Comparison of methods to assess the enzyme accessibility and hydrolysis of pretreated lignocellulosic substrates

Fiber size analysis, water retention value, and Simons’ stain measurements were assessed for their potential to predict the susceptibility of a given substrate to enzymatic hydrolysis. Slight modifications were made to the fiber size analysis and water retention protocols to adapt these measurements to evaluate substrates for cellulolytic hydrolysis rather than pulps for papermaking. Lodgepole pine was pretreated by the steam and ethanol-organosolv processes under varying conditions. The Simons’ stain procedure proved to be an effective method for indicating the potential ease of enzymatic hydrolysis of substrates pretreated by either process or when the pretreatment conditions were altered.     

Bovine chymosin: production by rDNA technology and application in cheese manufacture

Bovine chymosin, an aspartyl protease extracted from abomasum of suckling calves, is synthesized in vivo as preprochymosin and secreted as prochymosin which is autocatalytically activated to chymosin. Chymosin is bilobular, with Asp 32 and Asp 215 acting as the catalytic residues. Chymosin A and chymosin B have pH optima of 4.2 and 3.8, respectively, and act to initiate milk clotting by cleaving kappa-casein between Phe 105 and Met 106. The gene encoding chymosin has been cloned and expressed in suitable bacteria and yeast hosts under the control of lac, trp, trp-beta, gly A genes, and serine hydroxymethyl-transferase promoters. Protein engineering of chymosin has also been attempted. A number of companies are now producing recombinant chymosin for commercial use in cheese manufacture.     

Antimalarial evaluation of copper(II) nanohybrid solids: inhibition of plasmepsin II, a hemoglobin-degrading malarial aspartic protease from Plasmodium falciparum

A new class of copper(II) nanohybrid solids, LCu(CH₃COO)₂ and LCuCl₂, have been synthesized and characterized by transmission electron microscopy, dynamic light scattering, and IR spectroscopy, and have been found to be capped by a bis(benzimidazole) diamide ligand (L). The particle sizes of these nanohybrid solids were found to be in the ranges 5–10 and 60–70 nm, respectively. These nanohybrid solids were evaluated for their in vitro antimalarial activity against a chloroquine-sensitive isolate of Plasmodium falciparum (MRC 2). The interactions between these nanohybrid solids and plasmepsin II (an aspartic protease and a plausible novel target for antimalarial drug development), which is believed to be essential for hemoglobin degradation by the parasite, have been assayed by UV–vis spectroscopy and inhibition kinetics using Lineweaver–Burk plots. Our results suggest that these two compounds have antimalarial activities, and the IC₅₀ values (0.025–0.032 μg/ml) are similar to the IC₅₀ value of the standard drug chloroquine used in the bioassay. Lineweaver–Burk plots for inhibition of plasmepsin II by LCu(CH₃COO)₂ and LCuCl₂ show that the inhibition is competitive with respect to the substrate. The inhibition constants of LCu(CH₃COO)₂ and LCuCl₂ were found to be 10 and 13 μM, respectively. The IC₅₀ values for inhibition of plasmepsin II by LCu(CH₃COO)₂ and LCuCl₂ were found to be 14 and 17 μM, respectively. Copper(II) metal capped by a benzimidazole group, which resembles the histidine group of copper proteins (galactose oxidase, β-hydroxylase), could provide a suitable anchoring site on the nanosurface and thus could be useful for inhibition of target enzymes via binding to the S1/S3 pocket of the enzyme hydrophobically. Both copper(II) nanohybrid solids were found to be nontoxic against human hepatocellular carcinoma cells and were highly selective for plasmepsin II versus human cathepsin D. The pivotal mechanism of antimalarial activity of these compounds via plasmepsin II inhibition in the P. falciparum malaria parasite is demonstrated.     

Compacting effect of BBR3464, a new-generation trisplatinum anticancer agent,on DNA

BBR3464 is a trinuclear platinum compound of formula [{trans-PtCl(NH₃)₂}₂-μ-trans-Pt(NH₃)₂{NH₂(CH₂)₆NH₂}₂]⁴⁺. It is a new-generation platinum chemotherapeutic agent that exhibits cytotoxicity at ten to thousand times lower dose limit compared to the well-known platinum drug cisplatin, in cisplatin-sensitive as well as in cisplatin-resistant cells. DNA is thought to be the primary cellular target of BBR3464. In this work, we have applied high-resolution atomic force microscopy (AFM) for the first time, to obtain direct information on BBR3464-induced structural changes of DNA. It is found that the DNA molecules get compacted after treatment with BBR3464, for the drug:DNA molar ratio and the drug treatment period of 0.01 and 48 h, respectively. These values of molar ratio and incubation period have been obtained previously, as a result of biochemical optimization studies carried out for achieving maximum drug effects. The DNA structural changes, as observed in AFM topographs, have been correlated to the bulk level spectroscopic information. A remark on the significance of BBR3464-induced DNA compaction with respect to the available AFM reports on DNA modification by cisplatin has been made.     

Acclimatization of tissue cultured plantlets: from laboratory to land

The ultimate success of micropropagation on a commercial scale depends on the ability to transfer plants out of culture on a large scale, at low cost and with high survival rates. During field transfer the in vitro grown plantlets are unable to compete with soil microbes and to cope with the environmental conditions. The in vitro culture conditions result in the plantlets with altered morphology, anatomy and physiology. In order to increase growth and reduce mortality in plantlets at the acclimatisation stage, efforts are focused on the control of both physical and chemical environment and biohardening of micropropagated plantlets. This review describes the abiotic and biotic stresses and current developing methods for the acclimatization of microshoots.