A case-by-case evolutionary analysis of four imprinted retrogenes

Retroposition is a widespread phenomenon resulting in the generation of new genes that are initially related to a parent gene via very high coding sequence similarity. We examine the evolutionary fate of four retrogenes generated by such an event; mouse Inpp5f_v2, Mcts2, Nap1l5, and U2af1-rs1. These genes are all subject to the epigenetic phenomenon of parental imprinting. We first provide new data on the age of these retrogene insertions. Using codon-based models of sequence evolution, we show these retrogenes have diverse evolutionary trajectories, including divergence from the parent coding sequence under positive selection pressure, purifying selection pressure maintaining parent-retrogene similarity, and neutral evolution. Examination of the expression pattern of retrogenes shows an atypical, broad pattern across multiple tissues. Protein 3D structure modeling reveals that a positively selected residue in U2af1-rs1, not shared by its parent, may influence protein conformation. Our case-by-case analysis of the evolution of four imprinted retrogenes reveals that this interesting class of imprinted genes, while similar in regulation and sequence characteristics, follow very varied evolutionary paths.     

Improving piezoelectric cell printing accuracy and reliability through neutral buoyancy of suspensions

The sedimentation and aggregation of cells within inkjet printing systems has been hypothesized to negatively impact printer performance. The purpose of this study was to investigate this influence through the use of neutral buoyancy. Ficoll PM400 was used to create neutrally buoyant MCF‐7 breast cancer cell suspensions, which were ejected using a piezoelectric drop‐on‐demand inkjet printing system. It was found that using a neutrally buoyant suspension greatly increased the reproducibility of consistent cell counts, and eliminated nozzle clogging. Moreover, the use of Ficoll PM400 was shown to not affect cellular viability. This is the first demonstration of such scale and accuracy achieved using a piezoelectric inkjet printing system for cellular dispensing. Biotechnol. Bioeng. 2012; 109: 2932–2940. © 2012 Wiley Periodicals, Inc.     

The Importance of Exosite Interactions for Substrate Cleavage by Human Thrombin

Thrombin is a serine protease of the chymotrypsin family that acts both as a procoagulant and as an anticoagulant by cleaving either factor VIII, factor V and fibrinogen or protein C, respectively. Numerous previous studies have shown that electropositive regions at a distance from the active site, so called exosites, are of major importance for the cleavage by human thrombin. Upstream of all the known major cleavage sites for thrombin in factor VIII, factor V and fibrinogen are clusters of negatively charged amino acids. To study the importance of these sites for the interaction with the exosites and thereby the cleavage by thrombin, we have developed a new type of recombinant substrate. We have compared the cleavage rate of the minimal cleavage site, involving only 8-9 amino acids (typically the P4-P4’ positions) surrounding the cleavage site, with the substrates also containing the negatively charged regions upstream of the cleavage sites. The results showed that addition of these regions enhanced the cleavage rate by more than fifty fold. However, the enhancement was highly dependent on the sequence of the actual cleavage site. A minimal site that showed poor activity by itself could be cleaved as efficiently as an optimal cleavage site when presented together with these negatively charged regions. Whereas sites conforming closely to the optimal site were only minimally enhanced by the addition of these regions. The possibility to mimic this interaction for the sites in factor V and factor VIII by recombinant substrates, which do not have the same folding as the full size target, indicates that the enhancement was primarily dependent on a relatively simple electrostatic interaction. However, the situation was very different for fibrinogen and protein C where other factors than only charge is of major importance.     

Foliar Thiourea Confers Moisture Stress Tolerance in Rainfed Maize Through Elevated Antioxidative Defence System,Osmolyte Accumulation and Starch Synthesis Grown Under Different Planting Methods

Journal of Plant Growth Regulation - Moisture stress induced by intra-season and terminal dry spells has been addressed as principal constraint for maize cultivation across the rainfed environment....     

Genetic analysis of some seed quality characters in upland cotton (Gossypium hirsutum L.)

Summary A set of diallel crosses involving ten parents was evaluated over two locations to determine the genetic control of protein per cent, oil per cent, seed index, percentage of mature seeds and number of seeds per boll. The analysis of pooled data showed that percentage of mature seeds was controlled by additive (D) and non-additive (H₁ and H₂) genetic effects. Overdominance was noticed. For seed index the D component measuring additive, and the H₂ component measuring dominance, variation were significant. Protein content and oil content were primarily under the control of non-additive (H₁ and H₂) genetic effects, while for number of seeds per boll the variability was accounted for by dominance (H₁) effects only. The development of pure lines through appropriate methods is suggested.Part of the thesis of the senior author, submitted in partial fulfilment for the Ph.D. degree     

Electrophoretic variants of red cell phosphoglucose isomerase in some caste populations of Punjab

Hemolysates from a total of 640 blood samples from seven endogamous caste groups of the Patiala and Faridkot districts of Punjab, North-West India, were subjected to horizontal starch gel electrophoresis to study the phosphoglucose isomerase (PGI) types. In addition to the usual pattern PGI 1, the only rare phenotype encountered in the present material was PGI 3-1, present in four out of the seven groups investigated. Examination of the available data from Punjab suggests that apparently rare PGI types other than PGI 3-1 are lacking in populations studied.     

Bioconversion of hemicelluloses into fungal protein

Summary The utilization of cellulose from one ton of lignocellulose for ethanol production would yield 150–250 kg of hemicelluloses. The total soluble solids in the hemicellulose fraction (HF) obtained with the Université de Sherbrooke (UdeS) process contained about 56% carbohydrates. These carbohydrates were present in the form of oligomers of various sugars, predominantly xylose. All the test fungi,Chaetomium cellulolyticum, C. cellulolyticum (asporogenous mutant) andPleurotus sajor-caju, were capable of utilizing all the carbohydrates present in HF.C. cellulolyticum gave the highest amount of protein (7 g/l) from 19 g carbohydrates/l. The yield of protein was higher than expected, indicating that carbon compounds other than reducing sugars present in HF might have been consumed for fungal growth. The inhibitory effect of toxic compounds on protein production increased with an increase in concentration of soluble solids in HF. The inhibitory effect was overcome by increasing the pH of the medium to 6.0 or 7.0. Fungal protein production from hemicelluloses will give extra revenue in our integrated approach for ethanol production from lignocelluloses.NRCC publication No. 26277.     

Xylanase ofChaetomium cellulolyticum: Its nature of production and hydrolytic potential

Summary Maximum xylanase production byChaetomium cellulolyticum was obtained in the culture supernatant after 30 h of growth at 37°C in basal medium containing 1% xylan at pH maintained between 6.5 and 7.5. Addition of 0.05% Tween 80 to the medium increased the enzyme production considerably. Xylanase production was found to be growth associated. The optimal conditions for enzymatic hydrolysis of xylan were found to be pH 6.0 and 50°C. During enzymatic hydrolysis, xylose, xylobiose and other xylooligosaccharides were liberated from xylan. The pH values for xylanase production and for xylan hydrolysis were closely related to the utilization of hemicelluloses of aspen wood for fungal protein production by this organism as reported in our earlier work.     

Production of xylanases byChaetomium cellulolyticum during growth on lignocelluloses

Summary Chaetomium cellulolyticum was able to produce xylanases on all the three lignocelluloses: wheat straw, corn stover and aspen wood. Wheat straw was the best. Solid state fermentation of lignocelluloses gave higher yields of xylanases than liquid state fermentation, but it took longer time of incubation.     

The effect of different implant biomaterials on the behavior of canine bone marrow stromal cells during their differentiation into osteoblasts

We investigated the effects of different implant biomaterials on cultured canine bone marrow stromal cells (BMSC) undergoing differentiation into osteoblasts (dBMSC). BMSC were isolated from canine humerus by marrow aspiration, cultured and differentiated on calcium phosphate scaffold (CPS), hydroxyapatite, hydroxyapatite in gel form and titanium mesh. We used the MTT method to determine the effects of osteogenic media on proliferation. The characteristics of dBMSC were assessed using alizarin red (AR), immunocytochemistry and osteoblastic markers including alkaline phosphatase/von Kossa (ALP/VK), osteocalcin (OC) and osteonectin (ON), and ELISA. The morphology of dBMSC on the biomaterials was investigated using inverted phase contrast microscopy and scanning electron microscopy. We detected expression of ALP/VK, AR, OC and ON by day 7 of culture; expression increased from day 14 until day 21. CPS supported the best adhesion, cell spreading, proliferation and differentiation of BMSCs. The effects of the biomaterials depended on their surface properties. Expression of osteoblastic markers showed that canine dBMSCs became functional osteoblasts. Tissue engineered stem cells can be useful clinically for autologous implants for treating bone wounds.