Micellar electrokinetic chromatography: a convenient alternative to colorimetric and high performance liquid chromatographic detection to monitor protease activity

High performance capillary electrophoresis (HPCE) has been exploited as an analytical method alternative to current procedures for the determination of proteolytic activity of elastases from different sources. Due to some drawbacks with capillary zone electrophoresis (CZE), the mode of operation employed for the assay of elastolytic activity was micellar electrokinetic chromatography (MEKC). Using a background electrolyte consisting of 35 mM sodium tetraborate, pH 9.3, containing 65 mM SDS and 15% v/v methanol, separation of intact peptide substrate from products of proteolytic reaction was easily achieved in a fused-silica capillary of 50 cm effective length x 75 microm ID. This allowed us to determine the rate of hydrolysis of substrates and to calculate the kinetic parameters Km and k(cat) of the proteases investigated. A comparison of these data with those obtained from high performance liquid chromatography (HPLC)-based experiments showed that MEKC is a convenient technique for studying protease kinetics.     

Brownian dynamics study of the interaction between plastocyanin and cytochrome f

The electrostatic interaction between plastocyanin (PC) and cytochrome f (cyt f), electron transfer partners in photosynthesis was studied using Brownian dynamics (BD) simulations. By using the software package MacroDox, which implements the BD algorithm of Northrup et al. (Northrup, S. H., J. O. Boles, and J. C. L. Reynolds. 1987. J. Phys. Chem. 91:5991-5998), we have modeled the interaction of the two proteins based on crystal structures of poplar PC and turnip cyt f at pH 7 and a variety of ionic strengths. We find that the electrostatic attraction between positively charged residues (K58, K65, K187, and R209, among others) on cyt f and negatively charged residues (E43, D44, E59, and E60, among others) on PC steers PC into a single dominant orientation with respect to cyt f, and furthermore, that the single dominant orientation that we observe is one that we had predicted in our previous work (Pearson, D. C., E. L. Gross, and E. S. David. 1996. Biophys. J. 71:64-76). This dominant orientation permits the formation of hydrophobic interactions, which are not implemented in the MacroDox algorithm. This proposed complex between PC and cyt f implicates H87, a copper ligand on PC, as the residue that accepts electrons from the heme on cyt f (and possibly through Y1 as we proposed previously). We argue for the existence of this single dominant complex on the basis of observations that the most favorable orientations of the interaction between PC and cyt f, as determined by grouping successful BD trajectories on the basis of closest contacts of charged residues, tend to overlap one another and have very close distances between the metal centers on the two proteins (copper on PC, iron on cyt f). We use this knowledge to develop a model for PC/cyt f interaction that places a reaction between the two proteins occurring when the copper-to-iron distance is between 16 and 17 A. This reaction distance gives a good estimate of the experimentally observed rate constant for PC-cyt f interaction. Analysis of BD results as a function of ionic strength predicts an interaction that happens less frequently and becomes less specific as ionic strength increases.     

Structure of N-myristoyltransferase with bound myristoylCoA and peptide substrate analogs

N-myristoyltransferase (Nmt) attaches myristate to the N-terminal glycine of many important eukaryotic and viral proteins. It is a target for anti-fungal and anti-viral therapy. We have determined the structure, to 2.9 A resolution, of a ternary complex of Saccharomyces cerevisiae Nmt1p with bound myristoylCoA and peptide substrate analogs. The model reveals structural features that define the enzyme's substrate specificities and regulate the ordered binding and release of substrates and products. A novel catalytic mechanism is proposed involving deprotonation of the N-terminal ammonium of a peptide substrate by the enzyme's C-terminal backbone carboxylate.     

Micellar electrokinetic capillary chromatography (MECC) of UV-absorbing constituents in normal urine: a chemometric optimisation of the separation

A micellar electrokinetic capillary chromatography (MECC) method when compared to free solution capillary electrophoresis (CZE) was shown to offer improved selectivity and resolution for the separation of UV-absorbing components of human urine. Some of the factors affecting MECC separation e.g. methanol concentration, sodium dodecyl sulphate (SDS) concentration, beta-cyclodextrin (beta-CD) concentration, voltage, pH, temperature and electrolyte additives (urea, beta-CD and Brij 35) were optimised using chemometric techniques. Three-level three-factor (3(3)) factorial designs and simplex optimisation were used to achieve optimised conditions with the goal of obtaining the maximum number of peaks in the shortest possible analysis time. Using a TSP CE2000 instrument with detection from 195-300 nm and fitted with a 75 microns x 44 cm (37 cm effective length) fused silica capillary the final optimum conditions were found to be, an electrolyte consisting of 30 mM sodium tetraborate, pH 10, containing 75 mM SDS and 10 mM beta-CD, 15 degrees C, 20 kV, 4 s hydrodynamic injection of filtered urine. These conditions were capable of separating 70 peaks from a normal human urine pool in less than 12 min. The separation of components in urine using the optimised MECC was simpler, more reproducible, faster and gave better resolution than gradient reversed-phase high performance liquid chromatography.     

Salt effects on hydrophobic interaction and charge screening in the folding of a negatively charged peptide to a coiled coil (leucine zipper)

The stability of a coiled coil or leucine zipper is controlled by hydrophobic interactions and electrostatic forces between the constituent helices. We have designed a 30-residue peptide with the repeating seven-residue pattern of a coiled coil, (abcdefg)n, and with Glu in positions e and g of each heptad. The glutamate side chains prevented folding at pH values above 6 because of electrostatic repulsion across the helix dimer interface as well as within the individual helices. Protonation of the carboxylates changed the conformation from a random coil monomer to a coiled coil dimer. Folding at alkaline pH where the peptide had a net charge of -7e was promoted by the addition of salts. The nature of the charge screening cation was less important than that of the anion. The high salt concentrations (>1 M) necessary to induce folding indicated that the salt-induced folding resulted from alterations in the protein-water interaction. Folding was promoted by the kosmotropic anions sulfate and fluoride and to a lesser extent by the weak kosmotrope formate, whereas chloride and the strong chaotrope perchlorate were ineffective. Kosmotropes are excluded from the protein surface, which is preferentially hydrated, and this promotes folding by strengthening hydrophobic interactions at the coiled coil interface. Although charge neutralization also contributed to folding, it was effective only when the screening cation was partnered by a good kosmotropic anion. Folding conformed to a two-state transition from random coil monomer to coiled coil dimer and was enthalpy driven and characterized by a change in the heat capacity of unfolding of 3.9 +/- 1.2 kJ mol-1 K-1. The rate of folding was analyzed by fluorescence stopped-flow measurements. Folding occurred in a biphasic reaction in which the rapid formation of an initial dimer (kf = 2 x 10(7) M-1 s-1) was followed by an equally rapid concentration-independent rearrangement to the folded dimer (k > 100 s-1).     

Regulation of endothelial production of C-type natriuretic peptide by interaction between endothelial cells and macrophages

We demonstrated endothelial production of C-type natriuretic peptide (CNP), the third member of the natriuretic peptide family, and its regulation by cytokines, including tumor necrosis factor-alpha (TNF alpha). We thus proposed that CNP can control vascular tone and growth as an endothelium-derived relaxing peptide. We also revealed the marked elevation of plasma CNP concentration in patients with septic shock, in which TNF alpha plays a significant part. As the interaction between endothelial cells (EC) and monocytes-macrophages plays a pivotal role in the pathogenesis of atherosclerosis, we investigated the effect of coculture of EC and macrophages on endothelial production of CNP. We used a human monocytic leukemia cell line, THP-1, which differentiates into macrophages when treated with phorbol 12-myristate 13-acetate. The coculture of EC and THP-1-derived macrophages enhanced CNP secretion by more than 10-fold compared with the single culture of EC or the coculture of EC and THP-1 without phorbol 12-myristate 13-acetate treatment. Prevention of direct contact between EC and THP-1-derived macrophages did not attenuate the increase in CNP secretion. Northern blotting revealed the augmentation of CNP messenger RNA expression in EC in the coculture. We detected TNF alpha in the conditioned medium from the coculture of EC and THP-1-derived macrophages. Furthermore, anti-TNF alpha antibody inhibited the stimulation of CNP secretion in the coculture. CNP at a concentration of 1 nM did not stimulate cGMP production in EC or THP-1-derived macrophages, but it elevated cGMP production significantly in vascular smooth muscle cells. These results indicate that endothelial production of CNP is stimulated mainly by TNF alpha released from THP-1-derived macrophages in the coculture. Endothelial CNP at the enhanced level may be one of the vascular mediators to regulate local vascular tone and growth through cGMP production by vascular smooth muscle cells, suggesting the potential significance of endothelial CNP in atherosclerosis.     

A pharmacokinetic study to determine the drug interaction between valproate and propranolol

A 40-year-old female receiving divalproex sodium (VPA) monotherapy for epilepsy developed a tremor secondary to the drug. Propranolol treatment was initiated. While receiving propranolol 40 mg, VPA clearance was reduced from 1.66 L/hr to 1.19 L/hr and dropped to 1.08 L/hr on propranolol 80 mg. The mechanism of this interaction is unknown. To evaluate the potential for a drug interaction between these two agents, 12 patients on VPA monotherapy, ages 19-55, were studied. The subjects were maintained on a constant dose of VPA. Each was then randomly assigned to receive placebo, or long-acting propranolol 60 mg/day or 120 mg/day. Mean VPA serum concentrations did not change among the three groups. Plasma half-life of VPA ranged from 7.3-18 hours and did not change with coadministration of propranolol. We concluded that VFA metabolism is not affected by coadministration of propranolol in this group of patients.     

Dielectric study of the interaction between DNA and an oligopeptide (lysine-tyrosine-lysine)

Interaction between Na-DNA and the oligopeptide lysine-tyrosine-lysine (LTL) is studied by a dielectric method. The comparison between conductivities (at the frequence of 5MHz) of LTL alone and of the complex LTL-DNA allows us to show up an electrostatic interaction between LTL and phosphates sites of DNA. During the formation of the complex LTL-DNA, a certain fraction of Na+ counter-ions is ejected from the phosphates sites.     

Analysis of the stabilization of hen lysozyme by helix macrodipole and charged side chain interaction

In the N-terminal region of the alpha-helix of the c-type lysozymes, two Asx residues exist at the 18th and 27th positions. Hen lysozyme has Asp18/Asn27 (18D/27N), and we prepared three mutant lysozymes, Asn18/Asn27 (18N/27N), Asn18/Asp27 (18N/27D), and Asp18/Asp27 (18D/27D). The stability of the wild-type (18D/27N) lysozyme supported the existence of a hydrogen bond between the side chain of Asp18 and the amide group at the N1 position in the alpha-helix, while the stability of the 18N/27D lysozyme supported the presence of the capping box between the Ser24 (N-cap) and Asp27 residues. Although electrostatic repulsion was observed between Asp18 and Asp27 residues in 18D/27D lysozyme, the dissociation of each residue contributed to stabilizing the B-helix in 18D/27D lysozyme through hydrogen bonding and charge-helix macrodipole interaction. This is the first evidence that two neighboring negative charges at the N-terminus of the helix both increased the stability of the protein.     

Synthesis and activity of partial retro-inverso analogs of the antimetastatic laminin-derived peptide, YIGSR-NH2

Very low birth weight (VLBW) children at school age show variability in their outcome, compared with normal birth weight children, although many early physical and health differences are equalized by middle childhood. Studies of nonhandicapped VLBW children have found a higher rate of school retention and school problems in this population. Differences in intelligence have been reported, although these are often confounded by socioeconomic factors such as educational level of the parent. Few studies today of children born in the late 1970s and early 1980s have related school age outcome to central nervous system (CNS) status, yet for learning disabilities or other neuropsychological deficits, this may be highly relevant. Better understanding of medical risk factors, however, will not affect the decisive influence of social factors on their expression in the school age child.     

Microcapsules of alginate-chitosan--I. A quantitative study of the interaction between alginate and chitosan

The binding of chitosan to alginate beads was studied quantitatively by using radioactive labelled fractions of chitosan. The alginate-chitosan capsules were made either by dropping a solution of sodium alginate into a solution containing chitosan or by incubating calcium alginate beads in a solution of chitosan. The first procedure yielded a binding of 0.015 microg chitosan per mm2 of capsule surface, while the latter procedure yielded over 2 microg mm(-2). The maximum obtained weight ratio of chitosan to alginate in a microcapsule after 24 h was 0.40. The binding of chitosan was markedly increased by reducing the number average molecular weight of chitosan below 20000 Da and by increasing the porosity of the alginate gel. The porosity was increased by producing homogeneous gels, and by adding calcium chloride to the chitosan solution during the membrane forming stage. The effect of calcium ions on the porosity of the gel was studied by experiments involving release of blue dextran from calcium alginate beads. The binding of chitosan was also found to increase with decreasing fraction of N-acetylations, FA, on chitosan in the range of FA = 0.3 to FA = 0, and with increasing pH in the range from pH 4 to 6. Capsules with a diameter of 500 microm had a higher weight ratio of chitosan to alginate after 24 h of binding than the capsules with the larger diameter of 1500 microm.     

Central interaction between atrial natriuretic peptide and angiotensin II in the control of sodium intake and excretion in female rats

We investigated the effects of estrogen on sodium intake and excretion induced by angiotensin II (ANG II), atrial natriuretic peptide (ANP) or ANG II plus ANP injected into the median nucleus (MnPO). Female Holtzman rats weighing 250-300 g were used. Sodium ingestion and excretion 120 min after the injection of 0.5 microliters of 0.15 M NaCl into the MnPO were 0.3 +/- 0.1 ml (N = 12) and 29 +/- 7 microEq in intact rats, 0.5 +/- 0.2 ml (N = 10) and 27 +/- 6 microEq in ovariectomized rats, and 0.2 +/- 0.08 (N = 11) and 36 +/- 8 microEq in estrogen-treated ovariectomized (50 micrograms/day for 21 days) rats, respectively. ANG II (21 microM) injection in intact, ovariectomized, and estrogen-treated ovariectomized rats increased sodium intake (3.8 +/- 0.4, 1.8 +/- 0.3 and 1.2 +/- 0.2 ml/120 min, respectively) (N = 11) and increased sodium excretion (166 +/- 18, 82 +/- 22 and 86 +/- 12 microEq/120 min, respectively) (N = 11). ANP (65 microM) injection in intact (N = 11), ovariectomized (N = 10) and estrogen-treated ovariectomized (N = 10) rats increased sodium intake (1.4 +/- 0.2, 1.8 +/- 0.3, and 1.7 +/- 0.3 ml/120 min, respectively) and sodium excretion (178 +/- 19, 187 +/- 9, and 232 +/- 29 microEq/120 min, respectively). Concomitant injection of ANG II and ANP into the MnPO of intact (N = 12), ovariectomized (N = 10) and estrogen-treated ovariectomized (N = 10) rats caused smaller effects than those produced by each peptide given alone: 1.3 +/- 0.2, 0.9 +/- 0.2 and 0.3 +/- 0.1 ml/120 min for sodium intake, respectively, and 86 +/- 9, 58 +/- 7, and 22 +/- 4 microEq/120 min for sodium excretion, respectively. Taken together, these results demonstrate that there is an antagonistic interaction of ANP and ANG II on sodium intake and excretion, and that reproductive hormones affect this interaction.     

Fluorimetric study of the interaction between ATP and ciprofloxacin-Y3+ complex and its application

A simple spectrofluorimetric method was developed for determination of trace amount of adenosine disodium triphosphate(ATP).Under the optimum conditions,we studied the interaction between CIP-Y3+-ATP complex by using absorption and fluorescence spectra.It was observed that ATP remarkably enhanced the fluorescence intensity of the CIP-Y3+ complex at λem=415 nm and the enhanced fluorescence intensity of Y3+ ion remained proportional to the concentration of ATP.The developed method was successfully applied for...     

Gas-liquid chromatographic properties of positional isomers of methyl thia, selena, and tellura laurate analogs

Gas-liquid chromatographic analyses of three complete series of synthetic positional isomers of methyl thia, selena, and tellura laurate analogs were carried on a nonpolar (SE-30) and a polar (SP-2330) stationary phase. The average ECL (equivalent chain length) values of the thia, selena, and tellura laurate on SE-30 stationary phase were 13.8, 14.8, and 15.7, respectively, while on SP-2330 the average values for the same series were 17.1, 19.0, and 19.1, respectively. Positional isomers with the heteroatom at the 2-position exhibited the lowest ECL values, while those with the heteroatom at the omega-1 position gave the highest ECL values and were readily separated from the other positional isomers of the same series of analogs by this technique.     

金属有机骨架与相变芯材相互作用的分子动力学

金属有机骨架材料（metal-organic frameworks, MOFs）由于具有规整的孔道结构,较高的孔隙率十分适合作为相变材料的载体,从而实现对相变芯材的有效封装。本文采用分子动力学方法,对Cr-MIL-101负载十八烷,十八酸,十八胺和十八醇等不同芯材而构筑的复合相变材料的结构特性进行了研究,主要包括相变芯材和金属有机骨架基材之间的相互作用,芯材在金属有机骨架材料孔道内的扩散特性以及空间分布特性等。研究表明:十八酸和金属有机骨架基体之间的相互作用最强,十八醇和十八胺次之,十八烷最弱,具体体现在相变芯材分子与金属有机骨架材料之间的相互作用能,回转半径,分子动能,自扩散系数以及热容等众多方面,此外,当芯材分子间相互作用和金属有机骨架材料与芯材之间的相互作用达到平衡时,芯材分子在孔道内处于较为自由的状态,有利于扩散的进行,进而有利于芯材的结晶。