Naturally occurring monoterpenoids in needles of Picea abies (L.) Karst

Summary An analysis was made of the effects of different sampling and extraction techniques on the amounts and pattern of monoterpenoids isolated from needles of Norway spruce. The following isolation and analysis procedure was finally adopted: liquid nitrogen-cooled needles were pulverized by a microdismembrator, extracted with pentane overnight at 2°–3°C and concentrated to a volume not less than 3 ml/g fresh weight on a Vigreux column. The crude extract was injected splitless (with solvent split) onto a cold programmed temperature vaporized (PTV) precolumn of a gas chromatograph and the vaporizable compounds heated to a capillary column. This method was tested for production of artefacts and quantitative extraction and applied to needles of eleven 80-year-old spruce trees.     

Concentration gradients of free sterols,steryl esters and lipid phosphorus in the trunkwood of Scot's pine (Pinus sylvestris L.)

The amounts of free sterols, steryl esters and lipid phosphorus were determined in the sapwood and heartwood of mature, and in the outer and inner sapwood of young Pinus sylvestris trees. In the mature trees (up to 70 years old) the heartwood contains significantly higher amounts of free sterols than the sapwood. No radial gradient can be demonstrated in the amounts of steryl esters. Lipids extracted from the sapwood contain higher amounts of phosphorus than those from the heartwood. Stems of young Pinus sylvestris trees (up to 13 years old) show in the inner sapwood higher amounts of both free sterols and steryl esters than the peripheral younger wood zone. The inner sapwood of the young stems shows slightly higher amounts of lipid phosphorus than the outer sapwood. The results indicate that Pinus sylvestris accumulates both free sterols and steryl esters in the stems at a very early stage of the life cycle. Sterol accumulation in the innermost parts of the stems seems not to depend on heartwood formation.     

Immunohistochemical procedures for the demonstration of peptide- and tyrosine hydroxylase-containing nerve fibers in cryostat sections of unfixed rapidly frozen tissue stored for long periods of time

Summary Traditional protocols for the immunohistochemical localization of peptides and tyrosine hydroxylase (TH) in nerve fibers in cryostat sections require the tissue to be thoroughly fixed and rinsed and to be processed for the cryostat sectioning and the immunohistochemical staining more or less directly after freezing. In the present study it was tested whether also unfixed, rapidly frozen tissue, conforming to guinea pig and bovine heart specimens, can be used for the visualization of neuropeptides [neuropeptide Y (NPY) and substance P (S P)] and TH in cryostat sections. The following observations were made: 1) NPY-immunoreactive (IR) and S P-IR nerve fibers could be clearly identified in both fixed and unfixed sections of this type of tissue. 2) TH-IR nerve fibers could be detected in unfixed tissue if the sections were post-fixed with aldehydes by the use of a two-step fixation process related to a sudden change of pH. However, the outlines of the nerve fibers were sometimes diffuse. 3) Storage of unfixed tissue for periods of up to 2.5 yeart at –80° C did not lead to a decrease in immunoreactivity. 4) Somewhat higher concentrations of primary antibodies had to be used for sections of unfixed tissue than for sections of fixed tissue when the FITC method was used. This waste of antibodies was partly overcome by use of the biotin-streptavidin method. The glyoxylic acid induced catecholamine(CA)-fluorescence method for demonstration of sympathetic nerve fibers was also applied and was found to give optimal results after storage of tissue for up to 2.5 years. The study shows that the use of unfixed rapidly frozen tissue represents a fast and realistic method for the demonstration of neuropeptide immunoreactivity, that it to some extent can be used for the visualization of TH-containing nerve fibers and that it is a suitable method to maintain longterm neuropeptide and TH immunoreactivity as well as long-term CA-fluorescence reaction.     

Binding of anti-fibronectin to early amphibian ectoderm does not result in inhibition of neural induction under in vitro conditions

Summary Antibodies directed to fibronectin (anti-FN) were injected into the blastocoel of late blastulae of Xenopus laevis. Two animal caps (ectoderm) were isolated, when control embryos reached the early gastrula stage, and were combined with untreated upper blastopore lip in the sandwich method. In two control series fibronectin or Holtfreter solution was injected into the blastocoel. The results of the experiments suggest that neural induction cannot be prevented by binding anti-FN to fibronectin, which covers the blastocoelic side of the ectoderm. The data support the view that extracellular matrix proteins are not themselves responsible for neural induction. However, in comparison with the control series a slight shift of the differentiation pattern in the spinocaudal direction could be observed in the anti-FN series. The possible role of extracellular proteins in the formation of a close juxtaposition of mesodermal and ectodermal target cells as a prerequisite for shortdistance transmission of neural inducers is discussed.     

Genetic variability in mitochondrial DNA in a regional population of the great tit (Parus major)

Genetic variation of mitochondrial DNA (mtDNA) in 18 great tits (Parus major) from three neighboring localities in Sweden was investigated with eight tetranucleotide restriction endonucleases. The 18 individuals could be separated into 13 different maternal lineages. The high number of female lineages present in this regional population contrasts with a low level of sequence divergence between the different mtDNA clones, with a mean of 0.19% sequence divergence between all individuals. There was no obvious spatial structuring of mtDNA clones among the three localities. The presence of a high number of different clones with a low degree of sequence divergence could be explained by the effects of a large long-term effective population size, with the mtDNA clones having diverged about 25,000–200,000 years ago.This study was supported by the Swedish Natural Science Research Council, the Erik Philip-Sörensen Foundation, and the Nilsson-Ehle Foundation.     

Inhibition of human vitamin-K-dependent protein-S-cofactor activity by a monoclonal antibody specific for a Ca2+-dependent epitope

Protein S is an anticoagulant vitamin-K-dependent plasma protein functioning as a cofactor to activated protein C in the degradation of factors Va and VIIIa. A murine monoclonal antibody, HPS 7, specific for a calcium-stabilized epitope in human protein S, is described. The epitope was available in intact protein S, both in its free form and when protein S was bound to C4b-binding protein. It disappeared upon reduction of disulfide bridges and also after thrombin of chymotrypsin cleavage of protein S. Thrombin cleaves protein S close to the calcium-binding region containing gamma-carboxyglutamic acid (Gla). The cleaved protein still contains the Gla region, linked by a disulfide bridge, but it has a lower affinity for calcium and no protein C cofactor activity. The thrombin-mediated cleavage of protein S could be inhibited by HPS 7. The Ka for the interaction between protein S and the monoclonal was estimated to be approximately 0.7 X 10(8) M-1. Half-maximal binding between HPS 7 and protein S was observed at a calcium concentration of 0.50 mM, indicating that saturation of the Gla region with calcium was required for the interaction. The recently reported Gla-independent high-affinity calcium binding did not induce the epitope. The calcium-dependent binding of protein S to phospholipid vesicles as well as the protein C cofactor activity was inhibited by HPS 7. The data suggests that the epitope for HPS 7 is located in the Gla region of protein S or in the closely positioned thrombin-sensitive region.     

Cytogenetic mapping of marker genes on the chromosome elements C and E of Drosophila pseudoobscura and D. subobscura

Enzyme loci, visible marker genes and -cloned DNA-sequences from a D. miranda library were mapped cytologically on the chromosome elements C and E of D. pseudoobscura and D. subobscura. New data are incorporated into the linkage maps of the two species. Homologous segments can now be localized in the polytene chromosomes with these markers. A comparison of the chromosome elements E of D. melanogaster and D. subobscura shows 12 conserved subsections which have been rearranged by paracentric inversions in the evolution of the two lineages.     

Synthesis of methyl 2-acetamido-2-deoxy-3-O-(β-d-galactopyranosyl)-α-d-galactopyranoside from a corresponding thioglycoside derivative

The title disaccharide glycoside was synthesized by halide ion-promoted glycosidation, using methanol and the disaccharide bromide derived from methyl 2-azido-3-O-(2,3,4,6-tetra-O-benzoyl--d-galactopyranosyl)-4,6-O-benzylidene-2-deoxy-1-thio--d-galactopyranoside. This derivative in turn was prepared by silver triflate-promoted condensation of monosaccharide derivatives.     

Carbohydrate receptor specificity of K99 fimbriae of enterotoxigenicEscherichia coli

K99 Fimbriae from enterotoxigenicEscherichia coli (ETEC) were found to bind specifically to sialic acid, as measured in a haemagglutination inhibition assay using the intact bacteria and human erythrocytes. The affinity forN-glycolylneuraminic acid was about twice that ofN-acetylneuraminic acid (NeuAc), and other monosaccharides were found to be at least ten-fold less effective as inhibitors. The specificity was found to depend on electrostatic interaction where the carboxyl group and its orientation plays an important role. 2--Benzyl-NeuAc was a better inhibitor than 2--methyl-NeuAc suggesting a hydrophobic patch near the binding site on the protein. Axially oriented hydroxyl groups as in 4-epi-NeuAc and 3-hydroxy-NeuAc seemed to participate in binding since these derivatives were better inhibitors thanN-acetylneuraminic acid. K99 was found to have a higher affinity for 4-O-acetyl-NeuAc and lower affinity forN-acetylneuraminic acid withO-substituents at C7-C9 as compared toN-acetylneuraminic acid. Hence, the degree ofO-acetylation of sialic acid in the mucosa of the small intestine may influence colonization and determine susceptibility to infection.     

Expression of binding sites for Dolichos biflorus agglutinin at the apical aspect of collecting duct cells in rat kidney

Summary To identify precisely the structural and functional cell type in the collecting duct of the rat kidney expressing binding sites for Dolichos biflorus agglutinin (DBA), we stained serial paraffin sections of kidney with horseradish peroxidase-labeled DBA and with immunocytochemical methods for localizing (Na⁺+K⁺)-ATPase and carbonic anhydrase II (CA II), enzymes found preferentially in principal and intercalated cells, respectively. Most principal cells expressing a strong basolateral staining for (Na⁺ + K⁺)-ATPase showed binding sites for DBA at their luminal surfaces. However, a minority of cells rich in CA II and showing morphologic characteristics of intercalated cells also expressed DBA binding sites at their luminal surface and apical cytoplasm. These data suggest that DBA cytochemistrycan provide a useful tool for studying the functional polarity of the main cell types of the collecting duct of the rat kidney.