Development of AC microelectrophoresis for rapid protein affinity evaluation

We have developed a new method for evaluating the affinity interactions between two different proteins by applying an alternating current (AC) voltage to a micro-flow channel. An AC voltage was applied to the protein-modified microspheres in the micro-flow channel, which resulted in the oscillation of the microspheres owing to their surface charges. The oscillation amplitude showed a linear relationship with the charge density of the microspheres. As an example for protein affinity measurement, the amplitude changes of a profilin-modified microsphere were measured by the addition of actin. In the same electrical condition, the oscillation amplitude of the profilin-modified microsphere increased by ≈175% by binding with actin. Similar results in the principle were obtained for the affinity interaction between biotin and streptavidin. The results showed that the higher the charge density of the microspheres induced by binding with different proteins, the higher the oscillation amplitude of the microspheres, thus, suggesting a possible application of the micro-flow channel and AC voltage on the protein property study, as well as on the biosensor application using the oscillation amplitude changes.     

An Atg4B mutant hampers the lipidation of LC3 paralogues and causes defects in autophagosome closure

In the process of autophagy, a ubiquitin-like molecule, LC3/Atg8, is conjugated to phosphatidylethanolamine (PE) and associates with forming autophagosomes. In mammalian cells, the existence of multiple Atg8 homologues (referred to as LC3 paralogues) has hampered genetic analysis of the lipidation of LC3 paralogues. Here, we show that overexpression of an inactive mutant of Atg4B, a protease that processes pro-LC3 paralogues, inhibits autophagic degradation and lipidation of LC3 paralogues. Inhibition was caused by sequestration of free LC3 paralogues in stable complexes with the Atg4B mutant. In mutant overexpressing cells, Atg5- and ULK1-positive intermediate autophagic structures accumulated. The length of these membrane structures was comparable to that in control cells; however, a significant number were not closed. These results show that the lipidation of LC3 paralogues is involved in the completion of autophagosome formation in mammalian cells. This study also provides a powerful tool for a wide variety of studies of autophagy in the future.     

High-performance liquid chromatographic analyses of hydroxymonocarboxylic acids and dicarboxylic acids in urine as their 2-nitrophenylhydrazides

Both hydroxymonocarboxylic acids and dicarboxylic acids in urine were converted into their 2-nitrophenylhydrazides without lengthy and cumbersome sample workup and were separated from each other by two-step extraction with diethyl ether at different pH values. HPLC analysis of each acid group was achieved isocratically within 30 min. By the use of a visible-range detector (400 nm) the detection limits ranged from 1 to 2 pmol and from 2 to 5 pmol per injection for the hydroxymonocarboxylic acids and dicarboxylic acids, respectively. The analytical results showed good recovery and reproducibility. Analysis profiles of the two acid groups in normal and diabetic subjects could be performed with 200 microliters of urine. The present method is superior over previously published methods because of its great simplicity and its time-, cost-, and labor-saving nature.     

Effects of hydrocortisone and aminophylline on plasma leukotriene C4 levels in patients during an asthmatic attack

To study the role of leukotriene C4(LTC4) and the effect of hydrocortisone and aminophylline on plasma LTC4 levels in patients with asthmatic attacks, we measured LTC4 in plasma of 18 asthmatics during a wheezing attack and of 7 normal subjects. Blood samples were obtained before and after treatment with aminophylline and/or hydrocortisone injections. We extracted LTC4 using a Sep-Pak C18 cartridge for the measurement of LTC4 by radioimmunoassay. The plasma levels of immunoreactive LTC4 (i-LTC4) of the normal subjects were 142 +/- 25 pg/ml (n = 7), while those of nonatopic type asthmatic patients with wheezing attacks were 208 +/- 68 pg/ml (n = 15) (p less than 0.01). Before and after treatment with both hydrocortisone succinate (100 mg) and aminophylline (250 mg), 6 asthmatic patients with wheezing attacks had a mean plasma level of i-LTC4 181 +/- 24 and 132 +/- 18 pg/ml (p less than 0.01), respectively. On the other hand, the treatment with aminophylline 250 mg alone increased the i-LTC4 levels from 178 +/- 19 pg/mg to 213 +/- 16 pg/mg (n = 6)(p less than 0.05), while treatment with hydrocortisone succinate 100 mg decreased the i-LTC4 level 0.05 from 284 +/- 99 pg/ml to 249 +/- 85 pg/ml (n = 4)(p less than 0.05). In conclusion, the present study shows that the i-LTC4 level in venous blood of patients with asthmatic attacks is decreased significantly by treatment with hydrocortisone succinate.     

Effects of sand burial depth on the root system of Salix cheilophila seedlings in Mu Us Sandy Land, Inner Mongolia, China

Salix cheilophila Schneid. is a naturally occurring Salix species in Mu Us Sandy Land, Inner Mongolia, China. We focused on the morphological adaptability of S. cheilophila to sand dune burial. For morphological measurements, 32 S. cheilophila seedlings were removed from a community which was in the process of being buried by a shifting sand dune. Each seedling collected included the entire root system. We measured the number, length, and biomass of the adventitious roots, primary lateral roots, and taproot, and compared the morphological characteristics of the root system, including adventitious roots, for seedlings buried to various levels in the sand. The growth range of adventitious roots increased as the length of the buried portion of the main shoot increased. In addition, the total dry weight of all current-year shoots tended to increase gradually with increasing total dry weight of the adventitious roots. These results suggest that S. cheilophila tends to make use of the sedimentary sand layer that accompanies shifting sand dunes. However, there was no correlation between biomass or number of adventitious roots and the length of the buried part of the main shoot. Thus, S. cheilophila does not grow adventitious roots proportional to the buried part. These morphological characteristics of the root system, including the adventitious roots, may indicate that S. cheilophila has poor morphological adaptability to sand dune burial.     

Preparation of human salivary alpha-amylase specific monoclonal antibody

A mouse hybridoma cell line which produced an anti-human salivary alpha-amylase monoclonal antibody was obtained by fusion between mouse spleen cells immunized with human salivary alpha-amylase and mouse myeloma cells, followed by screening the hybridoma cells by enzyme-linked immunosorbent assay. The hybridoma cell line (27-4-1) secreted IgG. The monoclonal antibody produced by the hybridoma showed no inhibitory effect on the activity of human salivary alpha-amylase. The specificity and reactivity of this monoclonal antibody were examined by determining the activities of human salivary and pancreatic alpha-amylases bound to the monoclonal antibody immobilized on polystyrene balls or by enzyme immunoassay with the monoclonal antibody conjugated with beta-D-galactosidase. The results revealed that the monoclonal antibody produced by the hybridoma cell line was specific for salivary alpha-amylase and absolutely unreactive to pancreatic alpha-amylase.     

Biphasic effects of alcohols on the phase transition of poly(L-lysine) between alpha-helix and beta-sheet conformations.

Poly(L-lysine) exists as a random-coil at neutral pH, an alpha-helix at alkaline pH, and a beta-sheet when the alpha-helix poly(L-lysine) is heated. The present Fourier-transform infrared (FTIR) study showed that short-chain alcohols (methanol, ethanol, and 2-propanol) partially transformed alpha-helix poly(L-lysine) to beta-sheet when their concentrations were low. At higher concentrations, however, these alcohols reversed the reaction, and the alcohol-induced beta-sheet was transformed back to alpha-helix structure. The reversal occurred at 1.40 M methanol, 0.96 M ethanol, and 0.55 M 2-propanol. The alcohol effects on the secondary structure were further investigated by circular dichroism (CD) on the thermally induced beta-sheet poly(L-lysine). Methanol, ethanol, and 1-propanol, but not 1-butanol, shifted the negative mean-residue ellipticity at 217 nm of the beta-sheet poly(L-lysine) to the positive side at low concentrations of the alcohols and to the negative side at high concentrations. With 1-butanol, only the positive-side shift was observed. The positive-side shift at low concentrations of alcohols indicates enhancement of the hydrophobic interactions among the side chains of the polypeptide in the beta-sheet conformation. The negative-side shift indicates a partial transformation to alpha-helix. The shift from the positive to negative side occurred at 7.1 M methanol, 4.6 M ethanol, and 3.1 M 1-propanol. The alcohol concentrations for the beta-to-alpha transition were higher in the CD study than in the IR study.(ABSTRACT TRUNCATED AT 250 WORDS)