Conformational states of (K+ + H+)-ATPase studied using tryptic digestion as a tool

The (K+ + H+)-ATPase from gastric mucosa has been treated by limited proteolytic digestion with trypsin to study the conformational states of the enzyme. The existence of a K+- and an ATP-form of the enzyme follows from the kinetics of inactivation and from the specific cleavage products. In the presence of K+ the 95 kDa chain is cleaved into two fragments of 56 and 42 kDa, whereas in the presence of ATP fragments of 67 and 35 kDa are formed. When Mg2+ is present during tryptic digestion cleavage products which are specific for both the ATP- and the K+-form of the enzyme are yielded. In analogy to ATP, Mg2+ is able to convert the enzyme from a K+-conformation to a more protected form. Moreover Mg2+ supports the protecting effect of ATP against tryptic inactivation. The K0.5 for ATP is lowered from 1.6 mM (no Mg2+) to 0.2 mM in the presence of 10 mM Mg2+. Mg2+, which in previous studies has been shown to induce a specific conformation, apparently induces a conformation different from the K+-form of the enzyme and has ATP-like effects on the enzyme. In addition it has been found that in the initial rapid phase of the digestion process the K+-ATPase activity is interrupted at a step which is very likely the interconversion of the phosphoenzyme forms E1P and E2P, since neither the K+-stimulated p-nitrophenylphosphatase activity nor the phosphorylation of the enzyme are inhibited in this phase. During the tryptic digestion in the presence of K+ there is a good correlation between the residual ATPase activity and the amount of the catalytic subunit left, suggesting that the latter is homogeneous. After tryptic digestion in the presence of K+, phosphorylation only occurs in the 42 kDa and not in the 56 kDa band. The same experiments in the presence of ATP yield only phosphorylation in the 67 kDa band and not in the 35 kDa band. A provisional model for the structure of the catalytic subunit is given.     

The effect of vitreous humour on prostaglandin production by cultured rabbit chorioretinal fibroblasts

Factors in vitreous humour which regulate prostaglandin production were investigated using cultured rabbit chorioretinal fibroblasts. These cells produced predominantly prostaglandin E2, 6-ketoprostaglandin F1 alpha, a compound likely to be a metabolite of prostaglandin E2 and 5-hydroxyeicosatetraenoic acid. The synthesis of 6-ketoprostaglandin F1 alpha was nearly completely inhibited by the cyclooxygenase inhibitor aspirin and partially inhibited by 10(-6) M dexamethasone (49%) and 10(-5) M forskolin (68%). Addition of 10% rabbit vitreous humour to subconfluent cells maintained in Dulbecco's modified Eagle's medium plus 1% fetal bovine serum resulted in stimulation of 6-ketoprostaglandin F1 alpha production by as much as 246% as measured by radioimmunoassay. Chorioretinal fibroblasts labelled by [3H]arachidonic acid incorporation into cellular phospholipids synthesised greater amounts of all labelled arachidonic acid metabolites in response to vitreous humour. It was concluded, therefore, that there are factors present in vitreous humour of molecular weight above 10 kDa which are capable of stimulating cellular cyclooxygenase activity. Confluent cells also responded to a factor(s) present in vitreous humour. The fraction of less than 10 kDa inhibited 6-ketoprostaglandin F1 alpha production by 50% when used at a concentration of 10%. Furthermore, 6-ketoprostaglandin F1 alpha production in confluent cells (but not subconfluent cells) was inhibited to 40% of control levels by vitamin C at a concentration of 1 mg/100 ml. The latter result points to an inhibitory role for vitamin C in vitreous humour. We conclude, therefore, that vitreous humour contains factors important for the regulation of prostaglandin metabolism in the eye.     

Isolation and characterization of a collagen fibril-associated dermatan sulphate proteoglycan from bovine lung

Dermatan sulphate proteoglycans have been extracted from bovine lung with 2.0 M CaCl2 and isolated using CsCl density gradient centrifugation, DEAE ion-exchange chromatography, gel chromatography and preparative sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Ultrastructurally these proteoglycans are specifically associated with collagen fibrils. Dermatan sulphate (Mr 15.10(3)-35.10(3), with a strong prevalence for the higher Mr) is link via an O-glycosidic bond to a protein core, which is rich in Asx, Glx and Leu. Of the total uronic acid, 91% is iduronic acid. A part of the glucuronic acid residues is located near the protein core and a large cluster of disaccharides is devoid of glucuronic acid residues. An inhibition enzyme immunoassay has been developed to quantitate the proteoglycan. A model for the interaction between dermatan sulphate proteoglycans and collagen fibrils is proposed.     

Effect of pH on the kinetics of Na+-dependent phosphate transport in rat renal brush-border membranes

The kinetics of Na+-dependent phosphate uptake in rat renal brush-border membrane vesicles were studied under zero-trans conditions at 37 degrees C and the effect of pH on the kinetic parameters was determined. When the pH was lowered it turned out to be increasingly difficult to estimate initial rates of phosphate uptake due to an increase in aspecific binding of phosphate to the brush border membrane. When EDTA or beta-glycerophosphate was added to the uptake medium this aspecific binding was markedly reduced. At pH 6.8, initial rates of phosphate uptake were measured between 0.01 and 3.0 mM phosphate in the presence of 100 mM Na+. Kinetic analysis resulted in a non-linear Eadie-Hofstee plot, compatible with two modes of transport: one major low-affinity system (Km approximately equal to 1.3 mM), high-capacity system (Vmax approximately equal to 1.1 nmol/s per mg protein) and one minor high-affinity (Km approximately equal to 0.03 mM), low-capacity system (Vmax approximately equal to 0.04 nmol/s per mg protein). Na+-dependent phosphate uptake studied far from initial rate conditions i.e. at 15 s, frequently observed in the literature, led to a dramatic decrease in the Vmax of the low-affinity system. When both the extra- and intravesicular pH were increased from 6.2 to 8.5, the Km value of the low-affinity system increased, but when divalent phosphate is considered to be the sole substrate for the low-affinity system then the Km value is no longer pH dependent. In contrast, the Km value of the high-affinity system was not influenced by pH but the Vmax decreased dramatically when the pH is lowered from 8.5 to 6.2. These results suggest that the low-affinity, high-capacity system transports divalent divalent phosphate only while the high-affinity, low-capacity system may transport univalent as well as divalent phosphate. Raising medium sodium concentration from 100 to 250 mM increased Na+-dependent phosphate uptake significantly but the pH dependence of the phosphate transport was not influenced. This observation makes it rather unlikely that pH changes only affect the Na+ site of the Na+-dependent phosphate transport system.     

Three-dimensional reconstruction of a human metaphase chromosome from electron micrographs

A complete human metaphase chromosome has been reconstructed from a series of electron microscopical projections obtained by tilting the specimen stage at 3 degree intervals from –60 to +60 degrees. The reconstructed structure is about 3.0 m long, 1.6 m wide, and 0.8 m thick. The mass distribution was fairly homogeneous within the chromatids and neither a hollow nor a dense core was observed. The distribution and course of fibers observed are most consistent with a looping model of chromosome structure.     

Novel anaerobic gas-lift reactor (AGLR) with retention of biomass: Start-up routine and establishment of hold up

A start-up routine for a novel type of anaerobic gas-lift reactor using sand as support particles for Bacterial adhesion and involving a dilution rate shift-up is shown to result in rapid formation of mixed-culture aggregates from freely suspended cells. Formation of aggregates changed the general metabolism from acetate-butyrate production to acetate-propionate production. This change is attributed to a selection by washout, favoring propionate-producing bacteria with superior adhesive properties. Sand is shown to be essential in establishing, but not in sustaining, elevated holdup ratios. The importance of maintenance processes and cellular lysis in deeper parts of aggregates are manifest from a reduced effluent biomass concentration and a pronounced production of valeric acid.     

Artifical diets and rearing of the nun moth, Lymantria monacha

Artificial diets were reviewed and two tested. The highest level of survival to the adult stage (72%) was obtained on a modified diet of Odell & Rollinson (1966). Rearing required 61 days for males and 70 days for females. Wing deformation occurred in 16% of the adults. The sex ratio ( : ) was 0.80. Average pupal weight was 281 mg for males and 368 mg for females. Females averaged 102 eggs (range 80–125 eggs). Neonates from field-collected egg masses started hatching on 6 December; frost was not necessary for hatching. Hatching could be postponed until at least 10 November of the following year by storing egg masses at-2°C. L. monacha can be maintained continuously in the laboratory.

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Zusammenfassung Eine Massenvermehrung der Nonne, Lymantria monacha (L.), trat in 1984 auf 500 ha Pinus sylvestris bei Budelerbergen (S.O. der Niederlande) auf und betraf 1985 eine Fläche von 2800 ha. Obwohl die Vermehrung durch eine Luftapplikation mit Dimilin 25 bekämpft werden konnte, wurden Untersuchungen begonnen zur Entwicklung von für die Umwelt sicheren Bekämpfungsmethoden, besonders die Anwendung von Virosen. Das Ziel bestand in der Virusproduktion in künstlichen Raupenzuchten. Die Nonne wurde bisher mit verschiedenen für andere Insektenarten oder speziell für diesen Zweck entwickelten künstlichen Nährböden gezüchtet. Da die Zusammensetzung dieser Nährböden und die Zuchtmethoden nicht veröffentlicht waren und da wenige Details über die Raupenentwicklung vorlagen, wurden modifizierten Nährböden von McMorran sowie von Odell & Rollinson für die Massenzucht der Nonne geprüft.

     

Amino-acid sequence of human alpha 2-antiplasmin

The amino-acid sequence of human alpha 2-antiplasmin was determined by Edman degradation of peptides purified from CNBr, tryptic and chymotryptic digests. Of the total sequence of 452 amino acids of mature alpha 2-antiplasmin, as deduced from the cDNA sequence [Holmes et al. (1987) J. Biol. Chem. 262, 1659-1664], 444 residues were identified by amino-acid sequencing. Two differences were found between the peptide and cDNA analyses (Gly instead of Leu at position 10 and Gly instead of Ser at position 369). alpha 2-Antiplasmin contains two disulfide bridges (Cys64-Cys104 and Cys31-Cys113) and four glucosamine-based carbohydrate chains attached to Asn87, Asn256, Asn270 and Asn277. alpha 2-Antiplasmin is homologous with 12 other proteins belonging to the serine protease inhibitor (serpin) superfamily.     

Chondroid bone arises from mesenchymal stem cells in organ culture of mandibular condyles

Mandibular condyles of fetal mice 19 to 20 days in utero comprising clean cartilage and its perichondrium were cultured for up to 14 days, and their capacity to develop osteoid and to mineralize in vitro was examined. After 3 days in culture the cartilage of the mandibular condyle appeared to have lost its inherent structural characteristics, including its various cell layers: chondroprogenitor, chondroblastic, and hypertrophic cells. At that time interval no chondroblasts could be seen; instead, most of the cartilage consisted of hypertrophic chondrocytes. By that time, the surrounding perichondrium, which contains pluripotential mesenchymal stem cells, revealed the first signs of extracellular matrix enclosing type I collagen, bone alkaline phosphatase, osteonection, fibronectin, and bone sialoprotein as demonstrated by immunofluorescent techniques. Electron microscopic examinations of the newly formed matrix revealed foci of mineralization within and along collagen fibers as is normally observed during bone development. The composition of the latter mineral deposits resembled calcium pyrophosphate crystals. Following 14 days in culture larger portions of the condyle revealed signs of osseous matrix, yet the tissue reacted positively for type II collagen. Hence, the condylar cartilage, a genuine representative of secondary-type cartilage, elaborated in vitro a unique type of bone that would be most appropriately defined as chondroid bone. Biochemical assays indicated that the de novo formation of chondroid bone was correlated with changes in alkaline phosphatase activity and 45Ca incorporation. The findings of the present study imply that mesenchymal stem cells that ordinarily differentiate into cartilage possess the capacity to differentiate into osteogenic cells and form chondroid bone.     

The surface distribution of low density lipoprotein receptors on cultured fibroblasts and endothelial cells. Ultrastructural evidence for dispersed receptors

The distribution of low density lipoprotein (LDL) receptors marked with colloidal gold-conjugated low density lipoproteins has been mapped on the surfaces of cultured human skin fibroblasts and bovine aortic endothelial cells viewed whole in the transmission electron microscope. A dispersed or scattered population of LDL receptors, in addition to and clearly distinct from clustered receptors was detected on the surfaces of both fibroblasts and dividing endothelial cells. No LDL receptors could be detected on contact-inhibited endothelial cells. Clustered receptors imaged in whole-mount preparations were often arranged in rings with an approximate diameter of 250 nm. In ultra-thin sections of marked cells, clustered receptors were localised in coated pits while the few dispersed receptors seen were restricted to non-coated membrane regions. Clustered receptors often appeared localised on the rims of coated pits whose central areas were not marked. The dispersed population of receptors was usually distributed diffusely amongst the clusters on dividing endothelial cells and normal fibroblasts. Only the dispersed population appeared on LDL receptor internalisation-defective mutant fibroblasts. The marginal zones of both fibroblasts and dividing endothelial cells were populated by dispersed receptors. Clusters appeared further "inland" and were rarely seen near the cell margins. These results indicate that LDL receptors on dividing endothelial cells and fibroblasts may be dispersed on the cell surface upon or soon after their insertion during recycling.