The effect of high-dose simvastatin on triglyceride-rich lipoprotein metabolism in patients with type 2 diabetes mellitus

Statins decrease triglycerides (TGs) in addition to decreasing low density lipoprotein-cholesterol. Although the mechanism for the latter effect is well understood, it is still unclear how TG decrease is achieved with statin therapy. Because hypertriglyceridemia is common in obese patients with type 2 diabetes mellitus, we studied triglyceride-rich lipoprotein triglyceride (TRL-TG) turnover in 12 such subjects using stable isotopically labeled glycerol. The diabetic subjects were studied after 12 weeks of placebo and after a similar course of therapy with simvastatin (80 mg daily) in a single-blind design. The results were compared with those from six nonobese nondiabetic control subjects. Simvastatin therapy reduced serum TGs by 35% in the diabetic subjects. Compared with the control subjects, TRL-TG secretion was almost 2-fold higher in the diabetic subjects (45.4 +/- 4.9 vs. 24.4 +/- 1.9 micromol/min; P < 0.002) and was unaffected by simvastatin therapy. However, TRL-TG clearance was significantly increased in the diabetic subjects during simvastatin treatment compared with placebo (0.25 +/- 0.03 vs. 0.16 +/- 0.02 pools/h; P < 0.002). This change was accompanied by a 49% increase in preheparin plasma lipase activity (P < 0.03) and a 21% increase in postheparin LPL activity (P < 0.01). Together, these findings provide strong evidence that the effect of statins on serum TGs is related to an increase in LPL activity, resulting in accelerated delipidation of TRL particles. The effect of high-dose simvastatin on triglyceride-rich lipoprotein metabolism in patients with type 2 diabetes mellitus.     

The changing face of glucagon fibrillation: structural polymorphism and conformational imprinting

We have established a time-resolved fluorescence assay to study fibrillation of the 29 residue peptide hormone glucagon under a variety of different conditions in a high-throughput format. Fibrils formed at pH 2.5 differ in fibrillation kinetics, morphology, thioflavin T staining and FTIR/CD spectra depending on salts, glucagon concentration and fibrillation temperature. Apparent fibrillar stability correlates with spectral and kinetic properties; generally, fibrils formed under conditions favourable for rapid fibrillation (ambient temperatures, high glucagon concentration or high salt concentration) appear less thermostable than those formed under more challenging conditions (high temperatures, low glucagon or low salt concentrations). Properties of preformed fibrils used for seeding are inherited in a prion-like manner. Thus, we conclude that the structure of fibrils formed by glucagon is not the result of the global energy minimization, but rather kinetically controlled by solvent conditions and seed-imprinting. Fibrillar polymorphism, which is being reported for an increasing number of proteins, probably reflects that fibrils have not been under evolutionary constraints to retain a single active conformation. Our results highlight the complexity of the fibrillation mechanism of glucagon, since even subtle changes in fibrillation conditions can alter the type of fibrils formed, or result in formation of mixtures of several types of fibrils.     

Surprisingly high correlation between early and late stages in non-two-state protein folding

We collected quantitative kinetic data on early and late stages of folding in non-two-state proteins from the literature, and studied the relationship between the kinetics of the two stages. There was a surprisingly high correlation between the rate constants of these stages. The correlation coefficient of the logarithmic rate constants was as high as 0.97, which could not be caused by chance. We also studied relationships of the logarithmic rate constants of the two stages with native three-dimensional structures represented by the residue-residue contact map. There were again surprisingly high correlations between the logarithmic rate constants and the number of non-local contact clusters obtained from the contact maps. Because the number of non-local contact clusters represents overall arrangement of substructures in a native protein, the results strongly suggested the importance of the arrangement of the substructures for the kinetics of both early and late stages of protein folding.     

Role of lysine versus arginine in enzyme cold-adaptation: modifying lysine to homo-arginine stabilizes the cold-adapted alpha-amylase from Pseudoalteramonas haloplanktis

The cold-adapted alpha-amylase from Pseudoalteromonas haloplanktis (AHA) is a multidomain enzyme capable of reversible unfolding. Cold-adapted proteins, including AHA, have been predicted to be structurally flexible and conformationally unstable as a consequence of a high lysine-to-arginine ratio. In order to examine the role of low arginine content in structural flexibility of AHA, the amino groups of lysine were guanidinated to form homo-arginine (hR), and the structure-function-stability properties of the modified enzyme were analyzed by transverse urea gradient-gel electrophoresis. The extent of modification was monitored by MALDI-TOF-MS, and correlated to changes in activity and stability. Modifying lysine to hR produced a conformationally more stable and less active alpha-amylase. The k(cat) of the modified enzyme decreased with a concomitant increase in deltaH# and decrease in K(m). To interpret the structural basis of the kinetic and thermodynamic properties, the hR residues were modeled in the AHA X-ray structure and compared to the X-ray structure of a thermostable homolog. The experimental properties of the modified AHA were consistent with K106hR forming an intra-Domain B salt bridge to stabilize the active site and decrease the cooperativity of unfolding. Homo-Arg modification also appeared to alter Ca2+ and Cl- binding in the active site. Our results indicate that replacing lysine with hR generates mesophilic-like characteristics in AHA, and provides support for the importance of lysine residues in promoting enzyme cold adaptation. These data were consistent with computational analyses that show that AHA possesses a compositional bias that favors decreased conformational stability and increased flexibility.     

Bicarbonate kinetics in Indian males

Measurement of rates ofin vivo substrate oxidation such as that of glucose, fatty acids and amino acids, are based on tracer (¹⁴C or¹³C) data, and often depend on the isotopic content of expired CO₂. The recovery of tracer-labelled CO₂ generated from the oxidation of¹³C labelled substrates may not be 100% over short term. This can lead to underestimation of oxidation rate of substrates, and consequently a correction for the incomplete recovery of tracer has to be applied by the determination of the recovery of¹³CO₂ in the breath during tracer bicarbonate infusions. We have studied the recovery of tracer-labelled bicarbonate using a bolus administration model, and further characterized kinetics of bicarbonate using a three-compartment model, to assess which compartmental fluxes changed during the change from a fasted state to fed state. Recovery of bicarbonate was lower at 69% and 67% (fasted and fed state) than the value of 71% and 74% found during earlier longer term of continuous infusions. During feeding, there was a 20-fold increase in the flux of bicarbonate between the central compartment and the compartment that was equivalent to the viscera. This study shows that the difference between the fasted and fed state recovery of tracer bicarbonate similar to that obtained with continuous infusions, and that bicarbonate fluxes show large changes between different compartments in the body depending on metabolic state.     

Reaction kinetics in intracellular environments with macromolecular crowding: simulations and rate laws

We review recent evidence illustrating the fundamental difference between cytoplasmic and test tube biochemical kinetics and thermodynamics, and showing the breakdown of the law of mass action and power-law approximation in in vivo conditions. Simulations of biochemical reactions in non-homogeneous media show that as a result of anomalous diffusion and mixing of the biochemical species, reactions follow a fractal-like kinetics. Consequently, the conventional equations for biochemical pathways fail to describe the reactions in in vivo conditions. We present a modification to fractal-like kinetics following the Zipf–Mandelbrot distribution which will enable the modelling and analysis of biochemical reactions occurring in crowded intracellular environments.     

Adaptive responses to static conditions in nutrient-rich cultures of luminous Ralstonia eutropha

The lux-gene fused Ralstonia eutropha, when adapting to static conditions, causes stratification of air-exposed and nutrient-rich cultures at above 0.15 mg biomass ml(-1). The O2 respiring biofilm (luminous neuston) phase, along with the dark sub-neustonic suspension phase, develops within 5-60 min. The instability of the biphasic static culture was identified as a reason for occasionally observable oscillatory bioluminescence.     

Phospholipid-stabilized nanoparticles of cyclosporine a by rapid expansion from supercritical to aqueous solution

The purpose of this research was to form stable suspensions of submicron particles of cyclosporine A, a water-insoluble drug, by rapid expansion from supercritical to aqueous solution (RESAS). A solution of cyclosporine A in CO₂ was expanded into an aqueous solution containing phospholipid vesicles mixed with nonionic surfactants to provide stabilization against particle growth resulting from collisions in the expanding jet. The products were evaluated by measuring drug loading with high performance liquid chromatography (HPLC), particle sizing by dynamic light scattering (DLS), and particle morphology by transmission electron microscopy (TEM) and x-ray diffraction. The ability of the surfactant molecules to orient at the surface of the particles and provide steric stabilization could be manipulated by changing process variables including temperature and suspension concentration. Suspensions with high payloads (up to 54 mg/mL) could be achieved with a mean diameter of 500 nm and particle size distribution ranging from 40 to 920 nm. This size range is several hundred nanometers smaller than that produced by RESAS for particles stabilized by Tween 80 alone. The high drug payloads (≈10 times greater than the equilibrium solubility), the small particle sizes, and the long-term stability make this process attractive for development.     

Decontamination of surfaces by lysozyme encapsulated in reverse micelles

Cells and enzymes can be used to decontaminate soil, water supplies, personal equipment, weapons and hospital equipment that have been exposed to bacteria, toxins or viruses. One of the problems associated with the use of microorganisms and enzymes for decontamination purposes is that the presence of water is not acceptable for some applications such as electronic equipment. One way of circumventing this problem is to allow the enzyme to distribute between a water phase and an organic phase-containing surfactant and then use the encapsulated enzyme in reverse micelles directly into the device to be clean. Reverse micelles were used to deliver the enzyme (lysozyme) to the cell-surface interface. They serve as a way to increase the local concentration of lysozyme and decrease the amount of water delivered. Specifically, we explored the lysis by free lysozyme and lysozyme encapsulated in reverse micelles of Klebsiella pneumoniae and Staphylococcus epidermidis attached to steel, glass, and hydroxyapatite. These two bacteria have been selected because they are known to be pathogenic and because of their differences in cell wall structure. Lysozyme was added to the surfaces in either reverse micelles or as a free solution and was tested under conditions of stirring and no stirring. Stirring was implemented to study the interplay between mass transfer limitations and surface roughness. We have shown that free lysozyme or lysozyme encapsulated in reverse micelles is capable of decontaminating surfaces of different texture. Lysis of the cells is slower when the encapsulated enzyme is used but lysis is more complete.     

Modulation of S6 fibrillation by unfolding rates and gatekeeper residues

We present a protein engineering analysis of the fibrillation of a protein from a thermophilic organism, the 101 residue S6 from Thermus thermophilus. When agitated, S6 fibrillates at pH 2.0 in 0.4 M NaCl. Under these solvent conditions, S6 has native-like secondary structure and also unfolds and refolds cooperatively. However, its tertiary structure appears to be more plastic than at neutral pH, and some regions of the protein may be partially unstructured. At 42 degrees C, there is a lag phase of several days after which fibrillation takes place over several hours. Data from the fibrillation behaviour of a comprehensive series of single and double mutants of S6 suggests that several factors control the onset of fibrillation. Firstly, there appears to be a contiguous region of "gatekeeper" residues that inhibit fibrillation, since their truncation significantly reduces the duration of the lag phase. This region overlaps extensively with the partially unstructured region of the protein, suggesting that residues with enhanced flexibility and solvent-accessibility are important for the initiation of fibrillation. Secondly, longer lag phases correlate with faster rates of unfolding. We interpret this to mean that kinetic stability also controls fibrillation but in the sense that the quasi-native state, rather than the denatured state, is the species that participates in nucleation. This implies that fibrillation can also occur from a quasi-native state as opposed to an ensemble of highly fluctuating structures, and highlights the delicate balance between flexibility and structure required to form organized assemblies of polypeptide chains.