Studies on Polyadenylic Acid-Containing RNA from Developing Nervous System of the Chicken the

Abstract: To investigate certain biochemical aspects of myelination, a study was undertaken of the messenger-like RNA in the nervous system of pre- myelinating 14-day embryos and of myelinating 17-day embryos and 3-day chicks. The central and peripheral nervous systems of the chick were found to contain and to actively synthesize poly(A)⁺ RNA. RNA species binding to oligo(dT)-cellulose contained a relatively high proportion of adenylate residues and were resistant to the actions of pancreatic and T₁ ribonucleases. Preparations labeled by incubation with adenosine in vitro showed a decrease in the proportion of poly(A)⁺ RNA as the age of the animal increased, while preparations labeled in vivo exhibited the opposite trend. Polyacrylamide gel electrophoretograms of both in vivo and in vitro labeled pqeparations showed that the poly(A)⁺ fractions contained mainly heterodisperse RNA species. The average molecular size of poly(A)⁺ RNAs of purified polysomal fractions of nerve RNA from 3-day chicks was smaller than 18S, whereas that of total poly(A)⁻ RNA was larger than 18s. The proportion of poly(A)⁺ molecules larger than 18s was lower in the rapidly myelinating nerve tissues of 17-day embryos and post-hatching chicks than in those of premyelinating 14-day embryos. Similar results were obtained for crude nuclear RNA fractions or RNA preparations fractionated under denaturing conditions. These results are consistent with previous work showing that the embryonic peripheral nerve contains a larger proportion of high-molecular-weight, messenger-like RNA molecules than does nerve tissue from young chicks or adults.     

Aqueous Hydration of Nucleic Acid Constituents: Monte Carlo Computer Simulation Studies

Abstract

Monte Carlo computer simulations were performed on dilute aqueous solutions of thymine, cytosine, uracil, adenine, guanine, the dimethyl phosphate anion in the gauche-gauche conformation and a ribose and deoxyribose derivative. The aqueous hydration of each molecule was analysed in terms of quasi-component distribution functions based on the Proximity Criterion, and partitioned into hydrophobic, hydrophilic and ionic contributions. Color stereo views of selected hydration complexes are also presented. A preliminary discussion of the transferability of functional group coordination numbers is given. The results enable to comment on two current problems related to the hydration of nucleic acids: a) the theory of Dickerson and coworkers on the role of water in the relative stability of the A and B forms of DNA and b) the idea of water bridges and filaments emerging from the computer simulation results on the hydration of DNA fragments by Clementi.     

Evaluation of the Adaptive Umbrella Sampling Method

Abstract

The adaptive umbrella sampling technique, introduced recently to improve the probability ratio method and found to perform more reliably than the customary harmonic umbrella sampling, is tested and compared with other free energy methods. One of the tests applies the method to a transition involving a chemical change: calculation of the hydration free energy difference between acetone and dimethylamine and the other test calculates the conformational free energy difference between the C ₇ and α_R conformations of the alanide dipeptide. The dipeptide problem is also treated by two types of thermodynamic integrations and by the perturbation method. The result for the acetone-dimethylamine problem is compared with previous calculations on the same system using the perturbation method, overlap ratio method and finite difference thermodynamic integration. Enhancements to the adaptive umbrella sampling method are also presented.     

Association free energy of dipalmitoylphosphatidylserines in a mixed dipalmitoylphosphatidylcholine membrane

Blood coagulation is strongly dependent on the binding of vitamin K-dependent proteins to cell membranes containing phosphatidylserine (PS) via gamma-carboxyglutamic acid (Gla) domains. The process depends on calcium, which can induce nonideal behavior in membranes through domain formation. Such domain separation mediated by Ca(2+) ions or proteins can have an important contribution to the thermodynamics of the interaction between charged peripheral proteins and oppositely charged membranes. To characterize the properties of lipid-lipid interactions, molecular dynamics, and free energy simulations in a mixed bilayer membrane containing dipalmitoylphosphatidylcholine and dipalmitoylphosphatidylserine were carried out. The free energy of association between dipalmitoylphosphatidylserines in the environment of dipalmitoylphosphatidylcholines has been calculated by using a novel approach to the dual topology technique of the PS-PC hybrid. Two different methods, free energy perturbation and thermodynamic integration, were used to calculate the free energy difference. In thermodynamic integration runs three schemes were applied to evaluate the integral at the limits of lambda --> 0 or lambda --> 1. Our studies show that the association of two PSs in the environment of PCs is repulsive in the absence of Ca(2+) and becomes favorable in their presence. We also show that the mixed component membrane should exhibit nonideal behavior that will lead to PS clustering.     

Multiplexing Spheroid Volume,Resazurin and Acid Phosphatase Viability Assays for High-Throughput Screening of Tumour Spheroids and Stem Cell Neurospheres

Three-dimensional cell culture has many advantages over monolayer cultures, and spheroids have been hailed as the best current representation of small avascular tumours in vitro. However their adoption in regular screening programs has been hindered by uneven culture growth, poor reproducibility and lack of high-throughput analysis methods for 3D. The objective of this study was to develop a method for a quick and reliable anticancer drug screen in 3D for tumour and human foetal brain tissue in order to investigate drug effectiveness and selective cytotoxic effects. Commercially available ultra-low attachment 96-well round-bottom plates were employed to culture spheroids in a rapid, reproducible manner amenable to automation. A set of three mechanistically different methods for spheroid health assessment (Spheroid volume, metabolic activity and acid phosphatase enzyme activity) were validated against cell numbers in healthy and drug-treated spheroids. An automated open-source ImageJ macro was developed to enable high-throughput volume measurements. Although spheroid volume determination was superior to the other assays, multiplexing it with resazurin reduction and phosphatase activity produced a richer picture of spheroid condition. The ability to distinguish between effects on malignant and the proliferating component of normal brain was tested using etoposide on UW228-3 medulloblastoma cell line and human neural stem cells. At levels below 10 µM etoposide exhibited higher toxicity towards proliferating stem cells, whereas at concentrations above 10 µM the tumour spheroids were affected to a greater extent. The high-throughput assay procedures use ready-made plates, open-source software and are compatible with standard plate readers, therefore offering high predictive power with substantial savings in time and money.     

Flavescin: A new 1-ketopolyhydroxypregnene from Marsdenia flavescens

A new polyhydroxy-1-ketosteroid, flavescin, was isolated from Marsdenia flavescens A. Cunn. Its structure was elucidated as 12-O-acetyl-3β,8β,12β,14β-20-pentahydroxy-Δ⁵- pregnene-1-one.     

Involement of supertwisted DNA in integrative recombination of bacteriophage lambda.

Negatively supertwisted closed circular DNA is the primary substrate for integrative recombination of phage λ DNA in vitro. Closed circular λ DNA without supertwists must be converted to the supertwisted form by the action of Escherichia coli DNA gyrase before efficient recombination can occur. When negatively supertwisted substrate is provided, E. coli DNA gyrase and its cofactors are dispensable components of recombination reaction mixtures. In the absence of DNA gyrase activity, circular DNA considerably less negatively twisted than naturally occurring supercoils is an effective substrate, but positively supertwisted DNA appears to be an ineffective substrate.The predominant products of integrative recombination in vitro are covalently closed circles. The closure of the recombined sites appears to occur without appreciable DNA synthesis and without the action of E. coli DNA ligase. No detectable difference can be observed between the degree of supertwisting of product DNA and that of unrecombined DNA. These facts suggest that the resealing of broken DNA strands is an integral part of the recombination reaction mechanism and is closely coupled with the breakage and realignment steps of recombination.