Assignment of position-specific error probability to primary DNA sequence data.

DNA sequence predicted from polyacrylamide gel-based technologies is inaccurate because of variations in the quality of the primary data due to limitations of the technology, and to sequence-specific variations due to nucleotide interactions within the DNA molecule and with the gel. The ability to recognize the probability of error in the primary data will be useful in reconstructing the target sequence of a DNA sequencing project, and in estimating the accuracy of the final sequence. This paper describes the use of linear discriminant analysis to assign position-specific probabilities of incorrect, over- and under-prediction of nucleotides for each predicted nucleotide position in primary sequence data generated by a gel-based DNA sequencing technology. Using this method, most of the error potential in primary sequence data can be assigned to a limited number of discrete positions. The use of probability values in the sequence reconstruction process, and in estimating the accuracy of consensus sequence determination is described.     

The effect of estrogens and progestagens on biochemical components of hemostasis, platelets, continuous intravascular coagulation, and tolerance to thrombin: Correction of their effect by antioxidants

Estrogen and progestin (ethinylestradiol and levonorgestrel, respectively) in a dose dependent manner accelerated platelet lipid peroxidation (LPO), activated platelets, increased continuous intravascular coagulation and reduced tolerance to thrombin. Antioxidants limited these effects.     

Physiological pH values in the digestive tract of perch <Emphasis Type="Italic">Perca fluviatilis</Emphasis> from different habitats

Physiological pH values are determined in the gastrointestinal tract of the perch Perca fluviatilis from lakes Chany and Baikal. Values of pH are detected to vary significantly along the digestive tract. Such factors as a segment of the digestive tract, presence/absence of food, season of the year, and habitat affect pH values. Environmental temperature is the main factor affecting pH values that is confirmed by the decrease in pH values upon water temperature rise in spring, lower pH values in summer compared to other seasons, and lower pH in fish from the waterbody (Lake Chany) with higher temperature of water.     

Characteristics of the effect of cestodes parasitizing the fish intestine on the activity of the host proteinases

The activity and spectrum of proteinases in the intestines of host fishes change upon infestation with cestodes. Serine proteinases are found to make a greater contribution to the total proteolytic activity. The reduction of proteolytic activity is associated with adsorption of the enzymes of the host on the surface of cestodes, and the increase in the activity is caused by the injury of the intestinal mucosa by the attachment apparatuses of cestodes. The inhibition of proteainase activity indicates the possible participation of microbiota enzymes in protein hydrolyses.     

Analysis of canonical and non-canonical splice sites in mammalian genomes

A set of 43 337 splice junction pairs was extracted from mammalian GenBank annotated genes. Expressed sequence tag (EST) sequences support 22 489 of them. Of these, 98.71% contain canonical dinucleotides GT and AG for donor and acceptor sites, respectively; 0.56% hold non-canonical GC-AG splice site pairs; and the remaining 0.73% occurs in a lot of small groups (with a maximum size of 0.05%). Studying these groups we observe that many of them contain splicing dinucleotides shifted from the annotated splice junction by one position. After close examination of such cases we present a new classification consisting of only eight observed types of splice site pairs (out of 256 a priori possible combinations). EST alignments allow us to verify the exonic part of the splice sites, but many non-canonical cases may be due to intron sequencing errors. This idea is given substantial support when we compare the sequences of human genes having non-canonical splice sites deposited in GenBank by high throughput genome sequencing projects (HTG). A high proportion (156 out of 171) of the human non-canonical and EST-supported splice site sequences had a clear match in the human HTG. They can be classified after corrections as: 79 GC-AG pairs (of which one was an error that corrected to GC-AG), 61 errors that were corrected to GT-AG canonical pairs, six AT-AC pairs (of which two were errors that corrected to AT-AC), one case was produced from non-existent intron, seven cases were found in HTG that were deposited to GenBank and finally there were only two cases left of supported non-canonical splice sites. If we assume that approximately the same situation is true for the whole set of annotated mammalian non-canonical splice sites, then the 99.24% of splice site pairs should be GT-AG, 0.69% GC-AG, 0.05% AT-AC and finally only 0.02% could consist of other types of non-canonical splice sites. We analyze several characteristics of EST-verified splice sites and build weight matrices for the major groups, which can be incorporated into gene prediction programs. We also present a set of EST-verified canonical splice sites larger by two orders of magnitude than the current one (22 199 entries versus approximately 600) and finally, a set of 290 EST-supported non-canonical splice sites. Both sets should be significant for future investigations of the splicing mechanism.     

A novel type of RNase III family proteins in eukaryotes

The RNase III family of double-stranded RNA-specific endonucleases is characterized by the presence of a highly conserved 9 amino acid stretch in their catalytic center known as the RNase III signature motif. We isolated the drosha gene, a new member of this family in Drosophila melanogaster. Characterization of this gene revealed the presence of two RNase III signature motifs in its sequence that may indicate that it is capable of forming an active catalytic center as a monomer. The drosha protein also contains an 825 amino acid N-terminus with an unknown function. A search for the known homologues of the drosha protein revealed that it has a similarity to two adjacent annotated genes identified during C. elegans genome sequencing. Analysis of the genomic region of these genes by the Fgenesh program and sequencing of the EST cDNA clone derived from it revealed that this region encodes only one gene. This newly identified gene in nematode genome shares a high similarity to Drosophila drosha throughout its entire protein sequence. A potential drosha homologue is also found among the deposited human cDNA sequences. A comparison of these drosha proteins to other members of the RNase III family indicates that they form a new group of proteins within this family.     

A Computational,Tissue-Realistic Model of Pressure Ulcer Formation in Individuals with Spinal Cord Injury

People with spinal cord injury (SCI) are predisposed to pressure ulcers (PU). PU remain a significant burden in cost of care and quality of life despite improved mechanistic understanding and advanced interventions. An agent-based model (ABM) of ischemia/reperfusion-induced inflammation and PU (the PUABM) was created, calibrated to serial images of post-SCI PU, and used to investigate potential treatments in silico. Tissue-level features of the PUABM recapitulated visual patterns of ulcer formation in individuals with SCI. These morphological features, along with simulated cell counts and mediator concentrations, suggested that the influence of inflammatory dynamics caused simulations to be committed to “better” vs. “worse” outcomes by 4 days of simulated time and prior to ulcer formation. Sensitivity analysis of model parameters suggested that increasing oxygen availability would reduce PU incidence. Using the PUABM, in silico trials of anti-inflammatory treatments such as corticosteroids and a neutralizing antibody targeted at Damage-Associated Molecular Pattern molecules (DAMPs) suggested that, at best, early application at a sufficiently high dose could attenuate local inflammation and reduce pressure-associated tissue damage, but could not reduce PU incidence. The PUABM thus shows promise as an adjunct for mechanistic understanding, diagnosis, and design of therapies in the setting of PU.     

Increase of histidine content in Brassica rapa subsp. oleifera by over-expression of histidine-rich fusion proteins

An approach that enables the increase of the quantity of a specific amino acid in crop plants is reported. Oleosin gene from Arabidopsis thaliana or 30K movement protein gene of Tobacco mosaic virus (TMV; genus Tobamovirus) were cloned under the control of napin or hybrid promoters, and in fusion to synthetic poly-histidine (poly-His) sequences for transformation into spring turnip rape (Brassica rapa subsp. oleifera; synonym to B. campestris). The most stable expression cassettes for the poly-His production prior to the plant transformation were selected by analyzing the protein expression in in vitro translation and in transient plant expression systems using GFP as marker. Expression of the poly-His-constructs in transgenic Brassica rapa plants was analyzed using dot and western blotting and PCR. The constructs were stably expressed in the third generation of the transgenic plant lines. Histidine content was measured from the seeds of the transgenic plants, and some plant lines had more than 20% increase in histidine content compared to wild type. The methodology may be widely applicable to increase the content of any amino acid in crop plants including those encoded by rare codons.     

At-4/1, an interactor of the Tomato spotted wilt virus movement protein, belongs to a new family of plant proteins capable of directed intra- and intercellular trafficking

The Tomato spotted wilt virus (TSWV) encoded NSm movement protein facilitates cell-to-cell spread of the viral genome through structurally modified plasmodesmata. NSm has been utilized as bait in yeast two-hybrid interaction trap screenings. As a result, a protein of unknown function, called At-4/1, was isolated from an Arabidopsis thaliana GAL4 activation domain-tagged cDNA library. Using polyclonal antibodies against bacterially expressed At-4/1, Western blot analysis of protein extracts isolated from different plant species as well as genome database screenings showed that homologues of At-4/1 seemed to be encoded by many vascular plants. For subcellular localization studies, At-4/1 was fused to green fluorescent protein, and corresponding expression vectors were used in particle bombardment and agroinfiltration assays. Confocal laser scannings revealed that At-4/1 assembled in punctate spots at the cell periphery. The protein accumulated intracellularly in a polarized fashion, appearing in only one-half of a bombarded epidermal cell, and, moreover, moved from cell to cell, forming twin-structured bodies seemingly located at both orifices of the plasmodesmatal pore. In coexpression studies, At-4/1 colocalized with a plant virus movement protein TGBp3 known to reside in endoplasmic reticulum-derived membrane structures located in close vicinity to plasmodesmata. Thus, At-4/1 belongs to a new family of plant proteins capable of directed intra- and intercellular trafficking.