软骨血管生成抑制因子抑制血管生成的研究

小牛气管软骨经盐酸胍抽提,丙酮分级沉淀,膜超滤,柱层析等步骤得到软骨血管生成抑制因子(cartilage angiogenesis inhibiting factor,CAIF).SDS-聚丙烯酰胺凝胶电泳显示CAIF由单一组分组成,分子量为27700.通过[ ³H]-TdR掺入,活细胞检测等方法测定CAIF对内皮细胞、Hela细胞、QGY7703细胞与小鼠骨髓细胞、人皮肤成纤维细胞等的DNA合成的影响,以及细胞毒作用.采用鸡胚绒毛尿囊膜实验测定CAIF对血管生成的抑制效应.结果显示:CAIF对内皮细胞产生强的抑制作用,对Hela细胞抑制很弱,对QGY7703细胞、小鼠骨髓细胞、人皮肤成纤维细胞均无抑制作用;对鸡胚绒毛尿囊膜的血管生成产生明显的抑制作用.提示CAIF能较特异地抑制血管生成,CAIF达到电泳纯,是专一性较强的血管生成抑制因子.     

Removal of insect basal laminae using elastase

We have used the enzyme elastase to remove the basal lamina of epithelia from two insects: the upper Malpighian tubules of Rhodnius prolixus and imaginai discs of Drosophila melanogaster. Removal of the basal lamina was confirmed using scanning and transmission electron microscopy. Use of the technique on the Malphighian tubules of Rhodnius reveals for the first time the three-dimensional organization of the circumferential folds of the basal plasma membrane. Elastase is much more effective in removing the basal lamina than are the enzymes hyaluronidase, collagenase, and chymotrypsin, either alone or in combination. Following elastase treatment, cells of the Malpighian tubules dissociate with only mild mechanical agitation into single, viable cells. Treatment with elastase removes the basal laminae of imaginai discs of Drosophila and accelerates evagination as has been previously described for trypsin. To obtain single cell preparations from elastase-treated imaginai discs, mechanical stirring in Ringer low in Ca²⁺ was required. In addition to its usefulness in cell isolation, elastase treatment allows examination of the effect of removal of basal laminae on the physiology and development of insect epithelia.     

Structure and properties of pectin gels in plant cell walls

Abstract This review deals with recent advances in the structural characterization of pectins and the gels which they form, in relation to auxin-induced extension growth, the ripening of fruit, and cellular recognition. Pectins are block polysaccharides. Heavily branched, largely methyl-esterified blocks alternate with unbranched blocks of varying degrees of esterification. The unbranched, non-esterified blocks can aggregate through calcium binding to form the junction zones that hold a gel together. The aggregates are of two, or possibly four, chains at low calcium levels, and larger with excess calcium. The fall in wall pH during auxin-induced growth activates glycanase enzymes. These may attack some components of the pectic fraction, as well as xyloglucans. Pectin-bound calcium ions may be displaced but this probably has little effect on gel strength. Pectins may be cross-linked by diferulate esters when growth stops. The softening of ripe fruit is due to loss of cohesion in the pectin gel. In apples this results from replacement of the pectins by more esterified forms. In many other fruits it results from depolymerization by polygalacturonases, assisted by pectinesterases, so that the remaining segments are too short for effective calcium binding. Pectins have a further role in the recognition reactions between plant cells and some of their bacterial and fungal pathogens.     

Bioprocessing in space: Human cells attach to beads in microgravity

Attachment to a substrate and survival of human embryonic kidney (HEK) cells have been tested in an incubator installed in the flight-deck of the Space Shuttle ‘Challenger’ during its eighth mission.HEK cells are producing the enzyme urokinase and are presently investigated as candidates for electrophoretic separation in an apparatus developed and manufactured by McDonnell Douglas.Attachment of HEK cells to a substrate is mandatory for survival and production of urokinase after electrophoretic separation.Analysis of the samples shows that cells adhere, spread and survive in microgravity (< 10⁻³ ×g) conditions as well as the ground controls at 1 × g. This result represents an important step towards further bioprocessing in space.     

In vitro autoradiographic localization of [I]-angiotensin II binding sites in the rat and dog kidney

Light microscopic autoradiographic techniques have been utilized to demonstrate specific regions of the rat and dog kidney where angiotensin II receptors exist. Slide mounted tissue sections were labeled with [¹²⁵I]-angiotensin II using conditions which provided for highly specific binding. These angiotensin II binding sites were localized to several distinct renal structures. In the renal cortex, angiotensin II binding sites were found concentrated in all parts of the glomeruli including the vascular components, the macula densa and the juxtaglomerular apparatus. Angiotensin II binding in the medulla was more diffusely associated with the vasa recta, and to a lesser extent, the thick ascending segment of the loop of Henle. Binding sites specific for angiotensin II were also found in the smooth muscle laminae of the ureter. Scatchard analysis of the binding kinetics allowed the demonstration of two subpopulations of binding sites which differ slightly in their affinities for [¹²⁵I]-angiotensin II. These subpopulations appear to be associated with distinct components of the renal structure.     

Inhibition of surfactant release from isolated type II cells by compound 4880

Release of [³H]phosphatidylcholine from pulmonary Type II epithelial cells was stimulated by terbutaline, forskolin and cytochalasin D. Compound $\text{48}\text{80}$ inhibited both basal and agonist-stimulated release of [³H]PC. The IC₅₀ for inhibition by compound $\text{48}\text{80}$ was 1–2 μg/ml, and was similar for inhibition of both basal and stimulated release of [³H]phosphatidylcholine. Inhibitory effects of $\text{48}\text{80}$ were noted following a 1 h exposure to compound $\text{48}\text{80}$ and persisted up to 3 h. The inhibitory effect of compound $\text{48}\text{80}$ was entirely reversed by removing compound $\text{48}\text{80}$ from the external milieu. Compound $\text{48}\text{80}$ had no effect on cytosolic cyclic AMP levels or lactate dehydrogenase release. Inhibition of surfactant release produced by compound $\text{48}\text{80}$ was unaffected by changes in extracellular calcium concentrations. Compound $\text{48}\text{80}$ is a non-toxic inhibitor of phosphatidylcholine release from Type II epithelial cells.     

The proposed 5′-nucleotidase inhibitor in human leukemic cells is an artefact

It is now well established that human lymphoblastoid cell lines showing immaturity characters display ecto-5′-nucleotidase activities lower than normal levels. A recent paper (Sun, A.S., Holland, J.F. and Ohnuma, T. (1983) Biochim. Biophys. Acta 762, 577–584) mentioned that this phenomenon resulted from the presence of a 5′-nucleotidase inhibitor in these cell lines. We demonstrate here that the use of 5′-[³H]AMP as a substrate, and inadequate analysis of the products formed, led them to a misinterpretation. [³H]Adenosine derived from 5′-[³H]AMP hydrolysis was further transformed into [³H]inosine by the adenosine deaminase activity of the leukemic cell lines tested; [³H]inosine was precipitated with the excess substrate and was not taken into account in the ecto-5′-nucleotidase determination, which led the authors to confuse this adenosine deaminase activity with a 5′-nucleotidase inhibitor. We did not observe 5′-nucleotidase inhibition by leukemic cell cytosol when convenient assay methods were used and showed that the presence of such an inhibitor remains to be established.     

Fluorescence studies using synchrotron radiation on normal and differentiated cells labeled with parinaric acids

Changes in membrane properties during the differentiation process in K562 cells have been investigated. A decrease of lectin-induced agglutination has been detected. The agglutination assay revealed to be an early and sensitive test to monitor the induced differentiation of the K562 cells. Naturally occurring fluorescent fatty acids (cis- and trans-parinaric acids) and the recently developed multifrequency phase and modulation technique were used to study cell membrane properties. Changes in fluorescence lifetime and polarization are clearly associated with cell differentiation, suggesting the involvement of the cellular plasma membrane in the differentiation process.     

The effect of glyceraldehyde on red cells: Haemoglobin status,oxidative metabolism and glycolysis

Glyceraldehyde induces changes in the flux of glucose oxidised through the hexose monophosphate pathway, the concentrations of intermediates in the Embden-Meyerhoff pathway, the oxidative status of haemoglobin and levels of reduced and oxidised pyridine nucleotides and glutathione in red cells. Glyceraldehyde autoxidises in the cellular incubations, consuming oxygen and producing glyoxalase I- and II-reactive materials. Major fates of glyceraldehyde in red cells appear to be: (i) adduct formation with reduced glutathione and cellular protein; (ii) autoxidation and reaction with oxyhaemoglobin and pyridine nucleotides, and (iii) phosphorylation of d-glyceraldehyde and entry into the glycolytic pathway as glyceraldehyde 3-phosphate. The production of glycerol from glyceraldehyde by red cell l-hexonate dehydrogenase appears not to be a major reaction of glyceraldehyde in red cells. These results indicate that high concentrations of glyceraldehyde (1–50 mM) may induce oxidative stress in red cells by virtue of the spontaneous autoxidation of glyceraldehyde, forming hydrogen peroxide and α-ketoaldehydes (glyoxalase substrates). The implications of glyceraldehyde-induced oxidative stress for the in vitro anti-sickling effect of dl-glyceraldehyde and for the polyol pathway metabolism of glyceraldehyde are discussed.