Redox regulation of morphology,cell stiffness,and lectin-induced aggregation of human platelets

Redox regulation and carbohydrate recognition are potent molecular mechanisms which can contribute to platelet aggregation in response to various stimuli. The purpose of this study is to investigate the relationship between these mechanisms and to examine whether cell surface glycocalyx and cell stiffness of human platelets are sensitive to the redox potential formed by glutathione. To this end, human platelets were treated with different concentrations (0.05 μM to 6 mM) and ratios of reduced or oxidized glutathione (GSH or GSSG), and platelet morphological, mechanical, and functional properties were determined using conventional light microscopy, atomic force microscopy, and lectin-induced cell aggregation analysis. It was found that lowering the glutathione redox potential changed platelet morphology and increased platelet stiffness as well as modulated nonuniformly platelet aggregation in response to plant lectins with different carbohydrate-binding specificity including wheat germ agglutinin, Sambucus nigra agglutinin, and Canavalia ensiformis agglutinin. Extracellular redox potential and redox buffering capacity of the GSSG/2GSH couple were shown to control the availability of specific lectin-binding glycoligands on the cell surface, while the intracellular glutathione redox state affected the general functional ability of platelets to be aggregated independently of the type of lectins. Our data provide the first experimental evidence that glutathione as a redox molecule can affect the mechanical stiffness of human platelets and induce changes of the cell surface glycocalyx, which may represent a new mechanism of redox regulation of intercellular contacts.     

Expression and characterization of the bacterial mechanosensitive channel MscS in Xenopus laevis oocytes

We have successfully expressed and characterized mechanosensitive channel of small conductance (MscS) from Escherichia coli in oocytes of the African clawed frog, Xenopus laevis. MscS expressed in oocytes has the same single-channel conductance and voltage dependence as the channel in its native environment. Two hallmarks of MscS activity, the presence of conducting substates at high potentials and reversible adaptation to a sustained stimulus, are also exhibited by oocyte-expressed MscS. In addition to its ease of use, the oocyte system allows the user to work with relatively large patches, which could be an advantage for the visualization of membrane deformation. Furthermore, MscS can now be compared directly to its eukaryotic homologues or to other mechanosensitive channels that are not easily studied in E. coli.     

A Model of Genetic Search for Beneficial Mutations: Estimating the Constructive Capacities of Mutagenesis

We attempted to answer the following question: What evolutionary conditions are required to generate novel genetic modules? Our broad formulation of the problem allows us to simultaneously consider such issues as the relationship between the stage of "genetic search" and the rate of adaptive evolution; the theoretical limits to the generative capacities of spontaneous mutagenesis; and the correlation between genome organization and evolvability. We show that adaptive evolution is feasible only when the mutation rate is fine-tuned to a specific range of values and the structures of the genome and genes are optimized in a certain way. Our quantitative analysis has demonstrated that the rate of evolution of novelty depends on several parameters, such as genome size, the length of a module, the size of the adjacent nonfunctional DNA spacers, and the mutation rate at various genomic scales. We evaluated the efficiency of some mechanisms that increase evolvability: bias in the spectrum of mutation rates towards small mutations, and the availability and size of nonfunctional DNA spacers. We show that the probability of successful duplication and insertion of a copy of a functional module increases by several orders of magnitude depending on the length of the spacers flanking the module. We infer that the adaptive evolution of multicellular organisms has become feasible because of the abundance of nonfunctional DNA spacers, particularly introns, in the genome. We also discuss possible reasons underlying evolutionary retention of the mechanisms that increase evolvability.     

A 24-residue presequence localizes human factor B to mitochondria

We reported previously that the human factor B precursor is a 215-amino acid polypeptide, the first 40 amino acid residues of which function as a mitochondrial targeting presequence [G.I. Belogrudov, Y. Hatefi, J. Biol. Chem. 277 (2002) 6097-6103]. Confocal microscopy of live HEK293 cells, transiently transfected with factor B constructs tagged at the C-terminus with green fluorescent protein (GFP) revealed that either a 40- or 25-residue presequence localized factor B to mitochondria. Indirect immunofluorescent labeling of fixed, permeabilized HEK293 cells that were transiently transfected with a construct lacking a presequence, showed diffuse, intracellular staining that was consistent with targeting of ectopically expressed factor B to cellular compartments distinct from the mitochondria. Mutants in which either Met(-25) or both Met(-25)/Met(-24) residues of the presequence were deleted exhibited decreased or undetectable levels, respectively, of the GFP-tagged factor B. The factor B presequence alone was shown to target a reporter polypeptide GFP to mitochondria. Our studies, therefore, demonstrate that a 24-residue presequence is sufficient to localize factor B to mitochondria, and suggest that the human factor B precursor is a 199-amino acid polypeptide.     

Targeting base excision repair to improve cancer therapies

Most commonly used cancer therapies, particularly ionizing radiation and certain classes of cytotoxic chemotherapies, cause cell death by damaging DNA. Base excision repair (BER) is the major system responsible for the removal of corrupt DNA bases and repair of DNA single strand breaks generated spontaneously and induced by exogenous DNA damaging factors such as certain cancer therapies. In this review, the physico-chemical properties of the proteins involved in BER are discussed with particular emphasis on molecular mechanisms coordinating repair processes. The aim of this review is to apply extensive knowledge that currently exists regarding the biochemical mechanisms involved in human BER to the molecular biology of current therapies for cancer. It is anticipated that the application of this knowledge will translate into the development of novel effective therapies for improving existing treatments such as radiation therapy and oxaliplatin chemotherapy.     

DNA polymerase delta-dependent repair of DNA single strand breaks containing 3'-end proximal lesions

Base excision repair (BER) is the major pathway for the repair of simple, non-bulky lesions in DNA that is initiated by a damage-specific DNA glycosylase. Several human DNA glycosylases exist that efficiently excise numerous types of lesions, although the close proximity of a single strand break (SSB) to a DNA adduct can have a profound effect on both BER and SSB repair. We recently reported that DNA lesions located as a second nucleotide 5′-upstream to a DNA SSB are resistant to DNA glycosylase activity and this study further examines the processing of these ‘complex’ lesions. We first demonstrated that the damaged base should be excised before SSB repair can occur, since it impaired processing of the SSB by the BER enzymes, DNA ligase IIIα and DNA polymerase β. Using human whole cell extracts, we next isolated the major activity against DNA lesions located as a second nucleotide 5′-upstream to a DNA SSB and identified it as DNA polymerase δ (Pol δ). Using recombinant protein we confirmed that the 3′-5′-exonuclease activity of Pol δ can efficiently remove these DNA lesions. Furthermore, we demonstrated that mouse embryonic fibroblasts, deficient in the exonuclease activity of Pol δ are partially deficient in the repair of these ‘complex’ lesions, demonstrating the importance of Pol δ during the repair of DNA lesions in close proximity to a DNA SSB, typical of those induced by ionizing radiation.     

The role of structural reorganization in charge carrier transfer in DNA molecule

A model of hole transfer in DNA molecules has been proposed, which takes into account changes in the reorganization energy and orbital coupling between the neighboring bases during the charge transfer in different molecular sequences. It is shown that the rate of hole transfer by the superexchange and hopping transfer mechanisms is limited by the relaxation of the geometries of nucleobases participating in charge migration and the dynamics of solvent molecules. The rate of charge transfer in the DNA molecule is found to be dependent on the height of the potential barriers between the nucleotide and the molecular sequences. The inclusion of the interchain charge transfer, which is characterized by weak coupling between the nucleotides located in opposite strands, does not affect the general charge transport in DNA. The increase in the number of the parallel components of the hopping mechanism leads to a rise in the charge transfer rate in the double helix.     

Complex Microsatellite Dynamics in the Myostatin Gene Within Ruminants

A microsatellite has previously been identified in myostatin in cattle. Sequencing of this region from other artiodactyls coupled with phylogenetic analysis has been used to uncover the potential origins of the microsatellite event, which appears either to have been born twice or to have been gained and lost within ruminants. While caprids and ovids share the ancestral state with pigs and other mammals, microsatellite activity (length polymorphism) is uncovered in both deer and bovids. The dynamic process of microsatellite evolution, including birth, is discussed here in light of several models. Finally, these models are evaluated in the context of patterns of microsatellite conservation between closely related mammalian genomes.     

Comparative analysis of the karyotype of the sculpin Myoxocephalus ochotensis (Pisces: Cottidae) from the Odyan Bay of the Sea of Okhotsk

The karyotype of the endemic Okhotsk Sea sculpin Myoxocephalus ochotensis Schmidt (Cottidae) from Odyan Bay was studied. The number and morphology of chromosomes were determined, 2n = 42 (2 metacentric, 20 subtelocentric, and 20 acrocentric chromosomes), NF = 44. Variability of chromosome number was not revealed; no difference between male and female karyotypes was found. The karyotype of the Okhotsk sculpin M. ochotensis was compared with karyotypes of the Far East Steller’s M. stelleri (Tilesius), snow M. brandti (Steindachner), and plain M. jaok (Cuvier) sculpins, and to the European shorthorn sculpin M. scorpius (Linnaeus) from White Sea. Their similarities and distinctions were shown.