Protein Interaction Analysis of Senataxin and the ALS4 L389S Mutant Yields Insights into Senataxin Post-Translational Modification and Uncovers Mutant-Specific Binding with a Brain Cytoplasmic RNA-Encoded Peptide

Senataxin is a large 303 kDa protein linked to neuron survival, as recessive mutations cause Ataxia with Oculomotor Apraxia type 2 (AOA2), and dominant mutations cause amyotrophic lateral sclerosis type 4 (ALS4). Senataxin contains an amino-terminal protein-interaction domain and a carboxy-terminal DNA/RNA helicase domain. In this study, we focused upon the common ALS4 mutation, L389S, by performing yeast two-hybrid screens of a human brain expression library with control senataxin or L389S senataxin as bait. Interacting clones identified from the two screens were collated, and redundant hits and false positives subtracted to yield a set of 13 protein interactors. Among these hits, we discovered a highly specific and reproducible interaction of L389S senataxin with a peptide encoded by the antisense sequence of a brain-specific non-coding RNA, known as BCYRN1. We further found that L389S senataxin interacts with other proteins containing regions of conserved homology with the BCYRN1 reverse complement-encoded peptide, suggesting that such aberrant protein interactions may contribute to L389S ALS4 disease pathogenesis. As the yeast two-hybrid screen also demonstrated senataxin self-association, we confirmed senataxin dimerization via its amino-terminal binding domain and determined that the L389S mutation does not abrogate senataxin self-association. Finally, based upon detection of interactions between senataxin and ubiquitin–SUMO pathway modification enzymes, we examined senataxin for the presence of ubiquitin and SUMO monomers, and observed this post-translational modification. Our senataxin protein interaction study reveals a number of features of senataxin biology that shed light on senataxin normal function and likely on senataxin molecular pathology in ALS4.     

The effects of chronic aerobic and anaerobic exercises n lymphocyte subgroups

Exercise is the strongest stress to which the body is ever exposed. The body responds to this stress through a set of physiological changes in its metabolic, hormonal and immunological systems. In this study, responses of the immune system to the long-term aerobic and anaerobic exercises have been investigated. Twenty-four sedentary male university students and officers participated in this study. Subjects were divided into two groups, each consisting of twelve people. Group-1 (age: 25.67 +/- 3.79 years, height: 174.83 +/- 5.15 cm, body mass: 72.17 +/- 8.05 kg) and Group-2 (age: 24.83 +/- 2.89 years, height: 175.3 +/- 6.68 cm, body mass: 70.67 +/- 6.15 kg). After physical examinations of the two groups, resting ECG, respiratory function tests and metabolic tests with the use of the breath by breath method were completed, and anerobic heart rates at the threshold level were determined. The first group was subjected to exercise using Monark ergometry cycles at a heart rate 10% below the threshold level for 8 weeks, 3 days a week, 30 min a day. The second group exercised at a heart rate 10% above the threshold level for 8 weeks, 3 days a week, 20 min a day. Heart rates were checked with the Polar Test during exercises. Pre-exercise (Ep) venous blood samples were taken from each group before their 1st and 24th exercises. Hb (gr), Hct (%), erythrocyte (x10(6)/microl), leukocyte (x10(6)/microl), leukocyte subpopulations (neutrophil, lymphocyte, monocyte, eosinophil, basophil %) and thrombocyte (x10(6)/microl) values were determined. CD3, CD4, CD8, CD19 and CD56 values were determined by Flow Cytometry method using monoclonal antibodies. The chronic effects of exercise were examined through a comparison of Ep blood samples at the 1st exercise with Ep blood samples at the 24th exercise. While the increase in the total leukocyte number was significant (p<0.05) in the first group, increase in the second group was found to be non-significant. When percentiles of leukocyte subpopulations were taken into consideration, changes in the first and second group were found to be non-significant. When lymphocyte subgroups were examined; in the first group a decrease in CD3 and CD4 percentiles to 7% and 12%, respectively (p<0.05) and a 65% increase (p<0.01) in the CD56 value were observed. In the second group a decrease in CD3 and CD4 percentiles to 13% and 17%, respectively (p<0.05) and a 73% increase (p<0.01) in the CD56 value were observed. The Sample-t Test and The Wilcoxon Test were used for statistical analysis.     

The Rapid Manufacture of Uniform Composite Multicellular-Biomaterial Micropellets,Their Assembly into Macroscopic Organized Tissues,and Potential Applications in Cartilage Tissue Engineering

We and others have published on the rapid manufacture of micropellet tissues, typically formed from 100–500 cells each. The micropellet geometry enhances cellular biological properties, and in many cases the micropellets can subsequently be utilized as building blocks to assemble complex macrotissues. Generally, micropellets are formed from cells alone, however when replicating matrix-rich tissues such as cartilage it would be ideal if matrix or biomaterials supplements could be incorporated directly into the micropellet during the manufacturing process. Herein we describe a method to efficiently incorporate donor cartilage matrix into tissue engineered cartilage micropellets. We lyophilized bovine cartilage matrix, and then shattered it into microscopic pieces having average dimensions < 10 μm diameter; we termed this microscopic donor matrix “cartilage dust (CD)”. Using a microwell platform, we show that ~0.83 μg CD can be rapidly and efficiently incorporated into single multicellular aggregates formed from 180 bone marrow mesenchymal stem/stromal cells (MSC) each. The microwell platform enabled the rapid manufacture of thousands of replica composite micropellets, with each micropellet having a material/CD core and a cellular surface. This micropellet organization enabled the rapid bulking up of the micropellet core matrix content, and left an adhesive cellular outer surface. This morphological organization enabled the ready assembly of the composite micropellets into macroscopic tissues. Generically, this is a versatile method that enables the rapid and uniform integration of biomaterials into multicellular micropellets that can then be used as tissue building blocks. In this study, the addition of CD resulted in an approximate 8-fold volume increase in the micropellets, with the donor matrix functioning to contribute to an increase in total cartilage matrix content. Composite micropellets were readily assembled into macroscopic cartilage tissues; the incorporation of CD enhanced tissue size and matrix content, but did not enhance chondrogenic gene expression.     

A combined treatment using ethylmethane sulfonate and ultraviolet light to compare amylase production by three Bacillus sp. isolates

Abstract

In this study, three Bacillus sp.-producing amylase enzymes were isolated from soil samples and identified using 16S rDNA sequence analysis. Amylase production and total protein productions were spectrophotometrically measured. The following media were tested to increase enzyme production: LB medium and molasses. Three Bacillus sp. were identified as follows: Bacillus subtilis subtilis, Bacillus thuringiensis, and Bacillus cereus. Amylase production levels were in the range of 10?U/mL, whereas total protein production levels were at 15?mg/mL. Higher amylase activity was found in the Bacillus subtilis isolate. Ethylmethane sulfonate (EMS) and ultraviolet (UV) mutagenesis in combination were applied to compare amylase production. Amylase activity was increased to around 58% in the treatment with 0.03?mL of EMS and UV when compared to the control group. A pilot scale bioreactor with a total working volume of 10 liters was used to produce amylase by B. subtilis subtilis. In conclusion, B. subtilis subtilis can be used to produce amylase enzyme for various industrial purposes, and, for the first time, the amylase activities of B. subtilis can be enhanced with EMS and UV treatment.     

Regulation of valproic acid induced EMT by AKT/GSK3β/β-catenin signaling pathway in triple negative breast cancer

Molecular Biology Reports - Valproic acid (VPA) is a selective histone deacetylation (HDAC) inhibitor and exerts anti-cancer properties in different types of cancer. The epithelial-to-mesenchymal...