Optimization of potent and selective dual mTORC1 and mTORC2 inhibitors: The discovery of AZD8055 and AZD2014

The optimization of a potent and highly selective series of dual mTORC1 and mTORC2 inhibitors is described. An initial focus on improving cellular potency whilst maintaining or improving other key parameters, such as aqueous solubility and margins over hERG IC₅₀, led to the discovery of the clinical candidate AZD8055 (14). Further optimization, particularly aimed at reducing the rate of metabolism in human hepatocyte incubations, resulted in the discovery of the clinical candidate AZD2014 (21).     

Occurrence of Amblycerus species in Cordia trichotoma seeds and their influence on germination

Forest species can have their seeds damaged by granivorous insects, especially by those in their larval stage. In this context, this study aims to report the occurrence of Amblycerus species in Cordia trichotoma seeds, to describe their main damage to seeds and effects on germination, as well as their associated hymenopteran parasitoids. Therefore, seven trees were selected in the municipality of Taquaruçu do Sul, RS, Brazil. Fruits were collected weekly from the medium third of the tree crown, from the beginning of their formation until total dehiscence. To examine the damage caused by granivorous insects within the fruits, 15 fruits from each tree were sectioned with a scalpel. Furthermore, 10 fruits from each tree were stored individually in clear plates to verify the occurrence and identification of granivorous insect species. Evidence of the damage caused to seeds was verified through the germination test by comparing preserved and damaged seeds, with four repetitions of 25 seeds each. The species Amblycerus longesuturalis and Amblycerus profaupar (Chrysomelidae: Bruchinae) were found associated with fruits of C. trichotoma. Female insects predominantly laid eggs on the superior part between the marcescent calyx and the fruit, and larvae perforated the fruit tegument to start consuming seed embryos and reserves. Bruquine larvae are parasitized by Hymenoptera of Bracon, Mirax, Omeganastatus and Triapsis genera. In conclusion, the germination of C. trichotoma seeds is significantly affected by emergence orifices caused by granivorous species.     

Lysozyme crystal growth, as observed by small angle X-ray scattering, proceeds without crystallization intermediates

A combination of small angle X-ray scattering and gel techniques was used to follow the kinetics of protein crystal growth as a function of time. Hen egg white lysozyme, at different protein concentrations, was used as a model system. A new sample holder was designed, in which supersaturation is induced in the presence of salt by decreasing the temperature. It had been shown previously that a decrease in temperature and/or an increase in crystallizing agent induces an increase in the attractive interactions present in the lysozyme solutions, the lysozyme remaining monomeric. In the present paper we show that similar behaviour is observed in NaCl when agarose gels are used. During crystal growth, special attention was paid to determine whether oligomers were formed as the protein in solution was incorporated in the newly formed crystals. From these first series of experiments, we did not find any indication of oligomer formation between monomer in solution and crystal. The results obtained are in agreement with the hypothesis that lysozyme crystals in NaCl grow by addition of monomeric particles. Received: 28 July 1997 / Revised version: 4 December 1997 / Accepted: 5 December 1997     

Stabilization of membranes in human platelets freeze-dried with trehalose

Human blood platelets are normally stored in blood banks for 3-5 days, after which they are discarded. We have launched an effort at developing means for preserving the platelets for long term storage. In previous studies we have shown that trehalose can be used to preserve biological membranes and proteins during drying and have provided evidence concerning the mechanism. A myth has grown up about special properties of trehalose, which we discuss here and clarify some of what is fact and what is misconception. We have found a simple way of introducing this sugar into the cytoplasm of platelets and have successfully freeze-dried the trehalose-loaded platelets, with very promising results. We present evidence that membrane microdomains are maintained intact in the platelets freeze-dried with trehalose. Finally, we propose a possible mechanism by which the microdomains are preserved.     

Conjugated linoleic acids: all the same or to everyone its own function?

Conjugated linoleic acid (CLA) is a generic term referring to a mixture of geometrical and positional isomers of linoleic acid in which up to 16 members have been identified. Many potentially beneficial health effects have been ascribed to these fatty acids when consumed as a mixture, and where generally 2 isomers dominate, e.g. the 9c, 11t-isomer, the so-called rumenic acid, and the 10t, 12c-isomer: anti-carcinogenic, immune modulator, anti-atherosclerotic, and anti-obesity among the most spectacular. The question arises as to whether the pleiotropic biological activity is supported by one or several of the isomers. Recent studies using pure individual isomers have started to elucidate this issue, but many others are required to ascribe a respective role to each CLA isomer (the main ones as well as the minor ones), such as those occurring in some complex mixtures already commercially available, or even in foodstuff. The aim of the present study was to focus on the CLA-isomer specific effects depicted in the literature up to now.     

Infection of brassica leaf protoplasts by turnip yellow mosaic virus

Summary Conditions optimal for the infection of Brassica leaf protoplasts by turnip yellow mosaic virus (TYMV) were found to be: pH 5.4–5.8 with citrate buffer; concentration of poly-L-ornithine, 0.8–1.0 g/ml; concentration of TYMV, 0.2–1.0 g/ml. More than 90% of Brassica rapa and B. sinensis protoplasts were infected under these conditions. TYMV replication in Brassica protoplasts was followed by quantitating the virus extracted from protoplasts and separated by sucrose density gradient centrifugation. Brassica protoplasts produced 1 to 2x10⁶ TYMV particles per cell within 48 hrs. Infection by TYMV induced the formation of polyplasts (aggregates of chloroplasts) in Brassica protoplasts. Polyplast formation paralleled the appearance of TYMV-specific immunofluorescence and could thus be used conveniently to determine the number of infected protoplasts. TYMV replication in Brassica protoplasts was resistant to actinomycin D and chloramphenicol, but was completely inhibited by cycloheximide.