Dominance of parental genomes in embryonic stem cell/fibroblast hybrid cells depends on the ploidy of the somatic partner

Two dozen hybrid clones were produced by fusion of diploid embryonic stem (ES) cells positive for green fluorescent protein (GFP) with tetraploid fibroblasts derived from DD/c and C57BL-I(I)1RK mice. Cytogenetic analysis demonstrated that most cells from these hybrid clones contained near-hexaploid chromosome sets. Additionally, the presence of chromosomes derived from both parental cells was confirmed by polymerase chain reaction (PCR) analysis of polymorphic microsatellites. All hybrid cells were positive for GFP and demonstrated growth characteristics and fibroblast-like morphology. In addition, most hybrid cells were positive for collagen type I, fibronectin, and lamin A/C but were negative for Oct4 and Nanog proteins. Methylation status of the Oct4 and Nanog gene promoters was evaluated by bisulfite genomic sequencing analysis. The methylation sites (CpG-sites) of the Oct4 and Nanog gene promoters were highly methylated in hybrid cells, whereas the CpG-sites were unmethylated in the parental ES cells. Thus, the fibroblast genome dominated the ES genome in the diploid ES cell/tetraploid fibroblast hybrid cells. Immunofluorescent analysis of the pluripotent and fibroblast markers demonstrated that establishment of the fibroblast phenotype occurred shortly after fusion and that the fibroblast phenotype was further maintained in the hybrid cells. Fusion of karyoplasts and cytoplast derived from tetraploid fibroblasts with whole ES cells demonstrated that karyoplasts were able to establish the fibroblast phenotype of the reconstructed cells but not fibroblast cytoplasts. Thus, these data suggest that the dominance of parental genomes in hybrid cells of ES cell/somatic cell type depends on the ploidy of the somatic partner.     

Synthesis and biological activity of 5-styryl and 5-phenethyl-substituted 2,3,4,5-tetrahydro-1H-pyrido[4,3-b]indoles

Syntheses, biological evaluation, and structure–activity relationships for a series of novel 5-styryl and 5-phenethyl analogs of dimebolin are disclosed. The novel derivatives and dimebolin share a broad spectrum of activities against therapeutically relevant targets. Among all synthesized derivatives, 2,8-dimethyl-5-[(Z)-2-phenylvinyl]-2,3,4,5-tetrahydro-1H-pyrido[4,3-b]indole and its 5-phenethyl analog are the most potent blockers of 5-HT₇, 5-HT₆, 5-HT_2C, Adrenergic α₂ and H₁ receptors. The general affinity rank order towards the studied receptors was Z-3(2) > 4(2) ? 4(3) ? dimebolin, all of them having highest affinities to 5-HT₇ receptors.     

Diversity of Glycosyl Hydrolases from Cellulose-Depleting Communities Enriched from Casts of Two Earthworm Species

The guts and casts of earthworms contain microbial assemblages that process large amounts of organic polymeric substrates from plant litter and soil; however, the enzymatic potential of these microbial communities remains largely unexplored. In the present work, we retrieved carbohydrate-modifying enzymes through the activity screening of metagenomic fosmid libraries from cellulose-depleting microbial communities established with the fresh casts of two earthworm species, Aporrectodea caliginosa and Lumbricus terrestris, as inocula. Eight glycosyl hydrolases (GHs) from the A. caliginosa-derived community were multidomain endo-β-glucanases, β-glucosidases, β-cellobiohydrolases, β-galactosidase, and β-xylosidases of known GH families. In contrast, two GHs derived from the L. terrestris microbiome had no similarity to any known GHs and represented two novel families of β-galactosidases/α-arabinopyranosidases. Members of these families were annotated in public databases as conserved hypothetical proteins, with one being structurally related to isomerases/dehydratases. This study provides insight into their biochemistry, domain structures, and active-site architecture. The two communities were similar in bacterial composition but significantly different with regard to their eukaryotic inhabitants. Further sequence analysis of fosmids and plasmids bearing the GH-encoding genes, along with oligonucleotide usage pattern analysis, suggested that those apparently originated from Gammaproteobacteria (pseudomonads and Cellvibrio-like organisms), Betaproteobacteria (Comamonadaceae), and Alphaproteobacteria (Rhizobiales).Microorganisms producing diverse glycosyl hydrolases (GHs) are widespread and typically thrive in environments where plant materials tend to accumulate and deteriorate (42, 73). The habitats of microorganisms with great GH diversity are the ruminant animal rumen, mouse bowel, and rabbit cecum (10, 24, 26, 28, 49, 74). Microorganisms associated with soil invertebrates in general and with soil earthworms in particular carry out metabolic processes that contribute to element cycling and are essential in sustaining processes which their hosts are unable to perform (20, 52, 72, 76). Although some species of earthworms produce cellulases (15, 55), they generally rely on microbes inhabiting their gastrointestinal (GI) tracts to perform cellulose utilization processes (31, 47, 77). Casts are of special interest in this respect. Considering that the overall numbers of cellulolytic microbes in earthworm casts are greater than those in soil (57), earthworm casts seem to play an important role in the decomposition of plant litter, serving as an inoculum for cellulosic substrates (9). It is important to note that microorganisms from preingested substratum (soil or plant litter) are predominant in the gut lumen (20); however, microbial populations in earthworm casts differ from those in soil in terms of diversity and the relative abundance of different taxa (29, 57, 63). It is anticipated that the enzymatic repertoire of such microbial communities must be especially broad toward diverse sugar-based polymeric, oligomeric, and monomeric substrates; however, among approximately 115 families of GHs with thousands of members known to date (12), none of the GHs have been derived from microorganisms of earthworm-associated microbial communities.The aim of the present work was therefore to examine the diversity of GHs in metagenome libraries derived from fresh casts of Aporrectodea caliginosa and Lumbricus terrestris earthworms via functional screening. Other important tasks of this work were to characterize individual enzymes and to gain insight into their structural-functional features. Finally, we performed sequence analysis of large contiguous DNA fragments of fosmids harboring the genes for GHs to associate them with the organism(s) that may produce them, which was complemented by conventional small-subunit (SSU) rRNA clone library sequencing analysis.     

Chips from chips: application to the study of antibody responses to methylated proteins

Peptide microarrays are useful tools for the characterization of humoral responses against peptide antigens. The study of post-translational modifications requires the printing of appropriately modified peptides, whose synthesis can be time-consuming and expensive. We describe here a method named "chips from chips", which allows probing the presence of antibodies directed toward modified peptide antigens starting from unmodified peptide microarrays. The chip from chip concept is based on the modification of peptide microspots by simple chemical reactions. The starting peptide chip (parent chip) is covered by the reagent solution, thereby allowing the modification of specific residues to occur, resulting in the production of a modified peptide chip (daughter chip). Both parent and daughter chips can then be used for interaction studies. The method is illustrated using reductive methylation for converting lysines into dimethyllysines. The rate of methylation was studied using specific antibodies and fluorescence detection, or surface-assisted laser desorption ionization mass spectrometry. This later technique showed unambiguously the efficient methylation of the peptide probes. The method was then used to study the humoral response against the Mycobacterium tuberculosis heparin-binding hemagglutinin, a methylated surface-associated virulence factor and powerful diagnostic and protective antigen.     

Barnase-barstar high affinity interaction phenomenon as the base for the heterogenous bioluminescence pseudorabies virus' immunoassay

The effective new variant of "sandwich" bioluminescent enzyme immunoassay (BEIA) for the sensitive detection of glycoprotein B (gB) of pseudorabies virus (PrV) was presently developed. The high affinity interaction of barnase-barstar protein pair and photoprotein obelin as bioluminescent marker were for the first time successfully applied to BEIA development. Preliminary the two monoclonal antibodies, 11/5 and 34/2, were raised against gB for ELISA PrV detection. Presently we used the same immuno-"sandwich" principle for BEIA. To do this the two different bioconjugates were elaborated. Recombinant barnase was chemically conjugated with monoclonal anti-PrV's gB IgG, and also barstar was fused in frame to obelin. The characteristics of BEIA method have been compared to ELISA PrV detection. We have shown the proposed here gB-BEIA was 40-fold more sensitive as opposed to gB-ELISA test. The construction might have a broad promise in multiple potential immunological applications.     

Structures of MART-126/27-35 Peptide/HLA-A2 complexes reveal a remarkable disconnect between antigen structural homology and T cell recognition

Small structural changes in peptides presented by major histocompatibility complex (MHC) molecules often result in large changes in immunogenicity, supporting the notion that T cell receptors are exquisitely sensitive to antigen structure. Yet there are striking examples of TCR recognition of structurally dissimilar ligands. The resulting unpredictability of how T cells will respond to different or modified antigens impacts both our understanding of the physical bases for TCR specificity as well as efforts to engineer peptides for immunomodulation. In cancer immunotherapy, epitopes and variants derived from the MART-1/Melan-A protein are widely used as clinical vaccines. Two overlapping epitopes spanning amino acid residues 26 through 35 are of particular interest: numerous clinical studies have been performed using variants of the MART-1 26-35 decamer, although only the 27-35 nonamer has been found on the surface of targeted melanoma cells. Here, we show that the 26-35 and 27-35 peptides adopt strikingly different conformations when bound to HLA-A2. Nevertheless, clonally distinct MART-1(26/27-35)-reactive T cells show broad cross-reactivity towards these ligands. Simultaneously, however, many of the cross-reactive T cells remain unable to recognize anchor-modified variants with very subtle structural differences. These dichotomous observations challenge our thinking about how structural information on unligated peptide/MHC complexes should be best used when addressing questions of TCR specificity. Our findings also indicate that caution is warranted in the design of immunotherapeutics based on the MART-1 26/27-35 epitopes, as neither cross-reactivity nor selectivity is predictable based on the analysis of the structures alone.     

Response of photosynthetic apparatus to moderate high temperature in contrasting wheat cultivars at different oxygen concentrations

The photosynthetic responses to moderately high temperatures (38 degrees C, imposed at 21% or 2% O(2) in air and 1500 mumol m(-2) s(-1)) were compared in wheat (Triticum aestivum L.) cultivars grown in northern regions of Ukraine and expected to be relatively sensitive to high temperatures ('North' cultivars) and in cultivars grown in southern regions and expected to be relatively heat-tolerant ('South' cultivars). Heating intact leaves in 21% O(2) for 1 h decreased CO(2) assimilation by c. 63% in 'North' cultivars and only c. 32% in 'South' cultivars, with a decrease in PSII activity being observed in only one of the 'North' cultivars. Carboxylation efficiency was decreased by about 2.7-fold in 'North' cultivars with no significant effect in 'South' cultivars. The maximum rates of carboxylation by Rubisco in vivo, V(cmax), estimated from Farquhar's model, increased more than 2-fold in 'South' cultivars and remained unchanged in 'North' cultivars while the maximum rate of RuBP regeneration, J(max), decreased by 53% and 21% in 'North' and 'South' cultivars, respectively. Where the heat treatment was imposed in 2% O(2) this increased (as compared with treatment at 21% O(2)) the inhibitory effect on CO(2) assimilation in tolerant cultivars, but decreased it in sensitive ones. The results suggested that differences in tolerance of moderately high temperatures in wheat relate to the stability of the Rubisco function and to RuBP regeneration activity rather than to the effects on PSII activity or stomatal control.     

Structural basis for the recruitment of ERCC1-XPF to nucleotide excision repair complexes by XPA

The nucleotide excision repair (NER) pathway corrects DNA damage caused by sunlight, environmental mutagens and certain antitumor agents. This multistep DNA repair reaction operates by the sequential assembly of protein factors at sites of DNA damage. The efficient recognition of DNA damage and its repair are orchestrated by specific protein-protein and protein-DNA interactions within NER complexes. We have investigated an essential protein-protein interaction of the NER pathway, the binding of the XPA protein to the ERCC1 subunit of the repair endonuclease ERCC1-XPF. The structure of ERCC1 in complex with an XPA peptide shows that only a small region of XPA interacts with ERCC1 to form a stable complex exhibiting submicromolar binding affinity. However, this XPA peptide is a potent inhibitor of NER activity in a cell-free assay, blocking the excision of a cisplatin adduct from DNA. The structure of the peptide inhibitor bound to its target site reveals a binding interface that is amenable to the development of small molecule peptidomimetics that could be used to modulate NER repair activities in vivo.     

Electrophysical characteristics of Azospirillum brasilense Sp245 during interaction with antibodies to various cell surface epitopes

This work was undertaken to examine the electrooptical characteristics of cells of Azospirillum brasilense Sp245 during their interaction with antibodies developed to various cell surface epitopes. We used the dependences of the cell suspension optical density changes induced by electroorientation on the orienting field frequency (740, 1000, 1450, 2000, and 2800kHz). Cell interactions with homologous strain-specific antibodies to the A. brasilense Sp245 O antigen and with homologous antibodies to whole bacterial cells brought about considerable changes in the electrooptical properties of the bacterial suspension. When genus-specific antibodies to the flagellin of the Azospirillum sheathed flagellum and antibodies to the serologically distinct O antigen of A. brasilense Sp7 were included in the A. brasilense Sp245 suspension, the changes caused in the electrooptical signal were slight and had values close to those for the above changes. These findings agree well with the immunochemical characteristics of the Azospirillum O antigens and with the data on the topographical distribution of the Azospirillum major cell surface antigens. The obtained results can serve as a basis for the development of a rapid test for the intraspecies detection of microorganisms.