Activity of "nonspecific pancreatic carboxylesterase" in rat serum in experimentally induced acute pancreatitis (preliminary results)

The aim of this study was to obtain more information on the serum level of "nonspecific pancreatic carboxylesterase" (PCE) in experimentally induced acute pancreatitis in rats. The effects of caerulein stimulation, hepatic duct ligation, bile-pancreatic duct ligation or the effect of retrograde injection of saline, 5% taurocholate and sunflower oil were investigated. The activity of PCE and amylase was measured in the serum, pancreatic tissue, pancreatic juice and ascitic fluid. The changes in PCE activity were greater (both in directions to increase or decrease) than that of amylase, produced by different experimental procedures. The results confirm the thesis that the serum activity of PCE is a more sensitive diagnostic method than that of amylase to detect the inflammatory process in the pancreas or the effect of obstruction of the pancreatic duct.     

Effects of postnatal age and diet on the fatty acid composition of plasma lipid fractions in preterm infants

Diet and postnatal age effect the fatty acid composition of plasma and tissue lipids. This work was designed as a transversal study to evaluate the changes in the fatty acid composition of plasma phospholipids, cholesteryl esters, triglycerides and free fatty acids in preterm infants (28-35 weeks gestational age), fed human milk (HM) and milk formula (MF) from birth to 1 month of life. Sixteen blood samples were obtained from cord, and 19 at 6-8 h after birth, 14 at 1 week and 9 at 4 weeks from HM-fed infants and 18 at 1 week and 14 at 4 weeks from MF-fed ones. Groups had similar mean birth weight, gestational age and sex ratio. The MF provided 69 kcal/dl and contained 16% of linoleic acid and 1.3% of alpha-linolenic acid on the total fat. Plasma lipid fractions were extracted and separated by thin-layer chromatography and fatty acid methyl esters were quantitated by gas liquid chromatography. In plasma phospholipids, linoleic acid (18:2 omega 6) continuously increased from birth to 1 month of age, but no changes were seen as related to type of diet; polyunsaturated fatty acids greater than 18 carbon atoms of both the omega 6 and omega 3 series (PUFA omega 6 greater than 18 C and omega 3 greater than 18 C) dropped from birth to 1 week and continued to decrease in MF-fed infants until 1 month; eicosatrienoic (20:3 omega 6), arachidonic (20:4 omega 6) and docosahexaenoic (22:6 omega 3) were the fatty acids implicated. In cholesteryl esters palmitoleic (16:1 omega 7) and oleic (18:1 omega 9) acids decreased from birth to 1 month and linoleic acid increased and arachidonic acid dropped, especially in MF fed infants. In triglycerides, palmitic, palmitoleic and stearic acid (18:0) decreased during the first month of life; oleic acid remained constant and linoleic acid increased in all infants, but arachidonic acid decreased only in those fed formula. Free fatty acids showed a similar behavior in fatty acids and in plasma triglycerides. Preterm neonates seem to have special requirements of long-chain PUFA and adapted MF should contain these fatty acids in similar amounts to those of HM to allow the maintenance of an adequate tissue structure and physiology.     

A similar protein portion for two exoglucanases secreted by Saccharomyces cerevisiae

Exoglucanase (exo-1,3-β-D-glucan glycohydrolase, EC 3.2.1.56) activity secreted by Saccharomyces cerevisiae into the culture medium was separated by ion exchange chromatography into two glycoprotein isoenzymes which contributed 10% (exoglucanase I) and 90% (exoglucanase II) towards the total activity. Analysis of the “in vitro” deglycosylated products by polyacrylamide gel electrophoresis under native or denaturing conditions indicated that the protein portions of both exoglucanases exhibited identical mobility, each one consisting of two polypeptides with M _r of 47000 and 48000. The same profile was shown by the exoglucanase secreted in the presence of tunicamycin. Antibodies raised against the protein portion of exoglucanase II did react with both native exoglucanases and their deglycosylated products with a pattern indicative of immunological identity. Digestion of the “in vitro” deglycosylated products of both exoglucanases with Staphylococcus aureus V-8 protease or trypsin generated the same proteolytic fragments in each case. Only exoglucanase II was secreted by protoplasts. These and previously reported results indicate that the protein portions of both isoenzymes may be the product of the same gene (or a family of related genes), and that exoglucanase I is a product of enzyme II, modified by a process occurring beyond the permeability barrier of the cell.     

Direct solid phase sequencing of genomic and plasmid DNA using magnetic beads as solid support.

Approaches to direct solid phase sequencing of genomic and plasmid DNA have been developed using magnetic beads, coated with streptavidin, as solid support. The DNA is immobilized through selective incorporation of biotin into one of the strands. A single stranded template, suitable for sequencing, is obtained through strand-specific elution. Using this concept, in vitro amplified plasmid DNA and chromosomal DNA were sequenced directly from single colonies. The solid phase approach ensures that the amplification and the sequencing reactions can be performed under optimal conditions. The system was found to be suitable for sequencing using both isotope- and fluorescent-labelled primers.     

Identification of a novel 7-cis-11-trans-lipoxin A4 generated by human neutrophils: total synthesis, spasmogenic activities and comparison with other geometric isomers of lipoxins A4 and B4

Addition of (15S)-hydroxy-5,8,11-cis-13-trans-eicosatetraenoic acid (15-HETE) and the ionophore A23187 (2.5 microM) to human neutrophils led to the formation of both lipoxin A4 and lipoxin B4 as well as a novel 5,6,15-trihydroxyeicosatetraenoic acid. The new compound was identified using an improved isolation and detection system and its basic structure was determined by physical methods. On the basis of biosynthetic considerations, geometric isomers of lipoxin A4 and lipoxin B4 were prepared by total synthesis. Comparison of these synthetic materials with the neutrophil-derived product showed that the new compound is (5S,6R,15S)-trihydroxy-9,11,13-trans-7-cis-eicosatetraenoic acid or the 7-cis-11-trans-isomer of LXA4 (7-cis-11-trans-LXA4). LXA4, 11-trans-LXA4, 7-cis-LXA4 and 7-cis-11-trans-LXA4 all evoked dose-dependent (0.1-10 microM) contractions of the guinea pig lung strip, whereas 6-cis-LXB4 and 6-cis-8-trans-LXB4 relaxed this preparation. LXA4 and 7-cis-LXA4 were approx. 10-times more potent than the compounds with 11-trans geometry. However, all four double-bond isomers of LXA4 caused contractions which, based upon pharmacological evidence, appeared to involve specific activation of the same site as cysteinyl-containing leukotrienes. In conclusion, 7-cis-11-trans-LXA4 was isolated and identified as a novel biologically active eicosanoid formed by human neutrophils.     

The use of functional analysis of the ribosome as a tool to determine archaebacterial phylogeny

Forty different antibiotics with diverse kingdom and functional specificities were used to measure the functional characteristics of the archaebacterial translation apparatus. The resulting inhibitory curves, which are characteristic of the cell-free system analyzed, were transformed into quantitative values that were used to cluster the different archaebacteria analyzed. This cluster resembles the phylogenetic tree generated by 16S rRNA sequence comparisons. These results strongly suggest that functional analysis of an appropriate evolutionary clock, such as the ribosome, is of intrinsic phylogenetic value. More importantly, they indicate that the study of the nexus between genotypic and phenotypic (functional) information may shed considerable light on the evolution of the protein synthetic machinery.     

Thiol-directed immobilization of recombinant IgG-binding receptors

A genetic engineering approach is described for directed immobilization of IgG-binding receptors to a thiol-containing matrix using a single cysteine residue. The cysteine residue is introduced into the C-terminal part of receptors based on staphylococcal protein A. Receptors containing one, two or five IgG-binding domains were produced in Escherichia coli and subsequently immobilized on thiopropyl-Sepharose. A high amount, 5 mumol/ml gel (45 mg/ml), of recombinant receptor could be rapidly immobilized to the solid support and both the gel and the immobilized receptor could be regenerated by reduction and oxidation reactions. The gel was used for affinity purification of human IgG and analysis of IgG-binding capacity at different amounts of immobilized recombinant protein show the same maximal IgG-binding capacity (20-25 mg/ml) for all three immobilized receptors. However, at low substitution grade of receptors, the immobilized receptor molecules were shown to bind one (Z-Cys) and two (ZZ-Cys) IgG molecules, respectively. These results demonstrate that the immobilized protein molecules are in a functionally active form and that a two-domain receptor can bind two molecules of IgG without steric hindrance. Interestingly, the five-domain receptor (ZV-Cys), with a structure similar to native protein A, can only bind approximately two IgG molecules, despite the five-domain structure of the molecule.     

Biosynthesis of dolichol by rat liver peroxisomes

The ability of peroxisomes and microsomes to synthesize dolichol from [3H]mevalonate, [3H]isopentenyl-P2 or [3H]farnesyl-P2 in vitro was investigated. It was found that isoprenoid biosynthesis also occurs in peroxisomes and that this process demonstrates properties differing from those of isoprenoid biosynthesis by microsomes. The pH optimum in peroxisomes was 8.0 and, in contrast to microsomes, the peroxisomal biosynthesis was largely insensitive to detergents. After treatment with proteolytic enzymes, microsomes lost their capacity to incorporate [3H]mevalonate into dolichol, whereas proteolysis of intact peroxisomes did not influence their corresponding rate of incorporation. The soluble content of peroxisomes was separated from the membranes and found to demonstrate half of the biosynthetic capacity of the intact organelle. Fasting and cholestyramine treatment decreased only the microsomal incorporation of [3H]mevalonate into dolichol, while treatment with clofibrate, di-2-ethylhexyl phthalate or phenobarbital increased microsomal, but decreased peroxisomal labeling. After injection of [3H]mevalonate into the portal vein of rats, high initial labeling of dolichol was recovered both in isolated microsomes and peroxisomes, whereas when [3H]glycerol was administered, peroxisomal phospholipids became labeled later than the corresponding microsomal constituents. These results support the conclusion that dolichol is synthesized both in peroxisomes and the endoplasmic reticulum, but that the biosynthetic processes at these two locations have different properties.     

Biosynthesis of 1-alkenes in higher plants: stereochemical implications. A model study with Carthamus tinctorius (Asteraceae)

Odd numbered 1-alkenes, such as 1-pentadecene or 1,8,11,14-heptadecatetraene are formed from palmitic or linolenic acid by fragmentative decarboxylation. Incubation studies with germinating safflower (Carthamus tinctorius) and (2R,3R)-12-phenyl[2,3-2H2]dodecanoic acid, (2S,3S)-12-phenyl[2,3-2H2]dodecanoic acid, (2R)-12-phenyl[2-2H]dodecanoic acid and (2S)-12-phenyl[2-2H]dodecanoic acid instead of the natural alpha-linolenic acid precursor revealed the fragmentation to be an overall anti elimination of the 3-pro(S) hydrogen and the carboxyl group (anti-periplanar transition state geometry). Externally offered 3-hydroxy acids are not fragmented to 1-alkenes. The most probable mechanistic alternatives are in agreement with abstraction of the 3-pro(S) hydrogen as a radical followed by electron transfer and fragmentation, or transient insertion of oxygen into the 3-pro(S) C-H bond and subsequent fragmentation into an 1-alkene and CO2 after appropriate activation. The mechanism seems to be of general importance for the biosynthesis of vinylic substructures of natural products from oxygenated precursors.