A perm-selective model membrane exhibiting oscillatory behavior

An enzymic model membrane capable of simulating such permeability characteristics of chemically excitable membranes as generation of an action potential-like overshoot, selectivity over permeants, and saturation and hysteresis of transmembrane flow is constructed by means of coupling a nonlinear, interfacial flow regulating the attachment of permeants to the surface of the oligomeric membrane with a transmembrane allosteric conversion flow recently formulated by Blumenthal. Periods of sustained oscillation, as well as the predicted values of threshold, and height of an action potential-like overshoot are calculated for different choices of external and internal parameters of the membrane.     

Recognition of rat liver and kidney nuclear T3 receptors by an antibody against c-erb A peptide

It has been reported that c-erb A encodes nuclear T3 receptors (NT3R). Based on the sequence of c-erb A cDNA, we synthesized a polypeptide consisting of 15 amino acids, the sequence of which has high homology between c-erb A alpha 1 and beta. The antibody against this c-erb A peptide not only immunoprecipitated rat liver and kidney NT3R but also inhibited T3 binding to NT3R. In a displacement study, the inhibition of [125I]T3-binding by the antibody was parallel to that by T3 in terms of the concentration of the competitor added in the incubation mixture. Scatchard analysis revealed that the antibody decreased the value for the association constant in a dose dependent manner. The antibody did not bind T3 itself. The results show that the antibody against c-erb A peptide recognizes rat liver and kidney NT3R and that the sequence encoding this peptide, the closest carboxyl-terminal of c-erb A may be critical or at least closely related to the hormone binding.     

Extracellular thioredoxin: A therapeutic tool to combat inflammation

The manipulation of cellular redox status has emerged as a promising therapeutic strategy to prevent uncontrolled inflammatory response. Thioredoxin is an important regulator of cellular redox homeostasis, which catalyzes the reduction of disulfide bonds. Human thioredoxin, originally identified as a secretory protein ADF, has been implicated in a wide variety of redox regulations in both intracellular and extracellular compartments. This review includes a summary of the evidence available supporting the employment of the beneficial properties of thioredoxin to combat inflammation, an evaluation of the potential of redox-based therapy for the treatment of inflammatory diseases, and a discussion on the conceptual model of a redox-sensitive signaling complex, Redoxisome, consisting of thioredoxin and its redox partners.     

Quantitative Identification of Mutant Alleles Derived from Lung Cancer in Plasma Cell-Free DNA via Anomaly Detection Using Deep Sequencing Data

The detection of rare mutants using next generation sequencing has considerable potential for diagnostic applications. Detecting circulating tumor DNA is the foremost application of this approach. The major obstacle to its use is the high read error rate of next-generation sequencers. Rather than increasing the accuracy of final sequences, we detected rare mutations using a semiconductor sequencer and a set of anomaly detection criteria based on a statistical model of the read error rate at each error position. Statistical models were deduced from sequence data from normal samples. We detected epidermal growth factor receptor (EGFR) mutations in the plasma DNA of lung cancer patients. Single-pass deep sequencing (>100,000 reads) was able to detect one activating mutant allele in 10,000 normal alleles. We confirmed the method using 22 prospective and 155 retrospective samples, mostly consisting of DNA purified from plasma. A temporal analysis suggested potential applications for disease management and for therapeutic decision making to select epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKI).     

An In Vitro ES Cell-Based Clock Recapitulation Assay Model Identifies CK2α as an Endogenous Clock Regulator

We previously reported emergence and disappearance of circadian molecular oscillations during differentiation of mouse embryonic stem (ES) cells and reprogramming of differentiated cells, respectively. Here we present a robust and stringent in vitro circadian clock formation assay that recapitulates in vivo circadian phenotypes. This assay system first confirmed that a mutant ES cell line lacking Casein Kinase I delta (CKIδ) induced ∼3 hours longer period-length of circadian rhythm than the wild type, which was compatible with recently reported results using CKIδ null mice. In addition, this assay system also revealed that a Casein Kinase 2 alpha subunit (CK2α) homozygous mutant ES cell line developed significantly longer (about 2.5 hours) periods of circadian clock oscillations after in vitro or in vivo differentiation. Moreover, revertant ES cell lines in which mutagenic vector sequences were deleted showed nearly wild type periods after differentiation, indicating that the abnormal circadian period of the mutant ES cell line originated from the mutation in the CK2α gene. Since CK2α deficient mice are embryonic lethal, this in vitro assay system represents the genetic evidence showing an essential role of CK2α in the mammalian circadian clock. This assay was successfully applied for the phenotype analysis of homozygous mutant ES cells, demonstrating that an ES cell-based in vitro assay is available for circadian genetic screening.     

Unique active site formation in a novel galactose 1-phosphate uridylyltransferase from the hyperthermophilic archaeon Pyrobaculum aerophilum

A gene encoding galactose 1-phosphate uridylyltransferase (GalT) was identified in the hyperthermophilic archaeon Pyrobaculum aerophilum. The gene was overexpressed in Escherichia coli, after which its product was purified and characterized. The expressed enzyme was highly thermostable and retained about 90% of its activity after incubation for 10 minutes at temperatures up to 90°C. Two different crystal structures of P. aerophilum GalT were determined: the substrate-free enzyme at 2.33 Å and the UDP-bound H140F mutant enzyme at 1.78 Å. The main-chain coordinates of the P. aerophilum GalT monomer were similar to those in the structures of the E. coli and human GalTs, as was the dimeric arrangement. However, there was a striking topological difference between P. aerophilum GalT and the other two enzymes. In the E. coli and human enzymes, the N-terminal chain extends from one subunit into the other and forms part of the substrate-binding pocket in the neighboring subunit. By contrast, the N-terminal chain in P. aerophilum GalT extends to the substrate-binding site in the same subunit. Amino acid sequence alignment showed that a shorter surface loop in the N-terminal region contributes to the unique topology of P. aerophilum GalT. Structural comparison of the substrate-free enzyme with UDP-bound H140F suggests that binding of the glucose moiety of the substrate, but not the UDP moiety, gives rise to a large structural change around the active site. This may in turn provide an appropriate environment for the enzyme reaction.     

Single Quantum Dot Tracking Reveals that an Individual Multivalent HIV-1 Tat Protein Transduction Domain Can Activate Machinery for Lateral Transport and Endocytosis

The mechanisms underlying the cellular entry of the HIV-1 Tat protein transduction domain (TatP) and the molecular information necessary to improve the transduction efficiency of TatP remain unclear due to the technical limitations for direct visualization of TatP''s behavior in cells. Using confocal microscopy, total internal reflection fluorescence microscopy, and four-dimensional microscopy, we developed a single-molecule tracking assay for TatP labeled with quantum dots (QDs) to examine the kinetics of TatP initially and immediately before, at the beginning of, and immediately after entry into living cells. We report that even when the number of multivalent TatP (mTatP)-QDs bound to a cell was low, each single mTatP-QD first locally induced the cell''s lateral transport machinery to move the mTatP-QD toward the center of the cell body upon cross-linking of heparan sulfate proteoglycans. The centripetal and lateral movements were linked to the integrity and flow of actomyosin and microtubules. Individual mTatP underwent lipid raft-mediated temporal confinement, followed by complete immobilization, which ultimately led to endocytotic internalization. However, bivalent TatP did not sufficiently promote either cell surface movement or internalization. Together, these findings provide clues regarding the mechanisms of TatP cell entry and indicate that increasing the valence of TatP on nanoparticles allows them to behave as cargo delivery nanomachines.     

Inactivation of Bacteriophage ϕX174 by d-Fructose 6-Phosphate

The inactivation of bacteriophage ?X174 by d-fructose 6-phosphate was investigated. This inactivation was inhibited by EDTA or reducing agents, and stimulated by Cu²⁺ but other metal ions could not be substituted for Cu²⁺. The reaction was also inhibited by superoxide dismutase (EC 1.15.1.1), catalase (EC 1.11.1.6) and various free radical scavengers.

No detectable changes were observed in adsorption capacity of phage and in the conformation of the virion. The viral DNA in the virion was, however, found to be cleaved. This strand scission was also enhanced by Cu²⁺ and protected by catalase. Similar results were obtained when ?X174 DNA was directly treated with d-fructose 6-phosphate.

It is concluded that the inactivation of ?X174 is due to DNA strand scission in the virion by the free radical of d-fructose 6-phosphate or oxygen radicals generated during autoxidation of d-fructose 6-phosphate.     

Geometrical Isomers of Monohydroperoxides Formed by Autoxidation of Methyl Linoleate

Isomeric monohydroperoxides produced from autoxidized methyl linoleate were separated into two geometrical isomers (cis-trans and trans-trans) by silver nitrate TLC. Purified monohydroperoxides were converted into hydroxy octadecadienoates. Trimethylsilyl (TMS) derivatives of these compounds (four components) were separated into three peaks in the gas chromatogram; the mixture of 9-hydroxy-cis,trans-isomer and 13-hydroxy-cis,trans-isomer, 9-hydroxy-trans,trans-isomer and 13-hydroxy-trans,trans-isomer. The trans-trans isomers became more dominant than the cis-trans isomers in the later stage of autoxidation and with the rise of temperature. At the degradation of monohydroperoxides, the decrease of trans- trans isomers was apparently slower than that of cis-trans isomers. It is proposed that cis,trans isomerization of monohydroperoxides takes place at the process of autoxidation of methyl linoleate.