Long-chain acyl-CoA esters inhibit phosphorylation of AMP-activated protein kinase at threonine-172 by LKB1/STRAD/MO25.

Activation of the AMP-activated protein kinase (AMPK) results in acute changes in cellular metabolism and transcriptional events that make the cell more robust when encountering an energy challenge. AMPK is thought to be inhibited by glycogen, the major storage form of intracellular carbohydrate. We hypothesized that long-chain acyl-CoA esters (LCACEs) might also inhibit AMPK signaling. Cytosolic LCACEs are available for immediate transport and oxidation within the mitochondria and accordingly may be representative of the lipid energy charge of the cell. We found that LCACEs inhibited phosphorylation of AMPK by the recombinant AMPK kinase (AMPKK) LKB1/STRAD/MO25 in a concentration-dependent manner. Palmitoyl-CoA (PCoA) did not affect the activity of phosphothreonine-172 AMPK. PCoA potently inhibited AMPKK purified from liver. Conversely, PCoA stimulated the kinase activity of LKB1/STRAD/MO25 toward the peptide substrate LKB1tide. Octanoyl-CoA, palmitate, and palmitoylcarnitine did not inhibit AMPKK activity. Removal of AMP from the reaction mixture resulted in reduced AMPKK activity in the presence of PCoA. In conclusion, these results demonstrate that the AMPKK activity of LKB1/STRAD/MO25 is substrate specific and distinct from the kinase activity of LKB1/STRAD/MO25 toward the peptide substrate LKB1tide. They also demonstrate that LCACEs inhibit the AMPKK activity of LKB1/STRAD/MO25 in a specific manner with a dependence on both a long fatty chain and a CoA moiety. These results suggest that the AMPK signaling cascade may directly sense and respond to the lipid energy charge of the cell.

Endurance training increases LKB1 and MO25 protein but not AMP-activated protein kinase kinase activity in skeletal muscle.

LKB1 complexed with MO25 and STRAD has been identified as an AMP-activated protein kinase kinase (AMPKK). We measured relative LKB1 protein abundance and AMPKK activity in liver (LV), heart (HT), soleus (SO), red quadriceps (RQ), and white quadriceps (WQ) from sedentary and endurance-trained rats. We examined trained RQ for altered levels of MO25 protein and LKB1, STRAD, and MO25 mRNA. LKB1 protein levels normalized to HT (1 +/- 0.03) were LV (0.50 +/- 0.03), SO (0.28 +/- 0.02), RQ (0.32 +/- 0.01), and WQ (0.12 +/- 0.03). AMPKK activities in nanomoles per gram per minute were HT (79 +/- 6), LV (220 +/- 9), SO (22 +/- 2), RQ (29 +/- 2), and WQ (42 +/- 4). Training increased LKB1 protein in SO, RQ, and WQ (P < 0.05). LKB1 protein levels after training (%controls) were SO (158 +/- 17), RQ (316 +/- 17), WQ (191 +/- 27), HT (106 +/- 2), and LV (104 +/- 7). MO25 protein after training (%controls) was 595 +/- 71. Training did not affect AMPKK activity. MO25 but not LKB1 or STRAD mRNA increased with training (P < 0.05). Trained values (%controls) were MO25 (164 +/- 22), LKB1 (120 +/- 16), and STRAD (112 +/- 17). LKB1 protein content strongly correlated (r = 0.93) with citrate synthase activity in skeletal muscle (P < 0.05). In conclusion, endurance training markedly increased skeletal muscle LKB1 and MO25 protein without increasing AMPKK activity. LKB1 may be playing multiple roles in skeletal muscle adaptation to endurance training.

High-performance capillary electrophoretic separation of carbohydrates with indirect UV detection using diethylamine and borate as electrolyte additives.

The separation of underivatized carbohydrates was investigated using capillary zone electrophoresis with indirect UV detection based on sorbic acid or riboflavin. In comparison with sodium hydroxide, the separation of carbohydrates was greatly improved using diethylamine (DEA) as an electrolyte additive. Since the conductivity of DEA is much lower than that of sodium hydroxide, a stable baseline and a 3-5 times improvement in separation efficiency was observed at a pH as high as 12.6. It was found that DEA reduced the electrophoretic mobility of carbohydrates by formation of ion-pairs, and also reduced the electroosmotic flow by masking the silanol groups at the capillary surface. Many monosaccharides, including glucose, rhamnose, mannose, and fructose, which have very similar pKa values, were completely separated using 5% DEA at pH 12.6. The separation of di- and trisaccharides using indirect detection with a borate buffer at pH 9.5 was also investigated. The separation of disaccharides was greatly improved with borate complexation using riboflavin as a background electrolyte (BGE) over the sodium hydroxide and DEA electrolyte system with sorbic acid as BGE. The resolution of carbohydrates increased as pH increased, and sucrose, trehalose, raffinose, and maltotriose were successfully separated at pH 11.8.

A novel buffer system for separation of metal cations by capillary electrophoresis with indirect UV detection.

Generally, the buffers used for metal ion separations in capillary electrophoresis (CE) consist of a UV-active substance, pH-adjuster, and weak complexing reagent. This paper describes the successful separation of metal ions with a new buffer that contains no complexing reagent. Of several weakly basic compounds tested, 2-aminopyridine was selected as the most useful UV-active substance. It was used at a concentration of 15 mM with pH adjusted to 5.0 +/- 0.1 by acetic acid. The degree of protonation of the UV-active substance played an important role in detection. The stacking phenomenon was a significant contributor to efficiency in this buffer system, and water-diluted samples gave especially high efficiencies. When a 75-micron-i.d. fused-silica capillary was used, a separation efficiency of 1.8 x 10(5) was observed. Quantitative determinations of Ca2+, Mn2+, Zn2+, and Cd2+ were achieved with good linear calibration curves over the range of concentration from a few milligrams per liter to 100 mg/L. The detection limits were 0.2 mg/L for Ca2+, 0.4 mg/L for Mn2+ and Zn2+, and 0.6 mg/L for Cd2+, based on three times the baseline noise.

The use of macrocycles as electroosmotic flow modifiers in the separation of inorganic anions by capillary electrophoresis.

In a novel approach to the use of macrocycles in separation systems, we report the use of macrocyclic ligands as electroosmotic flow modifiers for the separation of inorganic anions by capillary electrophoresis (CE). Inorganic anions were successfully separated through the use of 18-crown-6 or cryptand 2.2.2. as electroosmotic flow modifiers. We found that for our CE system, use of a macrocycle as the modifier resulted in better baseline stability and better efficiency than the commonly used modifier DETA. In our system, the separation efficiency and electroosmotic flow varied according to the pH of the buffer and the affinity of the macrocycle for the buffer metal cation. Results for sodium- and potassium-based buffers show that for the cryptand, pH plays a role in determining the amount of macrocycle-metal complex formed. Separations of a 12 anion standard illustrate these results. In addition, we report the effect of concentration of both macrocycle and metal ion on complex formation and resultant electroosmotic flow modification. Relationships between the macrocycle-metal complex, the buffer medium, the efficiency of separation, and the migration times of the analytes are discussed, and theoretical interpretations presented.

Anaesthesia for thoracoscopic pulmonary lobectomy.

This report describes the anaesthetic management of a new surgical procedure--pulmonary lobectomy accomplished by minimal invasive surgery (MIS) techniques. Upper and middle pulmonary lobectomy were performed uneventfully during general anaesthesia in a 76-yr-old woman. No similar report has yet appeared in the literature. Important anaesthetic considerations include safety concerns, the need for differential lung ventilation, and the problems inherent in a lengthy operation. In the appropriate clinical setting, anaesthesia and surgery for major pulmonary resections accomplished by MIS techniques can be performed safely and effectively.

Ion chromatographic analysis of glucose, fructose, and sucrose concentrations in raw and processed vegetables.

To meet the demands for improved carbohydrate analysis techniques, a method of sugar extraction and analysis has been developed using ion chromatography. The sample preparation method, which uses filtration and extraction steps, has been shown to adequately isolate glucose, fructose, and sucrose from other food components in raw and processed vegetables. Resulting sample preparations were successfully analyzed by ion chromatography using pulsed amperometric detection, which is a sensitive, rapid, and accurate technique. Sugar concentrations in a number of different vegetable products were successfully determined in this way.

A comparison of gradient capacity anion chromatography using macrocycles D-2.2.2 and D-2.2.1 in constant or variable temperature mode.

The ability of macrocyclic ligands to complex alkali metal cations has been exploited to perform chromatographic separations of anions. Macrocycles adsorbed to reversed phase columns can complex eluent cations, thus generating anion exchange sites. Gradient separations of anions can be performed by changing the column capacity during the course of the separation, either by changing the eluent cation or by changing the column temperature. Gradient anion separations are performed by changing the eluent from sodium hydroxide to lithium hydroxide with the cryptand D-2.2.2, while similar anion separations are achieved with D-2.2.1 by a KOH-LiOH gradient. Since the complexation of cations by macrocycles is exothermic, increasing the column temperature decreases the anion column capacity, allowing temperature gradient separations. The experimentally measured DeltaH values for D-2.2.1 are higher than for D-2.2.2, leading to steeper gradients and thus better separations with D-2.2.1.

Calculation of site affinity constants and cooperativity coefficients for binding of ligands and/or protons to macromolecules. I. Generation of partition functions and mass balance equations.

The thermodynamics of binding of a ligand A and/or proton H to a macromolecule M is treated by the partition function method. In complex systems, the representation of the equilibria by means of cumulative constants beta PQR used as coefficients in partition functions ZM, ZA, and ZH is ill-suited to least-squares refinement procedures because the cumulative constants are interrelated by common cooperativity functions gamma j(i) and common site affinity constants kappa j. There is therefore the need to express ZM, ZA, ZH as functions of site constants kappa j and cooperativity coefficients bj. This is done by developing an algebra of partition functions based on the following concepts: (i) factorability of partition functions; (ii) binary generating function Jj = (1 + kappa j[Y])i tau for each class j of sites, represented by column (Jj) and row (Jj) vectors; (iii) cooperativity between sites of one class described by functions gamma j(i), represented by diagonal matrices gamma j; (iv) probability of finding microspecies represented by elements of tensor product matrix Ll = (J1)[J2]; (v) statistical factors mij obtained from Newton polynomials, Jj; (vi) power operators Oi', O(i-l)', and O(i tau-l)', transforming vectors Jj; and (vii) operators Oi or O(i-l) indicating tensor products of i or (i-l) vectors Jj. Vectors Jj combined in tensors Ll give rise to both an affinity/cooperativity space and a parallel index space. The partition functions ZM, ZA, and ZH and the total amounts TM, TA, and TH can be obtained as an appropriate sum of elements of matrices Ll, each of which is represented in an index space by a combination p1, p2,...q1, q2,...r1, r2,... of indices ij. From these indices the contribution of that element to partition function ZM, ZA, or ZH and to total amount TM, TA, or TH is calculated in the affinity/cooperativity space as product of factors: [i tau !/i !(i tau-i)!]kappa ij(exp[bj (i-1)i])[X]i, i being any index p, q, r and X any component M, A, or H. Future applications of this algorithm to practical problems of macromolecule-ligand-proton equilibria are outlined.

Calculation of site affinity constants and cooperativity coefficients for binding of ligands and/or protons to macromolecules. II. Relationships between chemical model and partition function algorithm.

The relationships between the chemical properties of a system and the partition function algorithm as applied to the description of multiple equilibria in solution are explained. The partition functions ZM, ZA, and ZH are obtained from powers of the binary generating functions Jj = (1 + kappa j gamma j,i[Y])i tau j, where i tau j = p tau j, q tau j, or r tau j represent the maximum number of sites in sites in class j, for Y = M, A, or H, respectively. Each term of the generating function can be considered an element (ij) of a vector Jj and each power of the cooperativity factor gamma ij,i can be considered an element of a diagonal cooperativity matrix gamma j. The vectors Jj are combined in tensor product matrices L tau = (J1) [J2]...[Jj]..., thus representing different receptor-ligand combinations. The partition functions are obtained by summing elements of the tensor matrices. The relationship of the partition functions with the total chemical amounts TM, TA, and TH has been found. The aim is to describe the total chemical amounts TM, TA, and TH as functions of the site affinity constants kappa j and cooperativity coefficients bj. The total amounts are calculated from the sum of elements of tensor matrices Ll. Each set of indices (pj..., qj..., rj...) represents one element of a tensor matrix L tau and defines each term of the summation. Each term corresponds to the concentration of a chemical microspecies. The distinction between microspecies MpjAqjHrj with ligands bound on specific sites and macrospecies MpAqHR corresponding to a chemical stoichiometric composition is shown. The translation of the properties of chemical model schemes into the algorithms for the generation of partition functions is illustrated with reference to a series of examples of gradually increasing complexity. The equilibria examined concern: (1) a unique class of sites; (2) the protonation of a base with two classes of sites; (3) the simultaneous binding of ligand A and proton H to a macromolecule or receptor M with four classes of sites; and (4) the binding to a macromolecule M of ligand A which is in turn a receptor for proton H. With reference to a specific example, it is shown how a computer program for least-squares refinement of variables kappa j and bj can be organized. The chemical model from the free components M, A, and H to the saturated macrospecies MpAQHR, with possible complex macrospecies MpAq and AHR, is defined first.(ABSTRACT TRUNCATED AT 250 WORDS)

The determination of nicotine and cotinine by ion pair reversed-phase chromatography.

An ion chromatographic method is described for the determination of nicotine and cotinine in aqueous solutions. This method is based on a type of reversed-phase chromatography involving ion pair formation of protonated nicotine, cotinine, pyridine, and pyridine derivatives. Detection is accomplished by measuring the UV absorption at 262 nm. Detection limits for nicotine and cotinine are 8 ng/mL and 2 ng/mL, respectively. Analyses of environmental samples and spiked environmental samples by both this ion chromatographic method and a previously reported gas chromatographic method have been used to demonstrate the accuracy and precision of this technique. The results of the analyses of both sets of samples by the two methods are in excellent agreement with a linear correlation coefficient of 0.97.

Anaesthetic considerations for major thermal injury.

Extensive thermal injury represents a major insult to body homeostasis. The anaesthetist, while providing anaesthesia for a multitude of debridement and reconstructive procedures, is also likely to assist in initial resuscitation and stabilization and subsequent intensive care management. A thorough understanding of the major systemic and end-organ effects after a major burn allow for a better appreciation of the many pertinent considerations for anaesthesia during the immediate post-burn phase as well as the later period of reconstruction and rehabilitation.

Anaphylactoid reaction to mannitol.

A 16-year old boy with a lesion of the right eye developed, during the preoperative administration of a mannitol infusion, an anaphylactoid reaction characterized by hypotension, periorbital oedema and bronchospasm. This quickly resolved following cessation of the infusion and appropriate therapeutic measures. There were no long-lasting effects. We considered mannitol the causative agent because of its temporal relationship to the reaction and our inability to seriously implicate any other medication. A history of childhood atopy may have been a predisposing factor.