Long double-stranded sequences (dsRNA-B) of nuclear pre-mRNA consist of a few highly abundant classes of sequences: evidence from DNA cloning experiments.

DNA preparations from about hundred randomly selected clones containing mouse DNA fragments were screened for the existence of sequences complementary to long double-stranded regions of pre-mRNA able to snap back after melting (dsRNA-B). Many clones containing such sequences were found. The cloned sequences can be subdivided into three groups: (1) those complementary to about a half (at least to 30-40%) of the total dsRNA, designated as sequences B1; (2) those complementary to a part of sequence B1; and (3) sequences complementary to about a quarter (at least to 15%) of the total dsRNA referred to as sequence B2. The size of DNA sequence complementary to dsRNA is about 400 base pairs.Melting experiments with hybrids show that the members of B1 family are very similar if not identical, while the divergence among B2 sequences is higher, but still the number of substitutions does not exceed 9% of bases. Thus, the major part of dsRNA-B consists of a small number of highly abundant sequences as was suggested earlier on the basis of renaturation kinetics /1-3/. Sequences B1 and B2 are represented by many copies in the mouse genome and in pre-mRNA, and many of them probably do not form hairpin-like structures.     

Bov-B-mobilized SINEs in vertebrate genomes

Two new short retroposon families (SINEs) have been found in the genome of springhare Pedetes capensis (Rodentia). One of them, Ped-1, originated from 5S rRNA, while the other one, Ped-2, originated from tRNA-derived SINE ID. In contrast to most currently active mammalian SINEs mobilized by L1 long retrotransposon (LINE), Ped-1 and Ped-2 are mobilized by Bov-B, a LINE family of the widely distributed RTE clade. The 3' part of these SINEs originates from two sequences in the 5' and 3' regions of Bov-B. Such bipartite structure of the LINE-derived part has been revealed in all Bov-B-mobilized SINEs known to date (AfroSINE, Bov-tA, Mar-1, and Ped-1/2), which distinguishes them from other SINEs with only a 3' LINE-derived part. Structural analysis and the distribution of Bov-B LINEs and partner SINEs supports the horizontal transfer of Bov-B, while the SINEs emerged independently in lineages with this LINE.     

Phylogeny of the order Rodentia inferred from structural analysis of short retrotransposon B1

A large-scale study of short retroposon (SINE) B1 has been conducted in the genome of rodents from most of the known families of this mammalian order. The B1 nucleotide sequences of rodents from different families exhibited a number of characteristic features including substitutions, deletions, and tandem duplications. Comparing the distribution of these features among the rodent families, the currently discussed phylogenetic relationships were tested. The results of analysis indicated (1) an early divergence of Sciuridae and related families (Aplodontidae and Gliridae) from the other rodents; (2) a possible subsequent divergence of beavers (Castoridae); (3) a monophyletic origin of the group Hystricognathi, which includes several families, such as porcupines (Hystricidae) and guinea pigs (Caviidae); (4) a possible monophyletic origin of the group formed by the remaining families, including six families of mouselike rodents (Myodonta). Various approaches to the use of short retroposons for phylogenetic studies are discussed.     

Barley microgreen incorporation in diet-controlled diabetes and counteracted aflatoxicosis in rats

Increased environmental pollution and unhealthy lifestyle are blamed for escalated chronic diseases. Exposure to aflatoxins was recently suggested to have a role in the increased incidence of type 2 diabetes mellitus. Diet modification and consumption of different functional food are now gaining attention, especially in diabetes management. This study investigates the effect of a diet containing barley microgreen against diabetes induced by streptozotocin with or without aflatoxin administration in rats. Barley microgreen was rich in 3′-Benzyloxy-5,6,7,4′-tetramethoxyflavone (48.8% of total) followed by 5β,7βH,10α-Eudesm-11-en-1α-ol (18.46%). Streptozotocin injection and/or aflatoxin administration significantly elevated glucose level, decreased insulin level, decreased β-cell function, deteriorated liver and kidney function parameters, and induced oxidative stress in the liver. Histopathology revealed irregular small-sized islets and decreased area % of insulin-positive beta cells in the pancreas, hepatic degeneration, nephropathy, and neuropathy in diabetic and/or aflatoxin administered rats compared to control. Barley microgreen diet fed to diabetic rats with or without aflatoxin alleviated all evaluated parameters. Barley microgreen diet also ameliorated the toxic effect of aflatoxin. In conclusion, exposure to aflatoxin aggravated diabetes and its complication. The incorporation of barley microgreen in the diet was able to control type 2 diabetes mellitus and the improved outcomes observed with barley microgreen treatments involved or occurred in conjunction with improved biomarkers of oxidative stress.     

Finding the best antibody dilution by repeated immunostaining of the same tissue section

One can find the optimal antibody dilution for immunostaining by repeated staining on the same tissue section by using a less dilute antibody for each attempt. Using secondary antibody and horseradish peroxidase conjugated to a dextran polymer, a section stained repeatedly with several dilutions of antibody appears as good as a section stained with only the last dilution.     

Repeated immunostaining of the same tissue section using alkaline phosphatase as a reporter

One can determine the best dilution of a primary antibody for immunohistochemistry that uses horseradish peroxidase conjugated to a secondary antibody by testing increasing concentrations sequentially on the same tissue section. When the same tissue section is incubated repeatedly with increasing concentrations of primary antibodies to epithelial membrane antigen, smooth muscle α-actin, or vimentin using alkaline phosphatase conjugated to a secondary antibody as the reporter, the best staining was obtained with a less concentrated primary antibody than was optimal for a single staining test. The best concentration of primary antibody for single run staining using an alkaline phosphatase reporting system is usually four times the best concentration for staining with multiple runs. The optimal concentration can be determined by denaturing the residual alkaline phosphatase and extracting residual stain by incubating the section in 4:1 diglyme:phosphate buffered saline for 20 min at 80^o C between tests of primary antibody concentrations. I tested the method for four chromogens from one supplier and one chromogen from a different supplier.