Isoforms of Linamarase in Cassava (Manihot esculenta)

Linamarase (EC. 3.2.1.21) was purified from different tissues of cassava (leaf, rind and tuber) to compare the kinetic properties and characteristics of the enzyme in these tissues. Purified enzyme preparation appeared as single band of average molecular size 70 kD in SDS-PAGE gels. The kinetic properties of linamarase with respect to pH and temperature indicated that tuber linamarase possessed a broader pH optimum and higher temperature stability as compared to leaf and rind enzymes. Differences in Km values for linamarin were observed with leaf linamarase having the highest Km value (500 μM) followed by rind (400 μM) and then tuber (250 μM) linamarases. Rind enzyme appeared to be less susceptible to urea denaturation than the leaf enzyme. Comparison of elution profiles from DEAE-Cellulose indicated that the relative amounts of the various ionic forms of the enzyme differed in the case of each tissue. Elution patterns of the enzyme from Con A-Sepharose also differed, suggesting difference in glycosylation among leaf, rind and tuber enzymes. This was confirmed by carbohydrate analysis which showed that the tuber linamarase contained significantly higher amount of protein bound carbohydrate. These results suggest the possible occurrence of different forms of linamarase in cassava.     

Advances in molecular cloning

“Molecular cloning” meaning creation of recombinant DNA molecules has impelled advancement throughout life sciences. DNA manipulation has become easy due to powerful tools showing exponential growth in applications and sophistication of recombinant DNA technology. Cloning genes has become simple what led to an explosion in the understanding of gene function by seamlessly stitching together multiple DNA fragments or by the use of swappable gene cassettes, maximizing swiftness and litheness. A novel archetype might materialize in the near future with synthetic biology techniques that will facilitate quicker assembly and iteration of DNA clones, accelerating the progress of gene therapy vectors, recombinant protein production processes and new vaccines by in vitro chemical synthesis of any in silico-specified DNA construct. The advent of innovative cloning techniques has opened the door to more refined applications such as identification and mapping of epigenetic modifications and high-throughput assembly of combinatorial libraries. In this review, we will examine the major breakthroughs in cloning techniques and their applications in various areas of biological research that have evolved mainly due to easy construction of novel expression systems.     

Quantification of Calcium Amount in a New Experimental Model: A Comparison between Ultrasound and Computed Tomography

Purpose

Calcification is an important prognostic factor in aortic valve stenosis. However, there is no ultrasound (US) method available to accurately quantify calcification in this setting to date. We aimed to validate a new US method for measuring the amount of calcium in an in vitro model, and compare it to computed tomography (CT), the current imaging gold standard.

Materials and Methods

An agar phantom (2% agar) was made, containing 9 different amounts of calcium-hydroxyapatite Ca₅(PO₄)₃OH (2 to 50mg). The phantoms were imaged with micro-CT and US (10 MHz probe). The calcium area (area_calcium) and its maximum pixel value (PV_max) were obtained. These values were summed to calculate CT and US calcium scores (∑(area_calcium × PV_max)) and volumes (∑area_calcium). Both US- and CT-calcium scores were compared with the calcium amounts, and with each other.

Results

Both calcium scores correlated significantly with the calcium amount (R² = 0.9788, p<0.0001 and R² = 0.8154, p<0.0001 for CT and US respectively). Furthermore, there was a significant correlation between US and CT for calcium volumes (R² = 0.7392, p<0.0001) and scores (R² = 0.7391, p<0.0001).

Conclusion

We developed a new US method that accurately quantifies the amount of calcium in an in vitro model. Moreover it is strongly correlated with CT.     

Histidine-15 and lytic activity of lysozyme

The literature data on the activity of histidine-15 modified hen egg white lysozyme are conflicting: the modified enzyme is reported to have more activity, similar activity or less activity by different authors. Amino acid analysis had shown modification of the single His-15. Detailed activity studies on His-15-modified (by iodoacetic acid or diethyl pyrocarbonate) lysozyme have shown that the contradicting reports are due to the specific choices of ionic strengths and cell wall substrate concentrations and can be attributed to the substrate being negatively charged. Our analysis suggests that even though histidine-15 is far removed from the active site of lysozyme, its chemical modification or binding of the negatively-charged substrate near it, changes the conformation around the active site. However, the change in the optimum activity on chemically modifying His-15 is small.     

Production of phosphatates by fungi isolated from desert soils

Twelve fungal cultures isolated from Indian desert soils belonging toAspergillus, Penicillium, Acrophialophora andAlternaria were found to produce both acid and alkaline phosphatases in liquid medium, their amounts varying from culture to culture. Maximum production of these enzymes was observed withA. niger. In general, acid phosphatase activity was much higher as compared to alkaline phosphatase. The optimum incubation period for the production of these enzymes was found to be 14 d and thereafter it started declining. There was a significant and positive correlation between biomass production and acid phosphatase activity but not with alkaline phosphatase.     

Direct mitogenic effects of human somatomedin on human embryonic lung fibroblasts

The ability of purified basic somatomedin to reinitiate cell division in nondividing cultures of human embryonic lung fibroblasts (WI-38) maintained in serum-free medium was determined in order to assess the direct mitogenic effect of this substance on mammalian tissue. Resting cultures were prepared by incubation of the cells in serum-free medium for 48–72 hours. Addition of partially purified somatomedin resulted in cellular hypertrophy, DNA synthesis and cell division with a time course similar to that seen when serum was added. Although cells divided in response to physiological concentrations of somatomedin, doses up to 100x in excess of this did not produce as much cell division as 10% fetal calf serum. Addition of fresh medium containing somatomedin to cells previously stimulated by somatomedin failed to induce further cell division. A highly purified somatomedin preparation also stimulated cell division.