Kinetic Process of β-Amyloid Formation via Membrane Binding

Recently we have studied thermodynamics of membrane-mediated β-amyloid formation in equilibrium experiments using penetratin-lipid mixtures. The results showed that penetratin bound to the membrane interface in the α-helical conformation when the peptide/lipid (P/L) ratios were below a lipid-dependent critical value P/L^∗. When P/L reached P/L^∗, small β-aggregates emerged, which served as the nuclei for large β-aggregates. Here we studied the corresponding kinetic process to understand the potential barriers for the membrane-mediated β-amyloid formation. We performed kinetic experiments using giant unilamellar vesicles made of 7:3 DOPC/DOPG. The observed time behavior of individual giant unilamellar vesicles, although complex, exhibited the physical effects seen in equilibrium experiments. Most interestingly, a potential barrier appeared to block penetratin from translocating across the bilayer. As a result, the kinetic value for the critical threshold P/L^∗ is roughly one-half of the value measured in equilibrium where peptides bind symmetrically on both sides of lipid bilayers. We also investigated the similarity and differences between the charged and neutral lipids in their interactions with penetratin. We reached an important conclusion that the bound states of peptides in lipid bilayers are largely independent of the charge on the lipid headgroups.     

The Relative Abundance of Mountain Pine Beetle Fungal Associates Through the Beetle Life Cycle in Pine Trees

The mountain pine beetle (MPB) is a native bark beetle of western North America that attacks pine tree species, particularly lodgepole pine. It is closely associated with the ophiostomatoid ascomycetes Grosmannia clavigera, Leptographium longiclavatum, Ophiostoma montium, and Ceratocystiopsis sp.1, with which it is symbiotically associated. To develop a better understanding of interactions between beetles, fungi, and host trees, we used target-specific DNA primers with qPCR to assess the changes in fungal associate abundance over the stages of the MPB life cycle that occur in galleries under the bark of pine trees. Multivariate analysis of covariance identified statistically significant changes in the relative abundance of the fungi over the life cycle of the MPB. Univariate analysis of covariance identified a statistically significant increase in the abundance of Ceratocystiopsis sp.1 through the beetle life cycle, and pair-wise analysis showed that this increase occurs after the larval stage. In contrast, the abundance of O. montium and Leptographium species (G. clavigera, L. longiclavatum) did not change significantly through the MPB life cycle. From these results, the only fungus showing a significant increase in relative abundance has not been formally described and has been largely ignored by other MPB studies. Although our results were from only one site, in previous studies we have shown that the fungi described were all present in at least ten sites in British Columbia. We suggest that the role of Ceratocystiopsis sp.1 in the MPB system should be explored, particularly its potential as a source of nutrients for teneral adults.     

Antiobesity and Antidiabetic β-agonists: Lessons Learned and Questions to be Answered

In several species of obese animals, a group of phenethanolamine β-agonists stimulates lipolysis and thermogenesis, resulting in the loss of body fat and weight. Brown adipose tissue is considered to be the major target tissue for the antiobesity activity of these compounds. Independent of this antiobesity activity, some of these compounds are also antidiabetic and increase muscle mass. Based on the pharmacological profile of these com-pounds, a npeceptor was proposed and character-ized in mouse, rat, and humans. The 133-receptor in brown adipose tissue has been suggested to mediate the antiobesity activity of these 13-agonists. Whether this receptor is responsible for the antidiabetic activ-ity and whether there is a linkage between the antiobesity/antidiabetic activity and the nutrient par-titioning activity is not clear. Clinical trials with these mixed 13-agonists showed marginal antiobesity effects when caloric intake of subjects was restricted. Insulin sensitivity was also improved in some of the trials designed to test the antidiabetic activity of these compounds. Side effects included tachycardia and tremor. To eliminate these side effects, a second generation of compounds was selected for its agonist activity on rat D3-receptors. Clinical trials with these compounds have shown lit-tle increase of energy expenditure even at high doses. Successful development of an antiohesity and antidi-abetic drug from this class of compounds will require the elucidation of the physiological role of the human 133-receptor and the regulatory mecha-nism between fuel efficiency and feeding behavior.     

Autoregulation of tubulin expression is achieved through specific degradation of polysomal tubulin mRNAs

We have utilized protein synthesis inhibitors to investigate the autoregulatory mechanism that uses the concentration of unpolymerized tubulin subunits to specify tubulin mRNA content in animal cells. Puromycin and pactamycin, both of which remove RNAs from polysomes, completely unlink tubulin RNA content from the level of free subunits, whereas pretreatment of cells with cycloheximide, which traps mRNAs onto stalled polyribosomes, enhances the specific degradation of tubulin RNAs in response to increases in the subunit content. Moreover, in the absence of protein synthesis inhibitors, the tubulin RNAs that are lost from cells with elevated free tubulin subunit levels are those that are associated with polyribosomes. Further, beta-tubulin mRNAs encoding a truncated translation product of only 26 amino acids (and that cannot be polyribosomal) are not substrates for autoregulation. We conclude that autoregulation of tubulin synthesis is achieved by specifically altering the stability of tubulin RNAs that are bound to polyribosomes.     

Oxygen consumption by portal vein-drained organs and by whole animal in conscious growing swine

A method was developed to measure simultaneously the O2 consumption (VO) by the whole animal and by the hepatic portal vein-drained organs (PVDO), including the gastrointestinal tract, spleen, and pancreas in conscious 3.5- to 4-month-old swine. The method was used to determine (i) the effect of feeding on hepatic portal vein blood flow rate (Qpv) and VO by PVDO and by the whole animal, and (ii) the significance of PVDO on the oxidative demand in the pig. Chronic cannulas were placed in the hepatic portal vein, carotid artery, and ileal vein. The Qpv was determined by an indicator dilution technique employing continuous constant infusion of 1% p-aminohippuric acid into the ileal vein. The VO2 by PVDO was estimated by multiplying Qpv by arterial-portal vein O2 difference measured with an arterial-venous O2 difference analyzer connected to the carotid artery and portal vein cannulas. Whole animal VO2 was measured with an open circuit indirect calorimeter. In seven pigs (3.5- to 4-month-old, 37.4 +/- 0.8 kg) trained to be fed once daily, feeding (1.2 kg of feed mixed with 1.2 liter of H2O) caused postprandial (6 hr) Qpv to increase more than 34 +/- 15% above the preprandial value of 34.5 +/- 4.2 ml.min-1.kg-1 body wt. The postprandial VO2 by PVDO was elevated more than 46 +/- 12% above the value of 1.52 +/- 0.20 ml.min-1.kg-1 body wt observed during the preprandial period. Whole animal VO2 increased 45 +/- 9 and 33 +/- 7% above the preprandial value of 6.23 +/- 0.57 ml.min-1.kg-1 body wt for the first 6 hr and the 7 to 12 hr after feeding, respectively. Although PVDO represent only 5% of body weight, they used 25% of whole body VO2. The study clearly illustrates the significance of PVDO on the whole animal oxidative demand in conscious growing swine.