Ultrasonication enhanced hydrolytic activity of lipase in water/isooctane two-phase systems

The hydrolysis of olive oil catalyzed by Chromobacterium viscosum lipase (EC 3.1.1.3) in a water/isooctane two-phase system was carried out both under ultrasound and conventional stirring. The maximum activity of lipase in the ultrasonicated system was 1.75 times higher than that in the stirred system. The lipase activity was dependent on ultrasonic power and volume ratio of isooctane to water. The optimum reaction temperature in both systems was around 25°C. The stability of lipase at 25°C in the ultrasonicated system decreased more rapidly than that in the stirred system. In the presence of exogenous oleic acid, however the half-life of lipase in the ultrasonicated system was improved to a value, which was respectively half and twice of that in stirred systems with and without oleic acid. The maximum reaction rate (V_max) was increased by ultrasonication whereas the Michaelis constant (K_m) remained unaltered.     

Jangomolide,a novel limonoid from flacourtia jangomas

Jangomolide, a novel limonoid has been isolated, together with limonin, from Flacourtia jangomas.     

Chi-dependent formation of linear plasmid DNA in exonuclease-deficient recBCD+ strains of Escherichia coli.

Escherichia coli strains carrying mutations in sbcB (exonuclease I) or xthA (exonuclease III) accumulate high-molecular-weight linear plasmid concatemers when transformed with plasmids containing the chi sequence, 5'-GCTGGTGG-3'. Chi-dependent formation of high-molecular-weight plasmid DNA is dependent on recA and recF functions. In addition, chi stimulation occurs only in cis. Our data are consistent with models in which RecA and RecF proteins bind to and protect the DNA ends produced by RecBCD-chi interaction.     

Mechanism and stereochemistry of enzymic reactions involved in porphyrin biosynthesis.

5-Aminolaevulinate synthetase cataylses the condensation of glycine and succinyl-CoA to give 5-aminolaevulinic acid. At least two broad pathways may be considered for the initial C--C bond forming step in the reaction. In pathway A the Schiff base of glycine and enzyme bound pyridoxal phosphate (a) undergoes decarboxylation to give the carbanion (b) which then condenses with succinyl-CoA with the retention of both the original C2 hydrogen atoms of glycine. In pathway B, loss of a C2 hydrogen atom gives another type of carbanion (c) that reacts with succinyl-CoA. Evidence has been presented to show that the initial C--C bond forming event occurs via pathway B which involves the removal of the pro R hydrogen atom of glycine. Subsequent mechanistic and stereochemical events occurring at the carbon atom destined to become C5 of 5-aminolaevulinate have also been delineated.(Carticle) Several mechanistic alternatices for the formation of the two vinyl groups of haem from the propionate residues of the precursor, coproporphyrinogen III, have been examined. (see article). It is shown that during the biosynthesis both the hydrogen atoms resident at the alpha positions of the propionate side chains remain undisturbed thus eliminating mechanisms which predict the involvement of acrylic acid intermediates. Biosynthetic experiments performed with precursors containing stereospecific labels have shown that the two vinyl groups of haem are formed through the loss of pro S hydrogen atoms from the beta-positions of the propionate side chains. In the light of these results, three related mechanisms for the conversion, propionate leads to vinyl, have been considered. In order to study the mechanism of porphyrinogen carboxy-lyase reaction, stereo-specifically deuterated, tritiated-succinate was incorporated into the acetate residues of uroporphyrinogen III which on decarboxylation generated asymmetric methyl groups in coproporphyrinogen III and then in haem. Degradation of the latter yielded chiral acetate deriving from C and D rings of haem. Configurational analysis of this derivate acetate shows that the carboxy-lyase reaction proceeds with a retention of configuration.     

Induction of colitis in cftr-/- mice results in bile duct injury

It is unknown why some patients with inflammatory bowel disease develop primary sclerosing cholangitis. We have recently shown that patients with primary sclerosing cholangitis have an increased prevalence of mutations in the gene responsible for cystic fibrosis (CFTR) compared with individuals with inflammatory bowel disease alone. Our aim was to examine whether induction of colitis by oral dextran leads to bile duct injury in mice heterozygous or homozygous for mutations in CFTR. The effect of oral administration of docosahexaenoic acid to correct a fatty acid imbalance associated with cystic fibrosis was also examined to determine whether this would prevent bile duct inflammation. Wild-type mice and mice heterozygous and homozygous for CFTR mutations were given dextran orally for 14 days to induce colitis. Bile duct injury was quantitated by blinded histological scoring and measurement of serum alkaline phosphatase activity. The effect of pretreatment with docosahexaenoic acid for 7 days was examined. Treatment of mice with 100 mg dextran/day for 9 days followed by 85 mg/day for 5 days resulted in a significant increase in bile duct injury as determined by histological scoring in homozygous cystic fibrosis mice compared with wild-type mice (P = 0.005). The bile duct injury seen in cystic fibrosis mice was reflected in a threefold increase in serum alkaline phosphatase (P = 0.0006). Pretreatment with oral docosahexaenoic acid decreased both histological evidence of bile duct injury and serum alkaline phosphatase levels. In the setting of colitis, loss of CFTR function leads to bile duct injury.     

Helix, sheet, and polyproline II frequencies and strong nearest neighbor effects in a restricted coil library

A central issue in protein folding is the degree to which each residue's backbone conformational preferences stabilize the native state. We have studied the conformational preferences of each amino acid when the amino acid is not constrained to be in a regular secondary structure. In this large but highly restricted coil library, the backbone preferentially adopts dihedral angles consistent with the polyproline II conformation rather than alpha or beta conformations. The preference for the polyproline II conformation is independent of the degree of solvation. In conjunction with a new masking procedure, the frequencies in our coil library accurately recapitulate both helix and sheet frequencies for the amino acids in structured regions, as well as polyproline II propensities. Therefore, structural propensities for alpha-helices and beta-sheets and for polyproline II conformations in unfolded peptides can be rationalized solely by local effects. In addition, these propensities are often strongly affected by both the chemical nature and the conformation of neighboring residues, contrary to the Flory isolated residue hypothesis.     

Computational model for cell migration in three-dimensional matrices

Although computational models for cell migration on two-dimensional (2D) substrata have described how various molecular and cellular properties and physiochemical processes are integrated to accomplish cell locomotion, the same issues, along with certain new ones, might contribute differently to a model for migration within three-dimensional (3D) matrices. To address this more complicated situation, we have developed a computational model for cell migration in 3D matrices using a force-based dynamics approach. This model determines an overall locomotion velocity vector, comprising speed and direction, for individual cells based on internally generated forces transmitted into external traction forces and considering a timescale during which multiple attachment and detachment events are integrated. Key parameters characterize cell and matrix properties, including cell/matrix adhesion and mechanical and steric properties of the matrix; critical underlying molecular properties are incorporated explicitly or implicitly. Model predictions agree well with experimental results for the limiting case of migration on 2D substrata as well as with recent experiments in 3D natural tissues and synthetic gels. Certain predicted features such as biphasic behavior of speed with density of matrix ligands for 3D migration are qualitatively similar to their 2D counterparts, but new effects generally absent in 2D systems, such as effects due to matrix sterics and mechanics, are now predicted to arise in many 3D situations. As one particular sample manifestation of these effects, the optimal levels of cell receptor expression and matrix ligand density yielding maximal migration are dependent on matrix mechanical compliance.     

Respiratory distress after intratracheal bleomycin: selective deficiency of surfactant proteins B and C

Intratracheal bleomycin in rats is associated with respiratory distress of uncertain etiology. We investigated the expression of surfactant components in this model of lung injury. Maximum respiratory distress, determined by respiratory rate, occurred at 7 days, and surfactant dysfunction was confirmed by increased surface tension of the large-aggregate fraction of bronchoalveolar lavage (BAL). In injured animals, phospholipid content and composition were similar to those of controls, mature surfactant protein (SP) B was decreased 90%, and SP-A and SP-D contents were increased. In lung tissue, SP-B and SP-C mRNAs were decreased by 2 days and maximally at 4--7 days and recovered between 14 and 21 days after injury. Immunostaining of SP-B and proSP-C was decreased in type II epithelial cells but strong in macrophages. By electron microscopy, injured lungs had type II cells lacking lamellar bodies and macrophages with phagocytosed lamellar bodies. Surface activity of BAL phospholipids of injured animals was restored by addition of exogenous SP-B. We conclude that respiratory distress after bleomycin in rats results from surfactant dysfunction in part secondary to selective downregulation of SP-B and SP-C.