Utility of peptide-protein affinity complexes in proteomics: identification of interaction partners of a tumor suppressor peptide.

We used a N-biotinylated peptide analog of the C-terminal domain of the tumor suppressor protein, p21cip1/waf1 to elucidate peptide/protein interacting partners. The C-terminal domain of p21cip1/waf1 protein spanning 141-160 amino acid residues is known to bind PCNA and this interaction is important in many biological processes including cell-cycle control. This C-terminal 20-mer efficiently extracts PCNA in the presence of a variety of N- or C-terminally attached affinity tags. Using difference silver stained 2D gels combined with in-gel tryptic digests, we identified the difference spots using MALDI-TOF mass spectrometry-based peptide mass fingerprinting followed by a database search using PROFOUND against NCBIs human nonredundant protein sequence data bank. Identified spots include the p48 subunit of chromatin assembly factor-1, the heat shock 70 protein analog BiP, calmodulin, nucleolin and a spot similar in size to dimeric PCNA. In contrast, microcapillary ion-trap LC-MS/MS analysis of a tryptic digest of entire affinity extracts derived from both control and experimental runs followed by database searches using SEQUEST confirmed the presence of most of the above proteins. This strategy also identified hnRNPA1, HPSP90alpha, HSP40 and T-complex protein 1, a protein similar to prothymosin, and a possible allelic variant of the p21cip1/waf1 protein. The use of N-biotinylated peptide derived from the C-terminal domain of p21cip1/waf1 protein in proteomic analysis exemplified here suggests that peptides obtained from intracellular functional screens could also potentially serve as efficient baits to discover new drug targets.     

Cloning and sequence analysis of two embryonic beta-like globin cDNAs (y and z) of hamster.

A cDNA library was prepared from poly(A) mRNA extracted from 9-day hamster-yolk-sac erythroid cells. Two clones containing inserts coding for embryonic beta-like z or y globin-chains were isolated. Their identity was confirmed by (a) translation of hybrid selected mRNAs and (b) nucleotide sequence analysis of the inserts and comparison to the embryonic beta-like globin genes of Balb/c mouse. Availability of sequences for embryonic and adult globin cDNAs will aid in investigations of the molecular mechanisms of the globin switch in hamster YSEC.     

Characteristics of carbohydrate metabolism in the rat liver in thiamine deficiency

Thiamine deficiency in rats induced by oxythiamine is accompanied by an increase in the free NADP+/NADPH ratio in liver tissue, which results in multifold stimulation of the metabolite flux in the oxidation branch of the pentose cycle. The increase in the intracellular concentrations of isocitrate and alpha-ketoglutarate with a simultaneous decrease of malate in the liver of vitamin-deficient rats points to the inhibition of alpha-ketoglutarate dehydrogenase responsible for the anomalous metabolism under conditions of thiamine deficiency. The decrease of the functional activity of the tricarboxylic acid cycle is concomitant with the activation of conversions in the oxidation branch of the pentose cycle, glucuronate and glycolytic pathways of carbohydrate metabolism, which is directed at eliminating the energy deficiency in rats with B1-hypovitaminosis.     

A method for flow cytometric cell cycle analysis of normal and psoriatic human epidermis based on a detergent/citric acid technique for suspension of nuclei

A new method is described for flow cytometric cell cycle analysis of normal and psoriatic human epidermis, based on non-enzymatic tissue disaggregation. The epidermis was isolated by treatment with acetic acid and stored by freezing. After thawing, the epidermis was disintegrated into a nuclear suspension by 3 steps: incubation with dithiotreitol, whirling in a buffer (pH 7.4) with the non-ionic detergent Nonidet P40, EGTA, RNase and spermine, and whirling after addition of citric acid to a final concentration of 1% (pH 2.4). The suspension was stained with propidium iodide and filtered before flow cytometry. The yield of suspended nuclei was approximately 70% of the original number of cells in the tissue. The detergent/citric acid method was found to be preferable to an ultrasonication method previously used on human epidermis. All cell cycle and cell maturation stages were represented in the detergent/citric acid suspension, in contrast to the selection of immature G1, S and G2 stages with enzymatic methods. In the analysis of psoriatic epidermis inadequately matured (parakeratotic) cells were present in the suspension and had to be discriminated by gating on light scattering intensity, as they were not susceptible to lysis and did not stain properly. The fraction of S phase nuclei was on average 1.9% in normal and 7.7% in psoriatic epidermis, thus confirming the results of other investigators using enzymes. The presence of mitotic figures in the suspension was demonstrated by flow sorting. In this way the mitotic fraction was estimated to 0.06% in normal and 0.22% in psoriatic epidermis, confirming histological data of other investigators.     

Molecular Reclassification of Crohn’s Disease: A Cautionary Note on Population Stratification

Complex human diseases commonly differ in their phenotypic characteristics, e.g., Crohn’s disease (CD) patients are heterogeneous with regard to disease location and disease extent. The genetic susceptibility to Crohn’s disease is widely acknowledged and has been demonstrated by identification of over 100 CD associated genetic loci. However, relating CD subphenotypes to disease susceptible loci has proven to be a difficult task. In this paper we discuss the use of cluster analysis on genetic markers to identify genetic-based subgroups while taking into account possible confounding by population stratification. We show that it is highly relevant to consider the confounding nature of population stratification in order to avoid that detected clusters are strongly related to population groups instead of disease-specific groups. Therefore, we explain the use of principal components to correct for population stratification while clustering affected individuals into genetic-based subgroups. The principal components are obtained using 30 ancestry informative markers (AIM), and the first two PCs are determined to discriminate between continental origins of the affected individuals. Genotypes on 51 CD associated single nucleotide polymorphisms (SNPs) are used to perform latent class analysis, hierarchical and Partitioning Around Medoids (PAM) cluster analysis within a sample of affected individuals with and without the use of principal components to adjust for population stratification. It is seen that without correction for population stratification clusters seem to be influenced by population stratification while with correction clusters are unrelated to continental origin of individuals.     

OGR1/GPR68 Modulates the Severity of Experimental Autoimmune Encephalomyelitis and Regulates Nitric Oxide Production by Macrophages

Ovarian cancer G protein-coupled receptor 1 (OGR1) is a proton-sensing molecule that can detect decreases in extracellular pH that occur during inflammation. Although OGR1 has been shown to have pro-inflammatory functions in various diseases, its role in autoimmunity has not been examined. We therefore sought to determine whether OGR1 has a role in the development of T cell autoimmunity by contrasting the development of experimental autoimmune encephalomyelitis between wild type and OGR1-knockout mice. OGR1-knockout mice showed a drastically attenuated clinical course of disease that was associated with a profound reduction in the expansion of myelin oligodendrocyte glycoprotein 35-55-reactive T helper 1 (Th1) and Th17 cells in the periphery and a reduced accumulation of Th1 and Th17 effectors in the central nervous system. We determined that these impaired T cell responses in OGR1-knockout mice associated with a reduced frequency and number of dendritic cells in draining lymph nodes during EAE and a higher production of nitric oxide by macrophages. Our studies suggest that OGR1 plays a key role in regulating T cell responses during autoimmunity.     

The evaluation and prognosis of the psychophysiological status of a human operator

In experiments on 56 healthy subjects (18-20 years old) the quality of their activity was determined during compensatory watching the mark at complicating regimes of work. Depending on the difficulty of the task five groups of subjects were singled out with optimum working capacity in one of four working conditions: normal, ordinary and strenuous work, model of stress situation. It is established that the change of the number of significant correlative connections between main parameters of psychophysiological state of man-operator reflects the condition of his functional systems. On the basis of computation of total range of organization values of both R-R intervals of the ECG and duration of expiration, the success of the man-operator work in complex conditions of activity is predicted.