Affinity-Bead Assisted Mass Spectrometry (Affi-BAMS): A Multiplexed Microarray Platform for Targeted Proteomics.

The ability to quantitatively probe diverse panels of proteins and their post-translational modifications (PTMs) across multiple samples would aid a broad spectrum of biological, biochemical and pharmacological studies. We report a novel, microarray analytical technology that combines immuno-affinity capture with Matrix Assisted Laser Desorption Ionization Mass Spectrometry (MALDI MS), which is capable of supporting highly multiplexed, targeted proteomic assays. Termed "Affinity-Bead Assisted Mass Spectrometry" (Affi-BAMS), this LC-free technology enables development of highly specific and customizable assay panels for simultaneous profiling of multiple proteins and PTMs.

Proton transfers in a channelrhodopsin-1 studied by Fourier transform infrared (FTIR) difference spectroscopy and site-directed mutagenesis.

Channelrhodopsin-1 from the alga Chlamydomonas augustae (CaChR1) is a low-efficiency light-activated cation channel that exhibits properties useful for optogenetic applications such as a slow light inactivation and a red-shifted visible absorption maximum as compared with the more extensively studied channelrhodopsin-2 from Chlamydomonas reinhardtii (CrChR2). Previously, both resonance Raman and low-temperature FTIR difference spectroscopy revealed that unlike CrChR2, CaChR1 under our conditions exhibits an almost pure all-trans retinal composition in the unphotolyzed ground state and undergoes an all-trans to 13-cis isomerization during the primary phototransition typical of other microbial rhodopsins such as bacteriorhodopsin (BR). Here, we apply static and rapid-scan FTIR difference spectroscopy along with site-directed mutagenesis to characterize the proton transfer events occurring upon the formation of the long-lived conducting P2 (380) state of CaChR1.

Comparison of the structural changes occurring during the primary phototransition of two different channelrhodopsins from Chlamydomonas algae.

Channelrhodopsins (ChRs) from green flagellate algae function as light-gated ion channels when expressed heterologously in mammalian cells. Considerable interest has focused on understanding the molecular mechanisms of ChRs to bioengineer their properties for specific optogenetic applications such as elucidating the function of specific neurons in brain circuits. While most studies have used channelrhodopsin-2 from Chlamydomonas reinhardtii (CrChR2), in this work low-temperature Fourier transform infrared-difference spectroscopy is applied to study the conformational changes occurring during the primary phototransition of the red-shifted ChR1 from Chlamydomonas augustae (CaChR1).

Retinal chromophore structure and Schiff base interactions in red-shifted channelrhodopsin-1 from Chlamydomonas augustae.

Channelrhodopsins (ChRs), which form a distinct branch of the microbial rhodopsin family, control phototaxis in green algae. Because ChRs can be expressed and function in neuronal membranes as light-gated cation channels, they have rapidly become an important optogenetic tool in neurobiology. While channelrhodopsin-2 from the unicellular alga Chlamydomonas reinhardtii (CrChR2) is the most commonly used and extensively studied optogenetic ChR, little is known about the properties of the diverse group of other ChRs.

Immune responses induced by T-cell vaccination in patients with rheumatoid arthritis.

Patients with rheumatoid arthritis (RA) were treated with a cellular vaccine, which consisted of autologous collagen-reactive T-cells. This study showed that antigen-specific proliferative activity of the peripheral blood mononuclear cells was significantly downregulated after T-cell vaccination in RA patients. T-cell vaccination resulted in a statistically significant decrease in plasma IFNγ levels and a concomitant increase in IL-4 levels in treated patients.

Conformational changes in the archaerhodopsin-3 proton pump: detection of conserved strongly hydrogen bonded water networks.

Archaerhodopsin-3 (AR3) is a light-driven proton pump from Halorubrum sodomense, but little is known about its photocycle. Recent interest has focused on AR3 because of its ability to serve both as a high-performance, genetically-targetable optical silencer of neuronal activity and as a membrane voltage sensor. We examined light-activated structural changes of the protein, retinal chromophore, and internal water molecules during the photocycle of AR3.

Near-IR resonance Raman spectroscopy of archaerhodopsin 3: effects of transmembrane potential.

Archaerhodopsin 3 (AR3) is a light driven proton pump from Halorubrum sodomense that has been used as a genetically targetable neuronal silencer and an effective fluorescent sensor of transmembrane potential. Unlike the more extensively studied bacteriorhodopsin (BR) from Halobacterium salinarum, AR3 readily incorporates into the plasma membrane of both E. coli and mammalian cells.

[Autoimmune states correction for pathologic component (link) elimination that hinder physiologic osteogenesis].

Technological progress widens the possibility to treat patients with serious diseases influencing the reparative processes in human organism. In their clinical practice stomatologists come across with the failure of healing up processes in bone and soft tissues of post operational wounds. Big group of patients was with autoimmune diseases.

Induction of antiidiotypic immune response with autologous T-cell vaccine in patients with multiple sclerosis.

Patients with different forms of multiple sclerosis were treated with a vaccine consisting of myelin-reactive T cells. It was found that after this treatment, lymphocytes from patients acquired the capacity to generate antiidiotypic proliferative response directed towards myelin-reactive T cells. The serum concentration of IFN-gamma decreased about 2-fold 1.

A high-throughput and sensitive method to measure global DNA methylation: application in lung cancer.

Background: Genome-wide changes in DNA methylation are an epigenetic phenomenon that can lead to the development of disease. The study of global DNA methylation utilizes technology that requires both expensive equipment and highly specialized skill sets.

Methods: We have designed and developed an assay, CpGlobal, which is easy-to-use, does not utilize PCR, radioactivity and expensive equipment.

N-terminal labeling of proteins using initiator tRNA.

Methodology based on tRNA mediated protein engineering is described for the introduction of fluorophores and other labels at the N-terminus of proteins produced in cell-free translation systems. One method for low-level (trace) N-terminal labeling is based on the use of an Escherichia coli initiator tRNA(fMet) misaminoacylated with methionine modified at the alpha-amino group. In addition to the normal formyl group, the protein translational machinery incorporates the fluorophore BODIPY-FL and the affinity tag biotin at an N-terminal end of the nascent protein.

Cell-free N-terminal protein labeling using initiator suppressor tRNA.

A highly efficient method for the introduction of fluorophores and other markers at the N terminus of proteins produced in a cell-free extract has been developed. The method utilizes an amber (CUA) initiator suppressor tRNA chemically aminoacylated with a fluorophore-amino acid conjugate which is introduced into an Escherichia coli S30 cell-free translation system. The DNA template contains a complementary amber (UAG) codon instead of the normal initiation (AUG) codon.

Methionine changes in bacteriorhodopsin detected by FTIR and cell-free selenomethionine substitution.

Bacteriorhodopsin (BR) is an integral membrane protein, which functions as a light-driven proton pump in Halobacterium salinarum. We report evidence that one or more methionine residues undergo a structural change during the BR-->M portion of the BR photocycle. Selenomethionine was incorporated into BR using a cell-free protein translation system containing an amino acid mixture with selenomethionine substituted for methionine.

Ultrasensitive fluorescence-based detection of nascent proteins in gels.

The most common method of analysis of proteins synthesized in a cell-free translation system (e.g., nascent proteins) involves the use of radioactive amino acids such as [(35)S]methionine or [(14)C]leucine.

[Device-aided determination of an effective dose in x-ray studies].

The paper proposes how to determine an effective radiation dose for patients undergone X-ray examinations, which includes the estimation of the size of a field while measuring the superficial dose and the assessment of an experiment protocol by taking into account the X-ray receiver used to measure exposure.

[The role of the enzymatic antioxidant system and Helicobacter pylori infection in the pathogenesis of peptic ulcer and their effect on treatment efficacy].

Aim: To assess activity of superoxide dismutase and catalase in acute ulcer and in the end of its treatment with antisecretory (omeprasole and zaran), anti-Helicobacter (metronidasole) and antioxidant (alpha-tocopherol) drugs in patients with duodenal ulcer.

Materials And Methods: 126 patients with duodenal ulcer were divided into 6 groups: group 1 patients had Helicobacter pylori (HP+) and were given omeprasole; group 2 patients had HP and were given omeprasole + metronizarole; group 3 patients were free of HP and received metronidasole + alpha-tocopherol; group 4 HP+ patients received zaran; group 5 HP+ patients got zaran = metronidasole; group 6 free of HP received zaran + alpha-tocopherol. The patients were examined using esophagogastroduodenoscopy, roentgenoscopy of the stomach, tests of the gastric juice.

[The characteristics of the lipid peroxidation and antioxidant activity of the duodenal mucosa in peptic ulcer patients].

Aim: Lipid peroxidation (LPO) and antioxidant defense (AOD) in ulcer and periulcer zones of duodenal mucosa were studied at different phases of ulcer.

Materials And Methods: 111 patients with duodenal ulcer exacerbation before treatment and in the course of the treatment were studied versus 23 healthy volunteers.

Results: In active ulcer there was high activity of LPO and depressed AOD.

[Effects of mono- and combined peptic ulcer therapy on lipid peroxidation and antioxidant defense in ulcerous zone].

Lipid peroxidation and antioxidant activity in the margin and periulcerous zones of duodenal mucosa were investigated in 111 patients with duodenal ulcer before treatment and in periscar zone after chemotherapy allowing for Helicobacter pylori infection. In active ulcer lipid peroxidation was high while antioxidant activity was depressed. Helicobacter pylori was found able to initiate peroxidation.

In vitro suppression as a tool for the investigation of translation initiation.

An in vitro protein synthesizing system that employs rabbit reticulocyte lysates has been employed for protein production from mRNAs containing nonsense (UAG) codons in the presence of misacylated suppressor tRNAs.The system includes a misacylated Escherichia coli tRNAAlaCUA that functions at least as efficiently as any suppressor tRNA transcript reported to date and which has been shown not to be a substrate for (re)activation by alanyl-tRNA synthetase. Application of the optimized system for preparation of dihydrofolate analogs has also permitted analysis of competing mechanisms that control the sites(s) of translation initiation.

Ethidium and azidoethidium oligonucleotide derivatives: synthesis, complementary complex formation and sequence-specific photomodification of the single-stranded and double-stranded target oligo- and polynucleotides.

Oligodeoxyribonucleotide derivatives containing ethidium or azidoethidium residues attached to 3' and/or 5' end were prepared. These derivatives formed tight specific complexes with complementary oligodeoxyribonucleotides where each attached ethidium residue led to an increase of complex Tm by 20-30 degrees C. Tandem complexes of two oligodeoxyribonucleotides containing ethidium residues with an oligodeoxyribonucleotide having two adjacent complementary sequences for these oligonucleotides were investigated.

[Site-directed chemical restriction of a single-stranded DNA fragment by an alkylating derivative of the tetranucleotide d(pApGpCpA) in the presence of tetranucleotide effectors].

Alkylation of a single-stranded DNA 302-mer by a 5'-O-phosphoryl-[4-(N-2-chloroethyl-N-methylamino)benzyl]amide derivative of the tetradeoxyribonucleotide d(pApGpCpA) in the presence of 3',5'-di-N-(2-hydroxyethyl) phenazinium derivatives of tetranucleotides as effectors led to specific chemical cleavage of the target at the guanosine residues of the sites ...

Functional topography of human ribosomes as studied by affinity labeling with reactive mRNA analogs.

Derivatives of 5'-32P labeled (pU)3 an (pU)6 bearing 4-(N-2-chloroethyl-N-methylamino)benzylmethylamine residue attached to 5'-phosphate via phosphamide bond and (Up)5U[32P]pC and (Up)11U[32P]pC bearing 4-(N-2-chloroethyl-N-methylamino)benzyl residue attached to 3'-end via benzylidene bond were applied for the affinity labeling of 80S ribosomes from human placenta in the presence of a cognate tRNA. The derivatives of 32P-labeled pAUG and pAUGU3 analogous to the 5'-phosphamides of (pU)n were used for affinity labeling of 40S subunits in the presence of ternary complex eIF-2.GTP.

[Affinity modification of 80S ribosomes from human placenta by derivatives of tri- and hexauridylates as mRNA analogs].

Derivatives of 5'-32P]labeled (pU)3 and (pU)6 bearing 4-(N-2-chloroethyl-N-methylamino)benzylmethylamine residues attached to 5'-phosphates via phosphamide bond were applied to the affinity labeling of 80S ribosomes from human placenta. The reagents had normal coding properties and were fixed in the ribosomal mRNA-binding region by codon-anticodon interaction with cognate Phe-tRNA(Rhe) at P site (in the case of (pU)3 derivative) or at both A and P sites (in the case of (pU)6 one). Both reagents were found to modify only the 40S subunit.

Sequence-specific cleavage of single-stranded DNA by oligonucleotides conjugated to bleomycin.

Cleavage of a single-stranded DNA fragment by complementary oligonucleotides conjugated to bleomycin A5 has been investigated. The conjugates efficiently cleave the DNA at the GT sequences near the oligonucleotide binding site. The temperature dependence of the reaction and the composition of the degradation products indicate that the oligonucleotide-linked bleomycin attacks the available double-stranded DNA regions within the oligonucleotide-DNA duplex and in the hairpin DNA region in the vicinity of the carrier oligonucleotide binding site.

[Complementary addressed alkylation of 16S rRNA of Escherichia coli by 2',3'-O-[4-N-methyl-N-(2-chloroethyl)-amino]benzylidene derivatives of oligodeoxyribonucleotides. V. Study of the factors affecting selectivity of modification].

We studied the effect of different factors (reagent concentration, temperature, presence of oligonucleotide-effector (3',5'-diphenazinium derivative of oligodeoxyribonucleotide) stabilizing duplex RNA.reagent) on the selectivity of the site-directed modification of 16S rRNA with 2,3'-O-[4-N-methyl-N-(2-chloroethyl)-amino]-benzylidene derivative of oligonucleotide p(dTTTGCTCCCC)rA (reagent I) under conditions of secondary structure stability. The constant of cooperative binding of the reagent and oligonucleotide-effector with 16s rRNA was determined.