The role of N-quinones in the regulation of glucose-6-phosphate metabolism

In experimental (white rats, rabbits) and clinical (erythrocytes, blood plasma) studies on 29 healthy subjects and patients it has been demonstrated that primary or secondary n-quinone deficiency is accompanied by increased tissue activity of glycolysis enzymes (aldolase, PGmutase) and aerobic pentose phosphate shunt (6 GPDH). Parallel rise in the amount of glycolysis metabolites (pyruvate and lactate) in the blood and the decline in blood plasma glucose level were observed. The changes in glucose-6-phosphate metabolism are, probably, secondary and reflect tissue structure alterations in the development of K and E avitaminosis.     

Occurrence of two structural types of mercury reductases among Gram-positive bacteria

Structural variants of mercury reductase containing the N-terminal domain, which is easily cleaved by trypsin, have been found in Gram-positive bacteria with a low genomic G + C content (Bacillus, Staphylococcus and, possibly, some other genera). Mercury reductases without the N-terminal domain and relatively resistant to limited proteolysis are typical for Gram-positive bacteria with a high genomic G + C content (Arthrobacter, Citreobacterium, Micrococcus, Mycobacterium, Rhodococcus). Both types of mercury reductase genes may be located on plasmids.     

A dual function of the CRISPR-Cas system in bacterial antivirus immunity and DNA repair

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) and the associated proteins (Cas) comprise a system of adaptive immunity against viruses and plasmids in prokaryotes. Cas1 is a CRISPR-associated protein that is common to all CRISPR-containing prokaryotes but its function remains obscure. Here we show that the purified Cas1 protein of Escherichia coli (YgbT) exhibits nuclease activity against single-stranded and branched DNAs including Holliday junctions, replication forks and 5'-flaps. The crystal structure of YgbT and site-directed mutagenesis have revealed the potential active site. Genome-wide screens show that YgbT physically and genetically interacts with key components of DNA repair systems, including recB, recC and ruvB. Consistent with these findings, the ygbT deletion strain showed increased sensitivity to DNA damage and impaired chromosomal segregation. Similar phenotypes were observed in strains with deletion of CRISPR clusters, suggesting that the function of YgbT in repair involves interaction with the CRISPRs. These results show that YgbT belongs to a novel, structurally distinct family of nucleases acting on branched DNAs and suggest that, in addition to antiviral immunity, at least some components of the CRISPR-Cas system have a function in DNA repair.     

Genetisch bedingte Entwicklungsstörungen der Genitalwege

Anomalies of the female and male genital tracts can be the cause of sterility and infertility. In this review disorders of the Mullerian and Wolffian structures which are responsible for the development of male and female genital tracts will be discussed.     

Electroneuromyographic studies of pain sensitivity

We analyze the published information on the techniques of studies of pain sensitivity using objective electroneuromyographic approaches and the results of these studies. In particular, we describe works where the H reflex, nociceptive flexor and blinking reflexes, and exteroceptive suppression of voluntary muscle activity were examined. Some aspects of using somatosensory evoked potentials in the studies of pain phenomena are also discussed.     

Structural requirements of tropomodulin for tropomyosin binding and actin filament capping

Regulation of actin filament dynamics underlies many cellular functions. Tropomodulin together with tropomyosin can cap the pointed, slowly polymerizing, filament end, inhibiting addition or loss of actin monomers. Tropomodulin has an unstructured N-terminal region that binds tropomyosin and a folded C-terminal domain with six leucine-rich repeats. Of tropomodulin 1's 359 amino acids, an N-terminal fragment (Tmod1(1)(-)(92)) suffices for in vitro function, even though the C-terminal domain can weakly cap filaments independent of tropomyosin. Except for one short alpha-helix with coiled coil propensity (residues 24-35), the Tmod1(1)(-)(92) solution structure shows that the fragment is disordered and highly flexible. On the basis of the solution structure and predicted secondary structure, we have introduced a series of mutations to determine the structural requirements for tropomyosin binding (using native gels and CD) and filament capping (by measuring actin polymerization using pyrene fluorescence). Tmod1(1)(-)(92) fragments with mutations of an interface hydrophobic residue, L27G and L27E, designed to destroy the alpha-helix or coiled coil propensity, lost binding ability to tropomyosin but retained partial capping function in the presence of tropomyosin. Replacement of a flexible region with alpha-helical residues (residues 59-61 mutated to Ala) had no effect on tropomyosin binding but inhibited the capping function. A mutation in a region predicted to be an amphipathic helix (residues 65-75), L71D, destroyed the capping function. The results suggest that molecular flexibility and binding to actin via an amphipathic helix are both required for tropomyosin-dependent capping of the pointed end of the actin filament.     

Using Bayesian multinomial classifier to predict whether a given protein sequence is intrinsically disordered

Intrinsically disordered proteins (IDPs) lack a well-defined three-dimensional structure under physiological conditions. Intrinsic disorder is a common phenomenon, particularly in multicellular eukaryotes, and is responsible for important protein functions including regulation and signaling. Many disease-related proteins are likely to be intrinsically disordered or to have disordered regions. In this paper, a new predictor model based on the Bayesian classification methodology is introduced to predict for a given protein or protein region if it is intrinsically disordered or ordered using only its primary sequence. The method allows to incorporate length-dependent amino acid compositional differences of disordered regions by including separate statistical representations for short, middle and long disordered regions. The predictor was trained on the constructed data set of protein regions with known structural properties. In a Jack-knife test, the predictor achieved the sensitivity of 89.2% for disordered and 81.4% for ordered regions. Our method outperformed several reported predictors when evaluated on the previously published data set of Prilusky et al. [2005. FoldIndex: a simple tool to predict whether a given protein sequence is intrinsically unfolded. Bioinformatics 21 (16), 3435-3438]. Further strength of our approach is the ease of implementation.     

CRISPR/Cas9-mediated gfp gene inactivation in Arabidopsis suspension cells

Molecular Biology Reports - Targeted genome editing using CRISPR/Cas9 is a promising technology successfully verified in various plant species; however, it has hardly been used in plant cell...     

Probing SWI/SNF remodeling of the nucleosome by unzipping single DNA molecules

Chromatin-remodeling enzymes can overcome strong histone-DNA interactions within the nucleosome to regulate access of DNA-binding factors to the genetic code. By unzipping individual DNA duplexes, each containing a uniquely positioned nucleosome flanked by long segments of DNA, we directly probed histone-DNA interactions. The resulting disruption-force signatures were characteristic of the types and locations of interactions and allowed measurement of the positions of nucleosomes with 2.6-base-pair (bp) precision. Nucleosomes remodeled by yeast SWI/SNF were moved bidirectionally along the DNA, resulting in a continuous position distribution. The characteristic distance of motion was approximately 28 bp per remodeling event, and each event occurred with a catalytic efficiency of 0.4 min(-1) per nM SWI/SNF. Remodeled nucleosomes had essentially identical disruption signatures to those of unremodeled nucleosomes, indicating that their overall structure remained canonical. These results impose substantial constraints on the mechanism of SWI/SNF remodeling.     

Ex vivo expansion of human CD8+ T cells using autologous CD4+ T cell help

Background

Using in vivo mouse models, the mechanisms of CD4⁺ T cell help have been intensively investigated. However, a mechanistic analysis of human CD4⁺ T cell help is largely lacking. Our goal was to elucidate the mechanisms of human CD4⁺ T cell help of CD8⁺ T cell proliferation using a novel in vitro model.

Methods/Principal Findings

We developed a genetically engineered novel human cell-based artificial APC, aAPC/mOKT3, which expresses a membranous form of the anti-CD3 monoclonal antibody OKT3 as well as other immune accessory molecules. Without requiring the addition of allogeneic feeder cells, aAPC/mOKT3 enabled the expansion of both peripheral and tumor-infiltrating T cells, regardless of HLA-restriction. Stimulation with aAPC/mOKT3 did not expand Foxp3⁺ regulatory T cells, and expanded tumor infiltrating lymphocytes predominantly secreted Th1-type cytokines, interferon-γ and IL-2. In this aAPC-based system, the presence of autologous CD4⁺ T cells was associated with significantly improved CD8⁺ T cell expansion in vitro. The CD4⁺ T cell derived cytokines IL-2 and IL-21 were necessary but not sufficient for this effect. However, CD4⁺ T cell help of CD8⁺ T cell proliferation was partially recapitulated by both adding IL-2/IL-21 and by upregulation of IL-21 receptor on CD8⁺ T cells.

Conclusions

We have developed an in vitro model that advances our understanding of the immunobiology of human CD4⁺ T cell help of CD8⁺ T cells. Our data suggests that human CD4⁺ T cell help can be leveraged to expand CD8⁺ T cells in vitro.