Poly AB for an Immobilized Trypsin

Pig polyclonal antibodies against the biospecific complex of trypsin with its inhibitor “antilysine” were prepared by affinity chromatography on trypsin-bound beaded cellulose. The antibodies were characterised by ion exchange FPLC and SDS PAGE as pure IgG. The catalytic activity of trypsin was not affected by interaction with these antibodies, even in the presence of excess of antibody. Trypsin, biospecifically bound to CNBr-activated Sepharose 4B, displayed full catalytic activity.     

Effects of starvation,chilling, and injury on endocrine gland function in Galleria mellonella

Starvation, chilling, and injury of last instar Galleria mellonella larvae typically elicit extra larval molts or a delay in pupation. The primary sites of action and the nature of the signals by which these treatments affect development are not known. However, since the connections of the brain to the nerve cord are crucial for the effects of starvation and chilling, these signals apparently affect the brain-centered program of developmental regulation via the nerve cord. Chilling, and occasionally starvation, cause extra larval molts in last instar larvae treated prior to the nervous inhibition of their corpora allata; release of a cerebral allatotropin, which stimulates the production of juvenile hormone, appears to be involved in this effect. After this time, a delay in pupation is the principal effect of starvation and chilling, and is apparently due to a temporal inhibition of the release of the prothoracicotropic hormone. Chilling also appears to inhibit unstimulated ecdysteroid production by the prothoracic glands. The effect of injury is not mediated by the nerve cord, but appears to involve an inhibitory humoral factor that affects either the brain or the prothoracic glands themselves. Injury also stimulates juvenile hormone production, an effect which is enhanced when the brain is separated from the nerve cord and which is evidenced by a delay of ecdysis and the occasional retention of some larval features in the ecdysed insects. None of the effects of these various treatments on the brain and the endocrine glands persist when the brains or glands are implanted into untreated hosts.     

Time resolved thermodynamics associated with ligand photorelease in heme peroxidases and globins: Open access channels versus gated ligand release

Heme proteins represent a diverse class of biomolecules responsible for an extremely diverse array of physiological functions including electron transport, monooxygenation, ligand transport and storage, cellular signaling, respiration, etc. An intriguing aspect of these proteins is that such functional diversity is accomplished using a single type of heme macrocycle based upon iron protoporphyrin IX. The functional diversity originates from a delicate balance of inter-molecular interactions within the protein matrix together with well choreographed dynamics that modulate the heme electronic structure as well as ligand entry/exit pathways from the bulk solvent to the active site. Of particular interest are the dynamics and energetics associated with the entry/exit of ligands as this process plays a significant role in regulating the rates of heme protein activity. Time-resolved photoacoustic calorimetry (PAC) has emerged as a powerful tool through which to probe the underlying energetics associated with small molecule dissociation and release to the bulk solvent in heme proteins on time scales from tens of nanoseconds to several microseconds. In this review, the results of PAC studies on various classes of heme proteins are summarized highlighting how different protein structures affect the thermodynamics of ligand migration. This article is part of a Special Issue entitled: Protein Dynamics: Experimental and Computational Approaches.     

Histochemical demonstration of non-specific esterase in wheat root

Azo-coupling methods were used for demonstrating non-specific esterase in the wheat root in all parts of a transverse section, usually with the exception of the woody parts of the vascular bundle. The central cylinder gave a more intense reaction than the primary cortex and the rhizoderm. The reaction was not inhibited by dodecyl sulphate. A weakening of the reaction intensity was observed after application of AgNO₃. The Tween method did not yield reliable results.     

Acridine derivatives inhibit lysozyme aggregation

We have screened a library of structurally distinct acridine derivatives (19 compounds) for their ability to inhibit lysozyme amyloid aggregation in vitro. Studied acridines were divided into three structurally different groups depending on the molecule planarity and type of the side chain-planar acridines, spiroacridines and tetrahydroacridines. Thioflavine T fluorescence assay and transmission electron microscopy were used for monitoring the inhibiting activity of acridines. We have found that both the structure of the acridine side chains and molecule planarity influence their antiamyloidogenic activity. The planar acridines inhibited lysozyme aggregation effectively. Spiroacridines and tetrahydroacridines had no significant effect on the prevention of lysozyme fibrillization, probably resulting from the presence of the heterocyclic 5-membered ring and non-planarity of molecule. Moreover, in the presence of some tetrahydroacridines the enhanced extent of aggregation was detected. We identified the most active acridine derivates from studied compound library characterized by low micromolar IC(50) values, which indicate their possible application for therapeutic purpose.     

Structure and dynamics of the N-terminal loop of PsbQ from photosystem II of Spinacia oleracea

Infrared and Raman spectroscopy were applied to identify restraints for the structure determination of the 20 amino acid loop between two beta-sheets of the N-terminal region of the PsbQ protein of the oxygen evolving complex of photosystem II from Spinacia oleracea by restraint-based homology modeling. One of the initial models has shown a stable fold of the loop in a 20 ns molecular dynamics simulation that is in accordance with spectroscopic data. Cleavage of the first 12 amino acids leads to a permanent drift in the root means square deviation of the protein backbone and induces major structural changes.