Surface localization of apolipoprotein AII in lipoprotein-complexes.

Lipoprotein particles reconstituted from the apolipoprotein AII (apo AII) component of human serum high density lipoprotein, phosphatidylcholine and lysophosphatidylcholine were covalently linked to the imidoester groups of a polystyrene residue. Apo AII was proteolytically digested with thermolysin after delipidation. The covalently bound peptides remaining at the resin were cleaved and separated by combined two-dimensional electrophoresis/chromatography. The peptides were isolated, hydrolyzed and their amino acid composition determined. They were assigned to the apo AII sequence. Since the imidoester groups on the surface of the resin carrier cannot react with buried lysine residues, this method gives strong chemical evidence for the spreading of the apo AII polypeptide chain over the surface of the lipoprotein particle, as far as the sequence carrying lysine residues between residue 22 and 55 of each symmetrical half is concerned.     

Chemical synthesis of D,L-3-dehydrosphinganine,its C14-, C16- and C20-homologues and the resolution into the enantiomeric forms

The chemical synthesis of D,L-3-dehydrosphinganine (3-keto sphinganine) and the resolution into its optical isomers via the mandelates is described. This procedure proved to be suitable for the homologous series. The products were characterized by elementary analysis, mass-spectroscopy, IR, NMR, gas chromatography and also by their mono-, di- and triacetyl derivatives respectively.     

Functional analysis of acid and neutral sphingomyelinases in vitro and in vivo

The molecular cloning and the elucidation of the gene structures of the acid (aSMase) and a neutral sphingomyelinases (nSMase) of mouse and human facilitated the structural and functional analysis of these enzymes responsible for the catabolism of sphingomyelin present ubiquitously in the membrane lipid bilayer of mammalian cells. The protein and enzymic properties of the glycoprotein aSMase and of a non-glycosylated nSMase residing in the membranes of the endoplasmic reticulum have been analysed in the native as well as in the recombinant shingomyelinases. Important insight was gained from gene targeting experiments in which an aSMase deficient mouse line was generated which mimics the neurovisceral form of the human Niemann-Pick disease. The availability of the cloned aSMase and nSMases discovered so far led to a genetic approach to the verification of the concept that these enzymes in the 'sphingomelin cycle' are responsible for the generation of ceramide regarded as a lipophilic second messenger in the intracellular signal cascades activated by e.g. TNF-alpha, Fas ligand or cellular stress. All the available evidence derived from the aSMase deficient mouse line and several cell lines overexpressing aSMase and nSMase questions a role of ceramide released by the mammalian sphingomyelinases known so far in intracellular signal transduction.     

Distribution of lactate dehydrogenase in healthy and degenerative canine stifle joint cartilage

In dogs, degenerative joint diseases (DJD) have been shown to be associated with increased lactate dehydrogenase (LDH) activity in the synovial fluid. The goal of this study was to examine healthy and degenerative stifle joints in order to clarify the origin of LDH in synovial fluid. In order to assess the distribution of LDH, cartilage samples from healthy and degenerative knee joints were investigated by means of light and transmission electron microscopy in conjunction with immunolabeling and enzyme cytochemistry. Morphological analysis confirmed DJD. All techniques used corroborated the presence of LDH in chondrocytes and in the interterritorial matrix of healthy and degenerative stifle joints. Although enzymatic activity of LDH was clearly demonstrated in the territorial matrix by means of the tetrazolium–formazan reaction, immunolabeling for LDH was missing in this region. With respect to the distribution of LDH in the interterritorial matrix, a striking decrease from superficial to deeper layers was present in healthy dogs but was missing in affected joints. These results support the contention that LDH in synovial fluid of degenerative joints originates from cartilage. Therefore, we suggest that (1) LDH is transferred from chondrocytes to ECM in both healthy dogs and dogs with degenerative joint disease and that (2) in degenerative joints, LDH is released from chondrocytes and the ECM into synovial fluid through abrasion of cartilage as well as through enhanced diffusion as a result of increased water content and degradation of collagen.     

Four-dimensional growth response of mature Larix decidua to stem burial under natural conditions

This paper illustrates the effects of abrupt stem burial (burial depth ~0.5 m) on tree growth in mature (46 ± 8 years) European larch (Larix decidua Mill.) trees. In contrast to the previous research, which was mostly carried out with saplings and on experimental sites where regular aggradation occurred through the transport of sand, this work focuses on the impact of natural, abrupt burial of mature trees with rocks contained in a sandy-silty matrix typical for debris flows in mountainous headwater catchments. The effect of burial is assessed radially and axially within the stem and over almost three decades after burial. The analysis of growth disturbances and their intensity was based on the 143 cross sections (572 growth series) taken at 10-cm intervals from 6 Larix decidua Mill. The results show quite clearly that abrupt burial causes massive suppression of radial growth as compared to pre-event conditions (mean 77 %, min 38 %, max 92 %, SD 7.2 %). The trees sampled were unable to resume pre-burial growth rates even after 25 years, but recovered to reference growth conditions (as measured in undisturbed, local reference trees) after 15 years (min 3 years, max 25 years, SD 9 years). The results differ only insignificantly between different heights along the tree axis and suppression is equally well expressed at different radial positions within the stem.     

3D analysis of anatomical reactions in conifers after mechanical wounding: first qualitative insights from X-ray computed tomography

The ability of trees to recover from damage beyond the last-formed periderm as well as the drivers and nature of associated wound reactions have been studied for more than two centuries using macroscopic (desiccation, aeration or discoloration of wood) and microscopic approaches (anatomical and chemical reactions). However, no studies currently exist which address large-scale macroscopic and microscopic reactions surrounding wounds in the tangential, axial, and radial directions over continuous segments of tree stems. This note explores the potential of 3D X-ray computed tomography in assessing effects of wounding under natural conditions in European conifers (Abies alba, Larix decidua, Picea abies). We present results from a pilot study and qualitatively evaluate the potential of the approach used in assessing and illustrating the formation and spread of de-differentiated xylem parenchyma cells, xylem decay compartmentalization, resin ducts, and stabilizing compression wood cells.     

Galactosphingolipids and axono-glial interaction in myelin of the central nervous system

The myelin of central and peripheral nervous system of UDP-galactose-ceramide galactosyltransferase deficient mice (cgt ^-/-) is completely depleted of its major lipid constituents, galactocerebrosides and sulfatides. The deficiency of these glycolipids affects the biophysical properties of the myelin sheath and causes the loss of the rapid saltatory conduction velocity of myelinated axons. With the onset of myelination, null mutant cgt ^-/- mice develop fatal neurological defects. CNS and PNS analysis of cgt ^-/- mice revealed (1) hypomyelination of axons of the spinal cord and optic nerves, but no apoptosis of oligodendrocytes, (2) redundant myelin in younger mice leading to vacuolated nerve fibers in cgt ^-/- mice, (3) the occurrence of multiple myelinated CNS axons, and (4) severely distorted lateral loops in CNS paranodes. The loss of saltatory conduction is not associated with a randomization of voltage-gated sodium channels in the axolemma of PNS fibers. We conclude that cerebrosides (GalC) and sulfatides (sGalC) play a major role in CNS axono-glial interaction. A close axono-glial contact is not a prerequisite for the spiraling and compaction process of myelin. Axonal sodium channels remain clustered at the nodes of Ranvier independent of the change in the physical properties of myelin membrane devoid of galactosphingolipids. Increased intracellular concentrations of free ceramides do not trigger apoptosis of oligodendrocytes.     

Proteolipid protein (PLP) of CNS myelin: positions of free, disulfide-bonded, and fatty acid thioester-linked cysteine residues and implications for the membrane topology of PLP.

Proteolipid protein (PLP), the major integral membrane protein of central nervous system myelin, contains 14 cysteine residues within its 276-residue polypeptide chain. We determined the state of all cysteine residues and localized four of them as free thiols at positions 24, 32, 34, and 168. Four cysteines are connected by disulfide bonds: Cys200-Cys219 and Cys183-Cys227. The remaining six cysteine residues at positions 5, 6, 9, 108, 138, and 140 are modified by long-chain fatty acids, mainly palmitic acid, in thioester linkage. The extreme hydrophobicity of PLP can therefore be explained by two structural features: a composition of approximately 50% apolar amino acid residues and a high degree of fatty acid acylation. A differential fluorescent-labeling technique was developed for the structural studies: the cysteine residues belonging to one of the three states were derivatized by N-(iodoacetylaminoethyl)-5-naphthylamine-1-sulfonic acid (I-AEDANS) either directly (a), after thioester cleavage with hydroxylamine (b), or after disulfide cleavage with dithiothreitol (c). The protein was then proteolytically digested with thermolysin, and the labeled peptides were isolated by reversed-phase HPLC followed by sequence analysis. The results were further confirmed by determination of the fatty acid to protein stoichiometry. The structural data not only demand the revision of our concept of the membrane topology of PLP but will also promote more sophisticated studies on the mechanism of myelination and new functions of PLP.     

Neutral sphingomyelinase 1 deficiency in the mouse causes no lipid storage disease

Sphingomyelin is a major lipid in the bilayer of subcellular membranes of eukaryotic cells. Different sphingomyelinases catalyze the initial step in the catabolism of sphingomyelin, the hydrolysis to phosphocholine and ceramide. Sphingomyelinases have been postulated to generate ceramide as a lipophilic second messenger in intracellular signaling pathways involved in cell proliferation, differentiation, or apoptosis. To elucidate the function of the first cloned Mg(2+)-dependent, neutral sphingomyelinase (nSMase 1) in sphingomyelin catabolism and its potential role in signaling processes in a genetic and molecular approach, we have generated an nSMase 1-null mutant mouse line by gene targeting. The nSMase 1-deficient mice show an inconspicuous phenotype and no accumulation or changed metabolism of sphingomyelin or other lipids, despite grossly reduced nSMase activity in all organs except brain. We also addressed the recent proposal that nSMase 1 possesses lysophospholipase C activity. The unaltered metabolism of lysophosphatidylcholine or lyso-platelet-activating factor excludes the proposed role of nSMase 1 as a lysophospholipase C.     

Sampling nucleotide diversity in cotton

Background  

Cultivated cotton is an annual fiber crop derived mainly from two perennial species, Gossypium hirsutum L. or upland cotton, and G. barbadense L., extra long-staple fiber Pima or Egyptian cotton. These two cultivated species are among five allotetraploid species presumably derived monophyletically between G. arboreum and G. raimondii. Genomic-based approaches have been hindered by the limited variation within species. Yet, population-based methods are being used for genome-wide introgression of novel alleles from G. mustelinum and G. tomentosum into G. hirsutum using combinations of backcrossing, selfing, and inter-mating. Recombinant inbred line populations between genetics standards TM-1, (G. hirsutum) × 3-79 (G. barbadense) have been developed to allow high-density genetic mapping of traits.