Extensional flow of erythrocyte membrane from cell body to elastic tether. I. Analysis.

This is the first of two papers on an analytical and experimental study of the flow of the erythrocyte membrane. In the experiment to be discussed in detail in the second paper, preswollen human erythrocytes are sphered by aspirating a portion of the cell membrane into a small micropipette; and long, thin, membrane filaments or "tethers" are steadily withdrawn from the cell at a point diametrically opposite to the point of aspiration. The aspirated portion of the membrane furnished a "reservoir" of material that replaces the membrane as it flows as a liquid from the nearly spherical cell body to the cylindrical tether. In this paper we show that an application of the principle of conservation of mass permits the tether radius (approximately 200 A or less) to be measured with the light microscope as the tether is formed and extended at a constant rate. A static analysis of the axisymmetric cell deformation and tether formation process reveals that the tether radius is uniquely determined by the isotropic tension in the membrane and the elastic constitutive (material) behavior of the tether itself. A dynamic analysis of the extensional flow process reveals that the tether radius must decrease as the velocity of the tether is increased and that the decrease depends on both the viscosity of the membrane and the elasticity of the tether. The analysis also shows that these two factors (membrane viscosity and tether elasticity) are readily decomposed and determined separately when flow experiments are performed at different isotropic tensions.     

Characteristics of the Photosystem II reaction centre. II. Electron donors

The properties of Photosystem II electron donation were investigated by EPR spectrometry at cryogenic temperatures. Using preparations from mutants which lacked Photosystem I, the main electron donor through the Photosystem II reaction centre to the quinone-iron acceptor was shown to be the component termed Signal II. A radical of 10 G line width observed as an electron donor at cryogenic temperatures under some conditions probably arises through modification of the normal pathway of electron donation. High-potential cytochrome b-559 was not observed on the main pathway of electron donation. Two types of PS II centres with identical EPR components but different electron-transport kinetics were identified, together with anomalies between preparations in the amount of Signal II compared to the quinone-iron acceptor. Results of experiments using cells from mutants of Scenedesmus obliquus confirm the involvement of the Signal II component, manganese and high-potential cytochrome b-559 in the physiological process leading to oxygen evolution.     

Identification of diacylglycerol-O-(N,N,N-trimethyl)-homoserine in the halotolerant alga, Dunaliella parva

An unusual zwitterionic polar lipid component isolated from the halotolerant alga, Dunaliella parva Lerche, has been identified as 1(3),2-diacylglyceryl-3(1)-O-4′-(N,N,N-trimethyl) homoserine by means of infrared spectrometry, ¹H- and ¹³C-NMR spectrometry and field desorption mass spectrometry of the intact lipid, as well as by its TLC mobilities and staining behaviour. Mass spectrometry of this lipid indicated the presence of the following major molecular species: 16:0–18:0 (24%); 18:3–18:3 (19%); 16:0–18:2 (16%); 16:0–18:1 (8%).     

Recombinant human [Cys281]insulin-like growth factor-binding protein 2 inhibits both basal and insulin-like growth factor I-stimulated proliferation and collagen synthesis in fetal rat calvariae.

It is recognized that insulin-like growth factors (IGFs) are bound to specific high-affinity insulin-like growth factor-binding proteins (IGFBPs). The role of IGFBPs in bone metabolism is not well established. The effect of recombinant human [Cys281]IGFBP-2 ([Cys281]rhIGFBP-2) on bone formation in 21-day-old fetal rat calvariae was investigated. [Cys281]rhIGFBP-2 was expressed in and purified from conditioned medium of a clonal Chinese hamster ovary cell line. IGF-I-stimulated cell proliferation was inhibited dose dependently by [Cys281]rhIGFBP-2, with half-maximal inhibition observed at 2 x 10(-8) M. Suppression of the IGF-I-stimulated DNA synthesis was observed at an apparent dose ratio of 1:10. [Cys281]rhIGFBP-2 (10(-6) M) also inhibited the basal incorporation of [3H]thymidine into DNA by up to 45%. Insulin-stimulated cell proliferation was not affected in the presence of the binding protein. In addition, [Cys281]rhIGFBP-2 inhibited bone collagen synthesis under basal and IGF-I-stimulated conditions. In contrast, [Cys281]rhIGFBP-2 did not alter the parathyroid hormone-stimulated bone cell proliferation rate. In conclusion, binding of hIGF-I to rhIGFBP-2 results in an inhibition of the actions of free IGF-I on bone cell replication and matrix synthesis. Parathyroid hormone-stimulated cell proliferation is not mediated by an increase in free IGFs.     

Protease specificity and heparin binding and activation of recombinant protease nexin I

Structural and functional properties of alpha-protease nexin I (alpha-PNI) expressed in Chinese hamster ovary cells were studied. All three cysteines were in the reduced form, showing that the potential disulfide bridge between residues Cys117 and Cys131 was not formed. Heparin association rate enhancements were from ka = 8.3 x 10(5) to 0.7-1.6 x 10(9) M-1 s-1 for the interaction of PNI with thrombin, from ka = 5.1 x 10(3) to 3.5 x 10(5) M-1 s-1 for interaction with Factor Xa, and from ka = 2.2 x 10(6) to 1.0 x 10(7) M-1 s-1 for interaction with trypsin; there was no rate enhancement of the plasmin interaction (ka = 1.0 x 10(5) M-1 s-1). The minimal heparin pentasaccharide had no effect on these interactions. Cleavage of the reactive center loop of PNI by three different proteases gave the typical stressed to relaxed change in thermal stability, but unlike with antithrombin III, there was no loss of heparin affinity. A similar difference from antithrombin was that PNI-thrombin complexes retained normal heparin affinity. These results are compatible with a role for protease nexin I as a cell-associated thrombin inhibitor that remains bound to the cell surface even after complexing with the protease, as compared with the role of antithrombin III as a circulating inhibitor of thrombin that becomes activated on binding to the microvasculature and is released on complex formation.     

The catalytic mechanism of the amidotransferase domain of the Syrian hamster multifunctional protein CAD. Evidence for a CAD-glutamyl covalent intermediate in the formation of carbamyl phosphate.

The multifunctional protein CAD catalyzes the first three steps in pyrimidine biosynthesis in mammalian cells, including the synthesis of carbamyl phosphate from bicarbonate, MgATP and glutamine. The Syrian hamster CAD glutaminase (GLNase) domain, a trpG-type amidotransferase, catalyzes glutamine hydrolysis in the absence of MgATP and bicarbonate (Km = 95 microM and kcat = 0.14 s-1). Unlike E. coli carbamyl phosphate synthetase (Wellner, V.P., Anderson, P.M., and Meister, A. (1973) Biochemistry 12, 2061-2066), a stable thioester intermediate did not accumulate when the mammalian enzyme was incubated with glutamine. However, a covalent adduct could be isolated when the protein was denatured in acid. The steady state concentration of the intermediate increased with increasing glutamine concentration to nearly one mole per mole of enzyme with half saturation at 105 microM, close to the Km value for glutamine. The adduct formed at the active site of the glutaminase domain. The rate of breakdown of the intermediate (k4), determined directly, was 0.17 s-1 and the rate of formation (k3) was estimated as 0.52 s-1. In the absence of MgATP and bicarbonate, k4 = kcat indicating that the decomposition of the intermediate is the rate-limiting step. The intermediate was chemically and kinetically competent, and the glutamine dissociation constant (330 microM) and rate constants were consistent with steady state kinetics and accurately predicted the steady state concentration of the intermediate. These studies suggest a mechanism similar to the cysteine proteases such as recently proposed by Mei and Zalkin (Mei, B., and Zalkin, H. (1989) J. Biol. Chem. 264, 16613-16619) who identified a catalytic triad in glutamine phosphoribosyl-5'-pyrophosphate amidotransferase, a purF-type enzyme. MgATP and bicarbonate increased kcat of the glutaminase reaction 14-fold by accelerating both the rate of formation and the rate of breakdown of the intermediate, and prevented the accumulation of the intermediate; however, the Km value for glutamine was not significantly altered. The instability of the thioester intermediate leads to appreciable hydrolysis of glutamine in the absence of the other substrates. However, bicarbonate alone spares glutamine by increasing the Km and Ks of glutamine to 600 and 8960 microM, respectively, thus reducing kcat/Km 3-fold when MgATP is limiting. In the absence of MgATP and bicarbonate, ammonia decreased the rate of hydrolysis and the accumulation of the thioester intermediate indicating that ammonia had direct access to the thioester at the GLNase domain active site.(ABSTRACT TRUNCATED AT 400 WORDS)     

Identification of human hookworm in failed-treatment cases using Chinese hamsters (Cricetulus griseus) and scanning electron microscopy

An attempt was made to identify the human hookworm involved in failed-treatment cases using abnormal hosts and scanning electron microscopy. Thirty-seven, 2 to 6 month old Chinese hamsters (Cricetulus griseus) from a closed, outbred, conventional colony, were each given between 20 and 120 filariform larvae per os. The larvae were cultured from faeces from mebendazole (Vermox) 500 mg single-dose, failed-treatment cases living in the lowveld farming area of the Transvaal Province, South Africa. About 60 to 78 days after inoculation, the animals were killed and adult worms were removed from their small intestines. Eleven (30%) of the 37 hamsters harboured a total of 31 adult worms (19 males and 12 females), while 26 hamsters were refractory to infection. The greatest number of worms recovered from a single animal was six. A total of 27 worms (17 males and 10 females) were subjected to examination by scanning electron microscopy. Micrographs showed male and female worms to be morphologically all of the Necator americanus species, as identified by a pair of ventral and dorsal cutting plates, a dorsal tooth and the fused terminus of spicules in the male bursa. The transverse cuticular striations were distinct and smooth. Several points of interest arose from the results of this study and are discussed.     

Culture and properties of cells derived from Kaposi sarcoma

We describe the establishment of four continuous cell cultures isolated from pleural or peritoneal fluid of patients with Kaposi sarcoma (KS) and show evidence that these cells are derived from vascular endothelium. Although provision of an extracellular matrix (fibronectin, laminin, or matrigel) was essential, the cell cultures were not dependent on exogenously added growth factors (platelet-derived growth factor, epidermal growth factor with or without heparin) for continuous culture. Specific staining for endothelial cell (EC) markers (factor VIII, Ulex europaeus type 1 lectin) and the secretion of endothelin, a vascular EC product, were demonstrated. The KS cells secreted large amounts of cytokines (granulocyte-macrophage-CSF, TNF-alpha, IL-1 beta, and especially IL-6). Conditioned media from the KS cells caused normal capillary EC to proliferate. The KS cells synthesized fibroblast growth activity in amounts sufficient to induce the proliferation of normal EC and fibroblasts. These data support the existence of a paracrine pathway of EC proliferation in KS and suggest that KS cells could sustain their own growth via an autocrine mechanism.