Cholesterol and anionic phospholipids increase the binding of amyloidogenic transthyretin to lipid membranes

Deposition of transthyretin (TTR) amyloid is a pathological hallmark of familial amyloidotic polyneuropathy (FAP). Recently we showed that TTR binds to membrane lipids via electrostatic interactions and that membrane binding is correlated with the cytotoxicity induced by amyloidogenic TTR. In the present study, we examined the role of lipid composition in membrane binding of TTR by a surface plasmon resonance (SPR) approach. TTR bound to lipid bilayers through both high- and low-affinity interactions. Increasing the mole fraction of cholesterol in the bilayer led to an increase in the amount of high-affinity binding of an amyloidogenic mutant (L55P) TTR. In addition, a greater amount of L55P TTR bound with high affinity to membranes made from anionic phospholipids, phosphatidylglycerol (PG) and phosphatidylserine (PS), than to membranes made from zwitterionic phospholipid phosphatidylcholine (PC). The anionic phospholipids (PS and PG) promoted the aggregation of L55P TTR by accelerating the nucleation phase of aggregation, whereas the zwitterionic phospholipid PC had little effect. These results suggest that cholesterol and anionic phospholipids may be important for TTR aggregation and TTR-induced cytotoxicity.     

Preparation of glycoconjugates by dialkyl squarate chemistry revisited

The methyl 6-hydroxyhexanoyl glycoside of lactose was treated with each of 1,2-diaminoethane or hydrazine hydrate, and the corresponding amino amide 4 and acyl hydrazide 13, were treated with each of squaric acid dimethyl, diethyl, dibutyl, and didecyl esters. The monoesters were conjugated to bovine serum albumin (BSA) at different concentrations of hapten using 0.05 and 0.5M pH 9 borate buffer. Maximum loading was achieved faster, and the conjugation efficiency was higher, when the conjugation was conducted at higher concentrations of both hapten and buffer. Conjugations involving haptens 14-17 prepared from hydrazide 13 were generally slower and less efficient than those with compounds 5-8, which were made from amino amide 4. Maintaining pH 9 during conjugation was found to be the most important factor in ensuring that the conjugation was a fast, highly efficient, and reproducible process. When the pH of the conjugation mixture fell during the reaction, resulting in decreased reaction rate or even termination of the conjugation process, the normal course of the conjugation process could be restored by addition of buffer salts. Hydrolysis studies with monoesters formed from amino amide 4 under conjugation conditions showed that decyl ester 8 was the most stable and that the methyl compound 5 was the one most readily hydrolyzed. The stability of monoesters prepared from hydrazide 13 was similar and comparable to the decyl ester prepared from 4. No definite advantage was found for the use of any of the four dialkyl squarate reagents (methyl-, ethyl-, butyl-, and decyl-) for conversion of carbohydrate derivatives to species amenable for conjugation. Nevertheless, dimethyl squarate seemed to be the most convenient reagent because it is a crystalline, easy to handle, and commercially available material with very good reactivity.     

Direct electron transfer of hemoglobin in layered alpha-zirconium phosphate with a high thermal stability

A heme protein hemoglobin (Hb) was reacted with preexfoliated layered alpha-zirconium phosphate (alpha-ZrP) platelets. An X-ray diffraction (XRD) pattern of small range showed that the exfoliated alpha-ZrP platelets reassembled after the addition of Hb molecules, with the protein intercalated between the layers. UV-Vis and Fourier transform infrared (FTIR) spectra analysis displayed that no significant denaturation occurred to the intercalated protein. The bioactivity of Hb was also investigated by testing the electrochemical properties of the Hb/alpha-ZrP composite. Results showed that the intercalation of Hb into the layered material not only improved the thermal stability of Hb but also enhanced the direct electron transfer ability between protein molecules and electrode. The protein still showed bioactivity after treatment at a temperature as high as 85 degrees C. A pair of well-defined redox peaks at approximately -0.37 and -0.32V was observed on the cyclic voltammograms (CVs) of the Hb/alpha-ZrP composite modified electrode, and the electrode reactions showed a surface-controlled process with a single proton transfer. The resultant biosensor constructed by the Hb/alpha-ZrP composite displayed an excellent response to the reduction of hydrogen peroxide (H(2)O(2)) with good reproducibility.     

A study of the genetic polymorphism of human inter-alpha-trypsin-inhibitor (ITI) in the Han population,Chengdu, China

Phenotypes of inter-alpha-trypsin-inhibitor (ITI) have been determined by isoelectric focusing on polyacrylamide gels followed by immunofixation. The phenotype frequencies of ITI in the Han population in Chengdu, P. R. China have been investigated using this method. In addition, family studies have been conducted in 21 families. The results show that ITI is polymorphic in the Han population in Chengdu, China. The allele frequencies are as follows: ITI*1 = 0.5763. ITI*2 = 0.4107, ITI*3 = 0.0130. ITI is thus a new and promising genetic marker that can be used in the field of forensic haematogenetics.     

Purification and characterization of chymotrypsinogen from pancreas of Japanese quail (Coturnix coturnix japonica)

1. A chymotrypsinogen from pancreas of Japanese quail (Coturnix coturnix japonica) was purified by acid extraction, salt fractionation and chromatographic separation on CM-cellulose and Sephadex G-100, and gave a single protein band on SDS-PAGE. 2. Quail chymotrypsinogen had a mol. wt of 26,100 calculated from amino acid composition data, an isoelectric point of 7.68, a Km of 3.1 mM and K0 of 40.7 sec-1 for tyrosine ester substrate. 3. The activated chymotrypsinogen of quail had a maximum activity at pH 7.0-8.0 and at 45 degrees C, and was stable at pH 4.0-6.0 below 55 degrees C. 4. Comparison of quail and bovine chymotrypsinogens indicates that the activities of the enzymes from quail and bovine are more constant than their physical characteristics.