of mitochondrial DNA,protects the endothelial monolayer and enhances oxidative phosphorylation

Damage-associated molecular patterns, including mitochondrial DNA (mtDNA) are released during hemorrhage resulting in the development of endotheliopathy. Tranexamic acid (TXA), an antifibrinolytic drug used in hemorrhaging patients, enhances their survival despite the lack of a comprehensive understanding of its cellular mechanisms of action. The present study is aimed to elucidate these mechanisms, with a focus on mitochondria. We found that TXA inhibits the release of endogenous mtDNA from granulocytes and endothelial cells. Furthermore, TXA attenuates the loss of the endothelial monolayer integrity induced by exogenous mtDNA. Using the Seahorse XF technology, it was demonstrated that TXA strongly stimulates mitochondrial respiration. Studies using Mitotracker dye, cells derived from mito-QC mice, and the ActivSignal IPAD assay, indicate that TXA stimulates biogenesis of mitochondria and inhibits mitophagy. These findings open the potential for improvement of the strategies of TXA applications in trauma patients and the development of more efficient TXA derivatives.