Effect of temperature on self-assembly of bovine beta-casein above and below isoelectric pH. Structural analysis by cryogenic-transmission electron microscopy and small-angle X-ray scattering

beta-Casein is one of the main proteins in milk, recently classified as an intrinsically unstructured protein. At neutral pH, it is composed of a highly polar N-terminus domain and a hydrophobic C-terminus tail. This amphiphilic block-copolymer-like structure leads to self-organization of the protein monomers into defined micelles. Recently, it has been shown that at room temperature, beta-casein also self-organizes into micelles in an acidic environment, but the effect of temperature on the micelles' formation and properties at the low pH regime were not explored. In the present study, we used two complementary techniques, cryogenic-transmission electron microscopy (cryo-TEM) and small-angle X-ray scattering (SAXS), to characterize at high-resolution the micelles' shape, dimensions, and aggregation numbers and to determine how these properties are affected by temperature between 1 and 40 degrees C. Two different regimes were studied: highly acidic pH where the protein is cationic, and neutral pH, where it is anionic. We found that flat disk-like micelles with low aggregation numbers formed at low temperature in the two pH regimes. Close to neutral pH increase in temperature involves a transition in the micelles' shape and dimensions from flat disks to bulky, almost spheroidal micelles, coupled with a sharp increase in the micelles' aggregation number. In contrast, no effects on the micelles' morphology or aggregation number were detected in the acidic environment within the entire temperature range studied. The self-organization into disk micelles and the lack of effect of temperature in the acidic environment are linked to the unstructured character of the protein and to the charge distribution map. The latter indicates that below the isoelectric pH (pI), beta-casein loses the distinct separation of hydrophobic and hydrophilic domains, thereby suggesting that it may no longer be considered as a classical head-tail block-copolymer amphiphile as in neutral pH.     

Composition and properties of freeze-dried products of nicotinic acid withβ-cyclodextrin and heptakis (2,6-0-dimethyl)-β-cyclodextrin

The purpose of the study was to examine the formation of inclusion compounds in the freeze-dried products obtained from aqueous solutions of nicotinic acid and -cyclodextrin (-CD), or heptakis (2,6-0-dimethyl)--cyclodextrin (DIMEB). The molar ratios used were 1:1 and 2:1. In addition two freezing temperatures (–40 and –196°C) and different secondary drying temperatures (+50 and +80°C) were used. Freeze-dried products with -CD obtained after low temperature freezing are of the same crystallographic structure as seen in a pure inclusion compound prepared by coprecipitation. Amorphous products were formed after fast freezing. The molar ratios of included nicotinic acid in the freeze-dried products vary — dependent on the preparation conditions — between 0.75:1 and 1:1. A factorial design proves that the included drug amount can be increased by enhancement of the amount of nicotinic acid used, by faster freezing, and by the combination of these two factors. The proof of inclusion formation was given by a combination of X-ray diffractography, differential scanning calorimetry, thermogravimetry and thermofractography.The freeze-dried preparations obtained with DIMEB were amorphous mixtures of the two components. No proof for inclusion of the nicotinic acid in the cyclodextrin cavity could be given. Higher (–40°C) or lower (–196°C) freezing temperatures and the running of the secondary drying process could not influence these results. The very low stability constant of the complex and steric reasons are responsible for this behavior.     

Oligodeoxynucleotide analogues containing 3′-deoxy-3′-C-threo-hydroxymethylthymidine: Synthesis, hybridization properties and enzymatic stability

Novel Oligodeoxynucleotide analogues containing 3′-C-threo-methylene phosphodiester internucleoside linkages were synthesized on automated DNA-synthesizers using the phosphoramidite approach. The sugar modified phosphoramidite building block 5 was obtained by phosphitylation of 1-(2,3-dideoxy-5-O-(4,4′-dimethoxytrityl)-3-C-hydroxymethyl-β-D-threo-pentofuranosyl)thymine (4) which was synthesized in only three steps from 5′-O-(4,4′-dimethoxytrityl)thymidine (1). The hybridization properties and enzymatic stability of the oligonucleotide analogues were studied by UV experiments. 17-Mers having one or three modifications in the middle or two modifications in each end hybridized to DNA with moderate lowered affinity compared to unmodified 17-mers (ΔT_m 1–3°C per modification). Furthermore, the end-modified and all-modified oligonucleotides were stable towards snake venom phosphodiesterase.     

Capillary electrophoretic detection in apolipoprotein E genotyping

We describe the application of capillary electrophoresis to detect DNA fragments, obtained after amplifying a part of the apolipoprotein E (apoE) gene with polymerase chain reaction (PCR). Compared to conventional agarose slab gel electrophoresis (AGE), CE appears the method of choice with regard to resolution and sensitivity, to detect DNA fragments in the range of 20-100 base pairs. Especially discrimination between apoE2/E2 and apoE2/E3 genotypes is more reliable with CE than with AGE, this being of great clinical value in the diagnosis of familiary dysbetalipoproteinemia.     

Electron distribution on an intramolecular hydrogen bond: A semiempirical AM1 study

Dipole moments of seven molecules were studied by AM1, each containing an intramolecular hydrogen bond between a hydroxyl group and a carbonyl or nitro group as hydrogen acceptors. The hydrogen bond causes two electron shifts: from H to O within the hydroxyl group and from C to O within the carbonyl group. The latter is accompanied by withdrawal of electrons from even more distant atoms. If the total electron density change is expressed as a vector, its direction is close to the direction of the O-H bond. This electron redistribution is in agreement with the previous, somewhat, puzzling experimental results. However, it differs from the commonly accepted picture according to which electron density changes on the hydrogen acceptor moiety are less important than those on the O-H bond.Dedicated to Professor Viktor Gutmann on the occasion of his 70th birthday.     

Oriental Lacquer. 10. The South East Asian Lacquer

Abstract

The family Anacardiaceae consists of 76 genera and about 600 species. A comparison of the species of Anacardiaceae is discussed with special emphasis on the importance of the exudates, the so-called sap, for their importance as lacquer to produce lacquer ware. Many exudates are poisonous. The individual chemical compounds which constitute the active ingredients of the oleoresin part of the sap and their function are described based on space-filling molecular models. Emphasis is also given to the organic components of lacquer trees of the South East Asian region, which is compared to other species of the Anacardiaceae family.