Extracellular and cell-bound lipase activity in relation to growth of Geotrichum candidum

Summary The imperfect fungus Geotrichum candidum produced extracellular lipase in a basic peptone-salt medium. By adding olive oil or Tween 80 to the basic medium the lipase yields could be enhanced and the maximal yields were found with Tween 80, which resulted in a sixfold increase in extracellular lipase activity as compared with basic medium. During the early phase of growth in medium with olive oil the proportion of cell-bound activity was higher than that of extracellular activity, and a delay in the secretion of extracellular lipase was found. The proportion of cell-bound activity from growth in basic medium and in basic medium with Tween 80 was lower than that of extracellular activity during the entire growth phase. Analyses by polyacrylamide gel electrophoresis showed that the lipase activity from growth in all three media could be ascribed to equivalent protein bands at 57 000 and 61 000 daltons. Immunodiffusion showed that the cell-bound preparation contained lipase that was immunologically identical with purified extracellular lipase from G. candidum.     

Chemical studies on the lipopolysaccharide of Rhizobium meliloti 10406 and its lipid A region

The structure of the lipopolysaccharide from Rhizobium meliloti 10406, a derivative of the wild-type strain MVII-1, was examined. The compositional analysis of its polysaccharide moiety demonstrated lack of heptose(s), but high contents in glucose, galacturonic acid and 2-keto-3-deoxy-octonate (dOclA) as characteristic features. The lipid A moiety consisted of a -1,6 linked glucosamine disaccharide carrying ester (at C-4) and glycosidically (at C-1) linked phosphate residues, both present exclusively as monoester phosphates but not as phosphodiesters. Ester- and amidelinked 3-hydroxy fatty acids were mostly present as non-3-O-acylated residues. Laser desorption mass spectrometry (LD-MS) revealed heterogeneity in the fatty acid substitution, as was also indicated by the non-stoichiometric ratios obtained by quantitative fatty acid analysis. The predominating lipid A structure contained at the reducing glucosamine residue ester-linked 3-hydroxy-tetradecanoic acid (3-OH-14:0) and amide-linked 3-OH-18:0, or 3-OH-18:1, respectively. The distal (non-reducing) glucosamine carried ester-bound the recently discovered 27-hydroxyoctacosanoic acid and 3-OH-14:0 and, as amide-linked fatty acid, mostly 3-hydroxy-stearic acid (3-OH-18:0).The isolated lipopolysaccharide exhibited a high extent of lethal toxicity in galactosamine-treated mice, comparable to that of enterobacterial lipopolysaccharide. The structural relationship of LPS and lipid A of Rhizobium meliloti to other rhizobial lipopolysaccharides and lipid A's with respect to questions of taxonomy and of phylogenetic relationships will be discussed.Abbreviations LPS lipopolysaccharide - dOclA 3-deoxy-D-mannooctulosonic acid (KDO) - GalA galacturonic acid - DOC sodium deoxycholate - PAGE polyacrylamide gel electrophoresis - LD-MS laser desorption-mass spectrometry     

Temperature jump study of charge translocation during the bacteriorhodopsin photocycle

Temperature jump experiments were carried out on purple membranes oriented and fixed in polyacrylamide gel. With green background illumination a relaxation of the photocurrent after an infrared laser pulse could be observed. To simulate the temperature jump signals different models of the bacteriorhodopsin photocycle were tested. The parameters of these models were obtained by measuring absorbance changes and photocurrent after excitation with a 575-nm laser flash.

A model with a temperature-dependent branching before the M state turned out to be satisfying. Other models, especially those with a late branching or without branching, could not reproduce the temperature jump measurements.

     

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.     

Characterization, size estimation and solubilization of α-macroglobulin complex receptors in liver membranes

Receptors for α₂-macroglobulin-proteinase complexes have been characterized in rat and human liver membranes. The affinity for binding of ¹²⁵I-labelled α₂-macroglobulin · trypsin to rat liver membranes was markedly pH-dependent in the physiological range with maximum binding at pH 7.8–9.0. The half-time for association was about 5 min at 37°C in contrast to about 5 h at 4°C. The half-saturation constant was about 100 pM at 4°C and 1 nM at 37°C (pH 7.8). The binding capacity was approx. 300 pmol per g protein for rat liver membranes and about 100 pmol per g for human membranes. Radiation inactivation studies showed a target size of 466 ± 71 kDa (S.D., n = 7) for α₂-macroglobulin · trypsin binding activity. Affinity cross-linking to rat and human membranes of ¹²⁵I-labelled rat α₁-inhibitor-3 · chymotrypsin, a 210 kDa analogue which binds to the α₂-macroglobulin receptors in hepatocytes (Gliemann, J. and Sottrup-Jensen, L. (1987) FEBS Lett. 221, 55–60), followed by SDS-polyacrylamide gel electrophoresis, revealed radioactivity in a band not distinguishable from that of cross-linked α₂-macroglobulin (720 kDa). This radioactivity was absent when membranes with bound ¹²⁵I-α₁-inhibitor-3 complex were treated with EDTA before cross-linking and when incubation and cross-linking were carried out in the presence of a saturating concentration of unlabelled complex. The saturable binding activity was maintained when membranes were solubilized in the detergent 3-[(3-cholamidopropyl)dimethylammonio]profane sulfonate (CHAPS) and the size of the receptor as estimated by cross-linking experiments was shown to be similar to that determined in the membranes. It is concluded that liver membranes contain high concentrations of an approx. 400–500 kDa α₂-macroglobulin receptor soluble in CHAPS. The soluble preparation should provide a suitable material for purification and further characterization of the receptor.     

The effect of ranitidine on cellular immunity in patients with multiple myeloma

Summary Multiple myeloma is characterized by an increased susceptibility to infections and to other malignancies. In a double-blind, placebo-controlled study the potential impact of immunomodulation by ranitidine was studied in 20 patients with multiple myeloma. Three patients were untreated, while 17 after previous cytotoxic therapy were in a stable phase of their disease. All were without clinical signs of infections and at that time had not been treated with other immunomodulating agents. The patients were randomized to oral ranitidine 300 mg twice a day for 21 days or placebo, and several immunological parameters related to multiple myeloma were studied. The blood monocyte chemotactic response was improved in patients treated with ranitidine, and superoxide anion production increased from 2.02 nmol/min to 3.86 nmol/min (median values), while it was unchanged in patients given placebo (2.19–2.25 nmol/min) (P <0.005 between groups). Among ranitidine-treated patients spontaneous NK cell activity was unchanged, while in vitro interleukin-2- and interferon--stimulated NK cell activity decreased (P <0.03, respectively). As production of oxygen radicals constitutes an important mechanism of monocyte killing activity against microorganisms and probably against malignant cells, it is suggested that ranitidine may be of beneficial impact in the treatment of multiple myeloma.     

Risk of nonocular cancer in first-degree relatives of retinoblastoma patients

Summary The increased risk of nonocular cancer seen consistently in studies of survivors of retinoblastoma may be caused in part by the presence of a retinoblastoma gene that also predisposes to other cancers. It has been claimed that this gene also increases the risk for cancer among unaffected relatives of genetic retinoblastoma probands. We report here a population-based study of the risk of nonocular cancer in parents and siblings of persons notified to the Danish Cancer Registry with retinoblastoma during 1943–84. No excess was observed among first degree relatives of 61 genetic retinoblastoma probands, whereas a slight (10%) excess was seen among the parents of 115 nongenetic probands. The latter was the result of significant excesses of malignant melanoma (4 observed, 0.4 expected), multiple myeloma (2 observed, 0.2 expected) and osteogenic sarcoma (1 observed, 0.03 expected). The observed risk pattern cannot be explained by the presence of the retinoblastoma gene.     

Molecular cloning of theEnvC gene ofEscherichia coli

DNA fragments complementing theenvC mutation could be isolated by cloning chromosomal DNA in the vector pUH84. When the frequencies of transformation and the frequencies of restoring theenvC ⁺ phenotype were compared, the high copy number hybrid plasmids complemented with a frequency of 10^–5. After subcloning theenvC-complementing DNA fragment into the low copy number plasmid pLG339, efficient complementation was achieved by spontaneous integration of the IS2 element ofEscherichia coli. By nucleotide sequence analysis, a potential promoter, a ribosome-binding site, and an unidentified reading frame were detected in the respective DNA fragment.     

Cytoskeletons of retinal pigment epithelial cells: Interspecies differences of expression patterns indicate independence of cell function from the specific complement of cytoskeletal proteins

Summary In vertebrate tissue development a given cell differentiation pathway is usually associated with a pattern of expression of a specific set of cytoskeletal proteins, including different intermediate filament (IF) and junctional proteins, which is identical in diverse species. The retinal pigment epithelium (RPE) is a layer of polar cells that have very similar morphological features and practically identical functions in different vertebrate species. However, in biochemical and immunolocalization studies of the cytoskeletal proteins of these cells we have noted remarkable interspecies differences. While chicken RPE cells contain only IFs of the vimentin type and do not possess desmosomes and desmosomal proteins RPE cells of diverse amphibian (Rana ridibunda, Xenopus laevis) and mammalian (rat, guinea pig, rabbit, cow, human) species express cytokeratins 8 and 18 either as their sole IF proteins, or together with vimentin IFs as in guinea pig and a certain subpopulation of bovine RPE cells. Plakoglobin, a plaque protein common to desmosomes and the zonula adhaerens exists in RPE cells of all species, whereas desmoplakin and desmoglein have been identified only in RPE desmosomes of frogs and cows, including bovine RPE cell cultures in which cytokeratins have disappeared and vimentin IFs are the only IFs present. These challenging findings show that neither cytokeratin IFs nor desmosomes are necessary for the establishment and function of a polar epithelial cell layer and that the same basic cellular architecture can be achieved by different programs of expression of cytoskeletal proteins. The differences in the composition of the RPE cytoskeleton further indicate that, at least in this tissue, a specific program of expression of IF and desmosomal proteins is not related to the functions of the RPE cell, which are very similar in the various species.     

Genetics of the quantitative Lp(a) lipoprotein trait

The Lp(a) lipoprotein is a complex particle composed of a low density lipoprotein (LDL)-like lipoprotein and the disulfide bonded Lp(a) glycoprotein. The complex represents a quantitative genetic trait. SDS gel electrophoresis under reducing conditions of sera followed by immunoblotting with affinity-purified polyclonal anti-Lp(a) demonstrated inter- and intra-individual size heterogeneity of the glycoprotein with apparent Mr in the range 400-700kDa. According to their relative mobilities compared to apo B-100 the Lp(a) patterns were categorized into phenotypes F, B, S1, S2, S3 und S4 and into the respective double-band phenotypes. This size heterogeneity seems to be controlled by multiple alleles designated LpF, LpB, LpS1, LpS2, LpS3, LpS4 and a null allele (LpO) at a single locus. Phenotype frequencies observed in 441 unrelated subjects were in good agreement with those expected from the genetic hypothesis. Comparison of Lp(a) lipoprotein concentrations in the different phenotypes revealed a highly significant association of phenotypes B, S1 and S2 with high, and phenotypes S3 und S4 with intermediate Lp(a) concentrations. A third mode is represented by the null phenotype were no Lp(a) band is detected upon immunoblotting and Lp(a) lipoprotein is low or absent. We conclude that the same gene locus is involved in determining Lp(a) glycoprotein phenotype and Lp(a) lipoprotein concentrations in plasma. This major gene seems to be the Lp(a) glycoprotein structural gene locus.