Similarities of physico-chimical and antigenic properties of neurocupreins and extremely acidic copper proteins from chromaffin granules

Extremely acidic copper-containing proteins, neurocupreins, were isolated from brains of various mammals (bovine, rabbit, pig and sheep). Neurocupreins from all these sources were found to have similar physico-chemical and antigenic properties. Using the immunological approach, it was shown that neurocuprein is located only in brain cytosol and synaptosomal fractions. Extremely acidic copper-containing proteins were also isolated from soluble and membranous fractions of chromaffin granules from bovine adrenal medulla. The soluble form of the protein from the granules has practically the same physico-chemical and antigenic properties as neurocupreins. The copper protein isolated from membranes of granules has slightly higher molecular weight and somewhat different amino acid composition, although their EPR spectra are identical. However, both copper proteins from chromaffin granules are immunoprecipitated with antibodies to neurocuprein. It is suggested that the membranous form differs from the soluble one in possessing a peptide which prolongs the protein chain without changes in its antigenic properties.     

Growth peculiarities of commensal Escherichia coli isolates from the gut microflora in Crohn’s disease patients

Verhulst’s logistic differential equation, popular in mathematical ecology, is used in modeling of population growth, neural networks, statistics, reaction models, Fermi distribution, modeling of tumor growth, etc. We used this function to characterize growth of commensal Escherichia coli isolates from gut microflora in Crohn’s disease patients. The results of our investigations show differences in growth parameters of commensal E. coli, isolated from the gut microflora in Crohn’s disease patients and healthy volunteers; it is most likely explained by the influence of chronic inflammatory processes on growth and reproduction of these bacteria. It has been established that the used mathematical model well characterizes growth of patients’ gut E. coli isolates, and it can be important for the expedient probiotics’ application during the disease.     

Low-Dose Electron-Beam Irradiation for the Improvement of Biofilm Formation by Probiotic Lactobacilli

The effects of 50–150 gray electron-beam irradiation on the biofilm-formation ability and cell surface hydrophobicity of the commercial strain, Lactobacillus acidophilus DDS®-1, from Lacto-G (a marketed synbiotic formulation) and the putative probiotic, L. rhamnosus Vahe, were evaluated. No significant changes in cell surface hydrophobicity were found after irradiation, while increases in biofilm-formation abilities were documented for both investigated microorganisms 0.22 ± 0.03 vs. 0.149 ± 0.02 (L. rhamnosus Vahe, 150 Gy) and 0.218 ± 0.021 vs. 0.17 ± 0.012 (L. acidophilus DDS®-1, 150 Gy). Given this, the use of electron-beam irradiation (50–100 Gy) for the treatment of L. rhamnosus Vahe and L. acidophilus DDS®-1 cells may be considered in product sterilization, quality improvement, and packaging practices.

     

Effect of diethylsulfoxide on the thermal denaturation of DNA

DNA thermal denaturation has been investigated in aqueous solutions of diethylsulfoxide (DESO) by means of UV-vis and densimetry methods. It is suggested that, on the one hand, the structural change of entire solutions and, on the other hand, a direct interaction of DESO with DNA are responsible for the observed peculiar behavior. The results obtained were compared with those of dimethylsulfoxide (DMSO), also known from literature.     

Quantification of Cardiovascular Biomarkers in Patient Plasma by Targeted Mass Spectrometry and Stable Isotope Dilution

Verification of candidate biomarkers requires specific assays to selectively detect and quantify target proteins in accessible biofluids. The primary objective of verification is to screen potential biomarkers to ensure that only the highest quality candidates from the discovery phase are taken forward into preclinical validation. Because antibody reagents for a clinical grade immunoassay often exist for a small number of candidates, alternative methodologies are required to credential new and unproven candidates in a statistically viable number of serum or plasma samples. Using multiple reaction monitoring coupled with stable isotope dilution MS, we developed quantitative, multiplexed assays in plasma for six proteins of clinical relevance to cardiac injury. The process described does not require antibodies for immunoaffinity enrichment of either proteins or peptides. Limits of detection and quantitation for each signature peptide used as surrogates for the target proteins were determined by the method of standard addition using synthetic peptides and plasma from a healthy donor. Limits of quantitation ranged from 2 to 15 ng/ml for most of the target proteins. Quantitative measurements were obtained for one to two signature peptides derived from each target protein, including low abundance protein markers of cardiac injury in the nanogram/milliliter range such as the cardiac troponins. Intra- and interassay coefficients of variation were predominantly <10 and 25%, respectively. The configured multiplex assay was then used to measure levels of these proteins across three time points in six patients undergoing alcohol septal ablation for hypertrophic obstructive cardiomyopathy. These results are the first demonstration of a multiplexed, MS-based assay for detection and quantification of changes in concentration of proteins associated with cardiac injury in the low nanogram/milliliter range. Our results also demonstrate that these assays retain the necessary precision, reproducibility, and sensitivity to be applied to novel and uncharacterized candidate biomarkers for verification of proteins in blood.Discovery of disease-specific biomarkers with diagnostic and prognostic utility has become an important challenge in clinical proteomics. In general, unbiased discovery experiments often result in the confident identification of thousands of proteins, hundreds of which may vary significantly between case and control samples in small discovery studies. However, because of the stochastic sampling of proteomes in discovery “omics” experiments, a large fraction of the protein biomarkers “discovered” in these experiments are false positives arising from biological or technical variability. Clearly discovery omics experiments do not lead to biomarkers of immediate clinical utility but rather produce candidates that must be qualified and verified in larger sample sets than were used for discovery (1).Traditional, clinical validation of biomarkers has relied primarily on immunoassays because of their specificity and sensitivity for the target analyte and high throughput capability. However, antibody reagents for a clinical grade immunoassay often only exist for a short list of candidates. The development of a reliable sandwich immunoassay for one target protein is expensive, has a long development time, and is dependent upon the generation of high quality protein antibodies. For the large majority of new, unproven candidate biomarkers, an intermediate verification technology is required that has shorter assay development time lines, lower assay cost, and effective multiplexing of dozens of candidates in low sample volumes. Ideally the approach should be capable of analyzing hundreds of samples of serum or plasma with good precision. The desired outcome of verification is a small number of highly credentialed candidates suitable for traditional preclinical and clinical validation studies.Multiple reaction monitoring (MRM)¹ coupled with stable isotope dilution (SID) MS has recently been shown to be well suited for direct quantification of proteins in plasma (2–4) and has emerged as the core technology for candidate biomarker verification. MRM assays can be highly multiplexed such that a moderate number of candidate proteins (in the range of 10–50) can be simultaneously targeted and measured in the statistically viable number of patient samples required for verification (hundreds of serum samples). However, sensitivity for unambiguous detection and quantification of proteins by MS-based assays is often constrained by sample complexity, particularly when the measurements are being made in complex fluids such as plasma.Many biomarkers of current clinical importance, such as prostate-specific antigen and the cardiac troponins, reside in the low nanogram/milliliter range in plasma and, until recently, have been inaccessible by non-antibody approaches. Our laboratory has recently shown for the first time that a combination of abundant protein depletion with limited fractionation at the peptide level prior to SID-MRM-MS provides robust limits of quantitation (LOQs) in the 1–20 ng/ml range with coefficient of variation (CV) of 10–20% at the LOQ for proteins in plasma (3).Here we demonstrate that this work flow can be extended to configure assays for a number of known markers of cardiovascular disease and, more importantly, can be deployed to measure their concentrations in clinical samples. We modeled a verification study comprising six patients undergoing alcohol septal ablation treatment for hypertrophic obstructive cardiomyopathy, a human model of “planned” myocardial infarction (PMI), and obtained targeted, quantitative measurements for moderate to low concentrations of cardiac biomarkers in plasma. This work provides additional evidence that MS-based assays can be configured and applied to verification of new protein targets for which high quality antibody reagents are not available.     

Covalent binding of the organophosphorus agent FP-biotin to tyrosine in eight proteins that have no active site serine

Organophosphorus (OP) esters are known to bind covalently to the active site serine of enzymes in the serine hydrolase family. It was a surprise to find that proteins with no active site serine are also covalently modified by OP. The binding site in albumin, transferrin, and tubulin was identified as tyrosine. The goal of the present work was to determine whether binding to tyrosine is a general phenomenon. Fourteen proteins were treated with a biotin-tagged organophosphorus agent called FP-biotin. The proteins were digested with trypsin and the labeled peptides enriched by binding to monomeric avidin. Peptides were purified by HPLC and fragmented by collision induced dissociation in a tandem ion trap mass spectrometer. Eight proteins were labeled and six were not. Tyrosine was labeled in human alpha-2-glycoprotein 1 zinc-binding protein (Tyr 138, Tyr 174 and Tyr 181), human kinesin 3C motor domain (Tyr 145), human keratin 1 (Tyr 230), bovine actin (Tyr 55 and Tyr 200), murine ATP synthase beta (Tyr 431), murine adenine nucleotide translocase 1 (Tyr 81), bovine chymotrypsinogen (Tyr 201) and porcine pepsin (Tyr 310). Only 1–3 tyrosines per protein were modified, suggesting that the reactive tyrosine was activated by nearby residues that facilitated ionization of the hydroxyl group of tyrosine. These results suggest that OP binding to tyrosine is a general phenomenon. It is concluded that organophosphorus-reactive proteins include not only enzymes in the serine hydrolase family, but also proteins that have no active site serine. The recognition of a new OP-binding motif to tyrosine suggests new directions to search for mechanisms of long-term effects of OP exposure. Another application is in the search for biomarkers of organophosphorus agent exposure. Previous searches have been limited to serine hydrolases. Now proteins such as albumin and keratin can be considered.     

Responses of neurons of the extrastriate cortex of the cat brain to moving stimuli of different forms

We examined responses of neurons of the field 21b of the cat brain cortex to presentation of moving visual stimuli of different forms. Characteristics of the responses of about 54% of the studied neurons showed that in these cases configurations of the contours of moving stimuli were to a certain extent discriminated. Most neurons selectively reacting to changes in the form of the stimulus were dark-sensitive units (they generated optimum responses to presentation of dark visual stimuli on the light background). Detailed examination of the spatial infrastructure of receptive fields (RFs) of the neurons and comparison of this structure with the selectivity of neuronal responses showed that there is no significant correlation between static organization of the RF and responses of the neuron to the movements of stimuli of different forms. We hypothesize that the dynamic infrastructure of the RF and the combined activity of functional groups of neurons, whose RFs spatially overlap the RF of the neuron under study, play a definite role in the mechanisms responsible for neuronal discrimination of the form of the visual stimulus. Neirofiziologiya/Neurophysiology, Vol. 38, No. 1, pp. 61–71, January–February, 2006.     

Background impulse activity of neurons of the fastigial cerebellar nucleus of intact and labyrinthectomized rats under conditions of vibration

In acute experiments on nembutal-anesthetized (40 mg/kg, i.p.) albino rats, we recorded extracellularly and analyzed the background impulse activity (BIA) of neurons of the fastigial nucleus of the cerebellum. Experiments were carried out on intact and labyrinthectomized rats in the norm and after long-lasting (up to 15 days) influence of general vertical vibration (60 Hz, 0.4 mm, 2-h-long everyday sessions). Distributions of the neurons according to the level of regularity of BIA, dynamics of spike trains, pattern of histograms of interspike intervals (ISIs), and different frequency ranges of BIA were plotted; the mean frequency of this activity and the coefficient of variation of ISIs were also calculated. Possible mechanisms of the effects of long-lasting vibration of different durations on the BIA generated by neurons of the fastigial cerebellar nucleus in intact animals and after switching off of labyrinth afferent inputs are discussed. Neirofiziologiya/Neurophysiology, Vol. 38, No. 1, pp. 32–39, January–February, 2006.