Optimized enzymatic synthesis of geranyl butyrate with lipase AY from Candida rugosa

Response surface methodology (RSM) and five-level, five-variable central composite rotatable design (CCRD) were used to evaluate the effects of synthetic variables, such as reaction time (1-9 h), temperature (25-65 degrees C), enzyme amount (10-50%), substrate molar ratio of geraniol to tributyrin (1:0.33-1:1), and added water amount (0-20%) on molar percent yield of geranyl butyrate, using lipase AY from Candida rugosa. Reaction time and temperature were the most important variables and substrate molar ratio had no effect on percent molar conversion. Based on contour plots, optimum conditions were: reaction time 9 h, temperature 35 degrees C, enzyme amount 50%, substrate molar ratio 1:0.33, and added water 10%. The predicted value was 100% and actual experimental value was 96.8% molar conversion. (c) 1996 John Wiley & Sons, Inc.     

Increased oxidazability of plasma low density lipoprotein from patients with coronary artery disease

Oxidative modification of lipoproteins may play a crucial role in the pathogenesis of atherosclerosis. This study was designed to examine whether increased lipid peroxides and/or oxidative susceptibility of plasma lipoproteins occur in patients with coronary artery disease. The levels of lipid peroxides, estimated as thiobarbituric acid-reactive substances (TBARS), were significantly greater in the plasma and very low density lipoprotein (VLDL) of symptomatic patients with coronary artery disease than in those of healthy persons, but the TBARS levels of low density lipoprotein (LDL) and high density lipoprotein (HDL) showed insignificant difference between patients and normals. To evaluate the oxidative susceptibility of lipoproteins, we employed in vitro Cu²⁺ oxidation of lipoproteins monitored by changes in fluorescenece, TBARS level, trinitrobenzene sulfonic acid (TNBS) reactivity, apolipoprotein immunoreactivity and agarose gel electrophoretic mobility. While VLDL and LDL of normal controls were oxidazed at 5–10 μM Cu²⁺, pooled VLDL and LDL of patients with coronary artery disease were oxidized at 1–2.5 μM Cu²⁺, i.e., at relatively lowver oxidative stress. At 5 μM Cu²⁺, VLDL and LDL of patients with coronary artery disease still showed at faster oxidation rate, judged by the rate of fluorescence increase, higher TBARS level, less TNBS reactivity, greater change in apo B immunoreactivity and higher electrophoretic mobility than those of normal controls. However, the difference on the oxidizability of HDL was insignificant for patients vs. normals. In conclusion, we have shown that plasm VLDL and LDL of patients with coronary artery disease are more susceptible to in vitro oxidative modification than those of health persons. The data suggest that enhanced oxidizability of plasma lipoproteins may be important factor influencing the development of coronary artery disease.     

Contribution of the NH2 terminus of Kv2.1 to channel activation

Opening and closing of voltage-operated channels requires theinteraction of diverse structural elements. One approach to theidentification of channel domains that participate in gating is tolocate the sites of action of modifiers. Covalent reaction of Kv2.1channels with the neutral, sulfhydryl-specificmethylmethanethiosulfonate (MMTS) caused a slowing of channel gatingwith a predominant effect on the kinetics of activation. These effectswere also obtained after intracellular, but not extracellular,application of a charged MMTS analog. Single channel analysis revealedthat MMTS acted primarily by prolonging the latency to first openingwithout substantially affecting gating transitions after the channelfirst opens and until it inactivates. To localize the channelcysteine(s) with which MMTS reacts, we generatedNH₂- and COOH-terminal deletion mutants and a construct in which all three cysteines in transmembrane regions were substituted. Only theNH₂-terminal deletion construct gave rise to currents that activated slowly and displayedMMTS-insensitive kinetics. These results show that theNH₂-terminal tail of Kv2.1 participates in transitions leading to activation through interactions involving reduced cysteine(s) that can be modulated from thecytoplasmic phase.

     

Multiorgan Detection and Characterization of Protease-Resistant Prion Protein in a Case of Variant CJD Examined in the United States

Background

Variant Creutzfeldt–Jakob disease (vCJD) is a prion disease thought to be acquired by the consumption of prion-contaminated beef products. To date, over 200 cases have been identified around the world, but mainly in the United Kingdom. Three cases have been identified in the United States; however, these subjects were likely exposed to prion infection elsewhere. Here we report on the first of these subjects.

Methodology/Principal Findings

Neuropathological and genetic examinations were carried out using standard procedures. We assessed the presence and characteristics of protease-resistant prion protein (PrP^res) in brain and 23 other organs and tissues using immunoblots performed directly on total homogenate or following sodium phosphotungstate precipitation to increase PrP^res detectability. The brain showed a lack of typical spongiform degeneration and had large plaques, likely stemming from the extensive neuronal loss caused by the long duration (32 months) of the disease. The PrP^res found in the brain had the typical characteristics of the PrP^res present in vCJD. In addition to the brain and other organs known to be prion positive in vCJD, such as the lymphoreticular system, pituitary and adrenal glands, and gastrointestinal tract, PrP^res was also detected for the first time in the dura mater, liver, pancreas, kidney, ovary, uterus, and skin.

Conclusions/Significance

Our results indicate that the number of organs affected in vCJD is greater than previously realized and further underscore the risk of iatrogenic transmission in vCJD.     

An improved alkaline lysis method for minipreparation of plasmid DNA

This study is to improve the digestion pattern of miniprepped plasmid analyzed on gel. Frequently, some ambiguous DNA bands, which are suspected to be denatured DNA molecules, appear during electrophoresis of enzyme digested miniprepped plasmids. By employing Southern hybridization of two identical gels, one had been treated with denaturation-neutralization step and another without such treatment, we confirmed that many of these ambiguous DNA bands were single-stranded (SS) DNA molecules. The presence of SS DNA was due to the use of excess amount of NaOH during plasmid DNA purification with the conventional alkaline lysis method. We, therefore, modified the procedure and recommend that a half amount of NaOH (0.1N instead of 0.2N) should be used when isolating small quantity of plasmid DNA with the method.     

Extracellular K+ elevates outward currents through Kir2.1 channels by increasing single-channel conductance

Outward currents through inward rectifier K+ channels (Kir) play a pivotal role in determining resting membrane potential and in controlling excitability in many cell types. Thus, the regulation of outward Kir current (IK1) is important for appropriate physiological functions. It is known that outward IK1 increases with increasing extracellular K+ concentration ([K+]o), but the underlying mechanism is not fully understood. A "K+-activation of K+-channel" hypothesis and a "blocking-particle" model have been proposed to explain the [K+]o-dependence of outward IK1. Yet, these mechanisms have not been examined at the single-channel level. In the present study, we explored the mechanisms that determine the amplitudes of outward IK1 at constant driving forces [membrane potential (Vm) minus reversal potential (EK)]. We found that increases in [K+]o elevated the single-channel current to the same extent as macroscopic IK1 but did not affect the channel open probability at a constant driving force. In addition, spermine-binding kinetics remained unchanged when [K+]o ranged from 1 to 150 mM at a constant driving force. We suggest the regulation of K+ permeation by [K+]o as a new mechanism for the [K+]o-dependence of outward IK1.