UDP-glucose: Glucan Synthetase in Developing Cotton Fibers: I. Kinetic and Physiological Properties

A uridine diphosphate(UDP)-glucose:glucan synthetase can be demonstrated in detached cotton fibers (Gossypium hirsutum L.) and in an isolated particulate fraction from such fibers. When assayed with detached fibers, the kinetics of the glucan synthetase activity with respect to variation in substrate concentration is complex and indicates activation of the enzyme by the substrate. Activity is stimulated by Ca(2+) or Mg(2+) and beta-linked glucosides; the effect of the beta-linked glucosides is to shift the range in which substrate activation occurs to lower concentrations of UDP-glucose. At concentrations of UDP-glucose below 50 mum, addition of uridine triphosphate, in addition to beta-linked glucoside, results in significant stimulation of activity. This effect can be explained by the conversion of uridine triphosphate to UDP-glucose by UDP-glucose pyrophosphorylase, thereby raising substrate concentration to the activating range. In detached fibers, glucan synthetase activity is high at all stages of fiber development. The properties of the glucan synthetase of the isolated particulate fraction closely resemble those of the enzyme assayed in detached fibers; however, in contrast to detached fibers, the ability to detect enzyme activity is more dependent on fiber age, showing maximal activity between 16 and 18 days postanthesis, coincident with the time of rapid onset of secondary wall cellulose deposition.     

Structural basis for chirality and directional motility of Plasmodium sporozoites

Plasmodium sporozoites can move at high speed for several tens of minutes, which is essential for the initial stage of a malaria infection. The crescent‐shaped sporozoites move on 2D substrates preferably in the same direction on circular paths giving raise to helical paths in 3D matrices. Here we determined the structural basis that underlies this type of movement. Immature, non‐motile sporozoites were found to lack the subpellicular network required for obtaining the crescent parasite shape. In vitro, parasites moving in the favoured direction move faster and more persistent than the few parasites that move in the opposite direction. Photobleaching experiments showed that sporozoites flip their ventral side up when switching the direction of migration. Cryo‐electron tomography revealed a polarized arrangement of microtubules and polar rings towards the substrate in Plasmodium sporozoites, but not in the related parasite Toxoplasma gondii. As aconsequence, secretory vesicles, which release proteins involved in adhesion, migration and invasion at the front end of the parasite, are delivered towards the substrate. The resulting chiral structure of the parasite appears to determine the unique directionality of movement and could explain how the sporozoite achieves rapid and sustained directional motility in the absence of external stimuli.     

Substrate Contact,Mucus, and Eugregregarine Gliding1

ABSTRACT. Experiments have been carried out on Gregarina garnhami to examine some of the factors that may be significant in gliding. Suspension of gregannes in Ficoll showed that substrate contact is essential. Reflection interference microscopy shows that there are fluctuating surface/substrate contacts, but it is not necessary for the whole of the undersurface of the cell to be in close contact with the substrate. Gliding is always accompanied by the formation of a mucus trail. The effects of the drugs amphetamine and ephedrine on mucus trail formation and gliding have been examined. Lateral undulations of the epicyte folds have previously been implicated in grcgarinc gliding, but G. garnhami does not exhibit lateral undulations of the epicyte folds as it moves. The folds are predominantly straight with indications of varicosities or swellings along the length of the folds. These observations are discussed in relation to gliding movement.     

Micromanipulation of adhesion of a Jurkat cell to a planar bilayer membrane containing lymphocyte function-associated antigen 3 molecules

Cell adhesion plays a fundamental role in the organization of cells in differentiated organs, cell motility, and immune response. A novel micromanipulation method is employed to quantify the direct contribution of surface adhesion receptors to the physical strength of cell adhesion. In this technique, a cell is brought into contact with a glass-supported planar membrane reconstituted with a known concentration of a given type of adhesion molecules. After a period of incubation (5-10 min), the cell is detached from the planar bilayer by pulling away the pipette holding the cell in the direction perpendicular to the glass-supported planar bilayer. In particular, we investigated the adhesion between a Jurkat cell expressing CD2 and a glass-supported planar bilayer containing either the glycosyl-phosphatidylinositol (GPI) or the transmembrane (TM) isoform of the counter-receptor lymphocyte function-associated antigen 3 (LFA-3) at a concentration of 1,000 molecules/microns 2. In response to the pipette force the Jurkat cells that adhered to the planar bilayer containing the GPI isoform of LFA-3 underwent extensive elongation. When the contact radius was reduced by approximately 50%, the cell then detached quickly from its substrate. The aspiration pressure required to detach a Jurkat cell from its substrate was comparable to that required to detach a cytotoxic T cell from its target cell. Jurkat cells that had been separated from the substrate again adhered strongly to the planar bilayer when brought to proximity by micromanipulation. In experiments using the planar bilayer containing the TM isoform of LFA-3, Jurkat cells detached with little resistance to micromanipulation and without changing their round shape.     

Calcium-dependent protein phosphorylation in Babesia bovis and its role in growth regulation

Intracellular growth of protozoan parasite Babesia bovis has been followed to study the effect of some chemical agents on growth regulation. Using an in vitro parasite culture system we present evidence that the normal growth of the parasite is dependent upon available calcium and a Ca2(+)-binding protein, calmodulin, because sequestration of either of these 2 components from the culture medium causes inhibition of parasitic growth. Further studies demonstrate that the parasite contains a protein kinase that can phosphorylate a 40-kDa parasitic protein and its activity is regulated by calcium and calmodulin. Both the enzyme and its substrate are present in the membrane of the parasite. In addition, the parasite also contains a highly active protein kinase C activity that is documented by phosphorylating histone, a known substrate for protein kinase C. These findings suggest a possible correlation between the growth of parasite and calcium/calmodulin-dependent protein phosphorylation activity.     

Carbon Nutrition in a Rafflesiaceae Holoparasite Cytinus hypocistis L. Fixed on,or Experimentally Isolated from the Host Cistus Monspeliensis L.

The host-parasite pair Cistus Monspeliensis-Cytinus hypocistis is fed, via the aerial organs of the host, with ¹⁴CO₂. The radioactivity measurements show that the main substances transfered from the host to the parasite are sucrose, glutamic acid, and aspartic acid. The parasite isolated from the host is also shown to be capable of fixing atmospheric ¹⁴C thus fixed represents about 12% of the ¹⁴C received from the host by the non-isolated parasite. The nature of the labelled products found in the detached parasite not only confirms the existence of PEP carboxylase activity, but also suggests the intervention of a RuBP carboxylase.     

Burrowing behaviour of the European eel (Anguilla anguilla): Effects of life stage

The European eel (Anguilla anguilla) is a fascinating species, exhibiting a complex life cycle. The species is, however, listed as critically endangered on the IUCN Red List due to an amalgam of factors, including habitat loss. This study investigated the burrowing behaviour and substrate preference of glass, elver and yellow stages of A. anguilla. Preference was determined by introducing eels in aquaria with different substrates and evaluating the chosen substrate for burrowing. In addition, burrowing was recorded using a camera in all substrate types and analysed for kinematics. The experiments showed that all of these life stages sought refuge in the sediments with particle sizes ranging from sand to coarse gravel. Starting from a resting position, they shook their head horizontally in combination with rapid body undulations until half of their body was within the substrate. High-speed X-ray videography revealed that once partly in the sediment, eels used only horizontal head sweeps to penetrate further, without the use of their tail. Of the substrates tested, burrowing performance was highest in fine gravel (diameter 1–2 mm; lower burrowing duration, less body movements and/or lower frequency of movements), and all eels readily selected this substrate for burrowing. However, glass eels and elvers were able to use coarse gravel (diameter >8 mm) because their smaller size allowed manoeuvring through the spaces between the grains. Further, burrowing performance increased with body size: glass eels required more body undulations compared to yellow eels. Interestingly, the urge to hide within the sediment was highest for glass eels and elvers. Documentation of substrate preference and burrowing behaviour of A. anguilla provides new information about their potential habitat use. Considering that habitat alterations and deteriorations are partly responsible for the decline of the eel, this information can contribute to the development of more effective conservation measures.     

Role of pleated septate junctions in the epithelium of miracidia of Schistosoma mansoni during transformation to sporocysts in vitro

The surfaces of miracidia of Schistosoma mansoni were examined ultrastructurally during in vitro transformation to sporocysts. Before transformation, the surface was composed of ciliated epithelial plates (EP) that were set into a reticulum of narrow syncytial ridges (SR). The EP were attached to SR by extensive pleated septate junctions that had 18-24 strands of intramembrane particles (IMP) on the protoplasmic faces and complementary pits on the ectoplasmic faces. These junctions also appeared to separate the EP plasma membrane into apical and basolateral domains with a larger number of IMPs on the latter. Transformation was induced by placing the miracidia in salt containing medium which also halted ciliary beating. In 2-5 hr, the SR expanded until they formed a syncytium covering the parasite surface, while the EP retracted and rounded up. During this time, the EP and SR were held in contact with one another by the septate junctions which became progressively convoluted. Subsequently, the EP detached from the parasite. When transforming miracidia were returned to fresh water, the cilia resumed beating and the EP detached from the parasite surface and exposed the underlying basement membrane. Those EP that remained attached were connected only by septate junctions to the expanded SR. These studies demonstrate that the formation of the syncytium occurs gradually with contact maintained between EP and SR via the septate junctions. Further, the septate junctions are similar to occluding junctions in mammalian epithelia since they segregate the plasma membrane of the EP and they have an adhesive function.     

Determination of electron density profiles and area from simulations of undulating membranes

The traditional method for extracting electron density and other transmembrane profiles from molecular dynamics simulations of lipid bilayers fails for large bilayer systems, because it assumes a flat reference surface that does not take into account long wavelength undulations. We have developed what we believe to be a novel set of methods to characterize these undulations and extract the underlying profiles in the large systems. Our approach first obtains an undulation reference surface for each frame in the simulation and subsequently isolates the long-wavelength undulations by filtering out the intrinsic short wavelength modes. We then describe two methods to obtain the appropriate profiles from the undulating reference surface. Most combinations of methods give similar results for the electron density profiles of our simulations of 1024 DMPC lipids. From simulations of smaller systems, we also characterize the finite size effect related to the boundary conditions of the simulation box. In addition, we have developed a set of methods that use the undulation reference surface to determine the true area per lipid which, due to undulations, is larger than the projected area commonly reported from simulations.     

Studies on the host parasite relationship between the parasitic prosobranch Thyca crystallina and the asteroid starfish Linckia laevigata

A study of the Blue starfish Linckia laevigata in the Banda Islands, Moluccas, Indonesia, revealed 62% infestation by the molluscan prosobranch parasite Thyca crystallina. Infestation rate varies directly with the degree of water movement. A marked difference in settlement pattern according to parasite size is observed. The smallest limpets tend to settle on the upper surface of the distal parts of the arms and are randomly oriented. The largest parasites are found on the oral arm surface on the right hand side of the ambulacral groove and facing the starfish mouth. All of the attached parasites were female. Most of the large females had a dwarf male, one tenth the length, attached, under the mantle, at the anterior end. Amongst the small limpets, less than half had made a proboscis hole in the host integument and none had made an attachment disc lesion; of the large parasites 90% had the proboscis embedded in the host and 80% left an attachment disc lesion on the host. Small parasites can move very slowly over the host surface. Larger limpets are probably permanently attached. A detached parasite of any size is probably unable to reattach. There is evidence of an extended parasite breeding period and repetitive settlement on the hosts, which could cause the observed overdispersion without invoking a mature female parasite pheromone.     

Sequestration and metabolism of host cell arginine by the intraerythrocytic malaria parasite Plasmodium falciparum

Human erythrocytes have an active nitric oxide synthase, which converts arginine into citrulline and nitric oxide (NO). NO serves several important functions, including the maintenance of normal erythrocyte deformability, thereby ensuring efficient passage of the red blood cell through narrow microcapillaries. Here, we show that following invasion by the malaria parasite Plasmodium falciparum the arginine pool in the host erythrocyte compartment is sequestered and metabolized by the parasite. Arginine from the extracellular medium enters the infected cell via endogenous host cell transporters and is taken up by the intracellular parasite by a high‐affinity cationic amino acid transporter at the parasite surface. Within the parasite arginine is metabolized into citrulline and ornithine. The uptake and metabolism of arginine by the parasite deprive the erythrocyte of the substrate required for NO production and may contribute to the decreased deformability of infected erythrocytes.     

How cells tiptoe on adhesive surfaces before sticking

Cell membranes are studded with protrusions that were thoroughly analyzed with electron microscopy. However, the nanometer-scale three-dimensional motions generated by cell membranes to fit the topography of foreign surfaces and initiate adhesion remain poorly understood. Here, we describe the dynamics of surface deformations displayed by monocytic cells bumping against fibronectin-coated surfaces. We observed membrane undulations with typically 5 nm amplitude and 5-10 s lifetime. Cell membranes behaved as independent units of micrometer size. Cells detected the presence of foreign surfaces at 50 nm separation, resulting in time-dependent amplification of membrane undulations. Molecular contact then ensued with apparent cell-membrane separation of 30-40 nm, and this distance steadily decreased during the following tens of seconds. Contact maturation was associated with in-plane egress of bulky molecules and robust membrane fluctuations. Thus, membrane undulations may be the major determinant of cell sensitivity to substrate topography, outcome of interaction, and initial kinetics of contact extension.     

Mitochondria DNA synthesis in oocytes of Xenopus laevis

The process of attachment was studied in primary mouse kidney epithelial cell cultures by means of reflexion contrast microscopy, a method developed for studying the cell membrane-substrate relationship. The first in a series of events is simple adherence to the substrate, called close contact. This phenomenon is associated with the greatest extension of lamellar cytoplasm and the fewest number of cell nuclei/unit area. The nuclei of such cells are in close contact with the bottom portion of the cell membrane. Approx. 24 h after planting, as the cultures become more crowded, cells develop a different kind of attachment to the substrate—focal contacts—that are correlated with a decrease in lamellar cytoplasm. Cells detached from the substrate after close contact formation readily reattach, while cells detached after formation of focal contacts do not reattach. After incubation for periods greater than 5 days, the dense cultures degenerate and cells lose their attachment to the glass surface.     

Parasite-induced fruit mimicry in a tropical canopy ant

Some parasites modify characteristics of intermediate hosts to facilitate their consumption by subsequent hosts, but examples of parasite-mediated mimicry are rare. Here we report dramatic changes in the appearance and behavior of nematode-parasitized ants such that they resemble ripe fruits in the tropical rain forest canopy. Unlike healthy ants, which are completely black, infected ants have bright red, berry-like gasters full of parasite eggs. The infected gasters are held in a conspicuous elevated position as the ants are walking, and they are easily detached from living ants, which also exhibit reduced defensive responses. This combination of changes presumably makes the infected ants attractive to frugivorous birds, which ingest the red gasters and pass the parasite eggs in their feces. The feces are collected by ants and fed to the developing brood, thus completing the cycle. This is the first documentation of parasites causing apparent fruit mimicry in an animal host to complete their life cycle.     

Centrifugal assessment of cell adhesion

The design of a chamber for determining the centrifugal force necessary to detach cells from various substrates is presented. Cells from an SV40-transformed murine peritoneal macrophage line and human erythrocytes were used to assess the feasibility of using the chamber for studies of cell adhesion. This work was confirmed the usefulness of the chamber and provided data concerning the force necessary to detach the cells. These data indicated that the percentage of cells detached from a glass substrate was not a function of force alone. The number of cells detaching increased with the impulse applied to the cells when they were exposed to a constant force. Similarly, when the impulse applied to the IC21 cells was maintained at a constant level, the percentage of cells detached by a centrifugal force increased with the magnitude of the force.     

Molecular and biochemical characterization of D-phosphoglycerate dehydrogenase from Entamoeba histolytica. A unique enteric protozoan parasite that possesses both phosphorylated and nonphosphorylated serine metabolic pathways.

A putative phosphoglycerate dehydrogenase (PGDH), which catalyzes the oxidation of d-phosphoglycerate to 3-phosphohydroxypyruvate in the so-called phosphorylated serine metabolic pathway, from the enteric protozoan parasite Entamoeba histolytica was characterized. The E. histolytica PGDH gene (EhPGDH) encodes a protein of 299 amino acids with a calculated molecular mass of 33.5 kDa and an isoelectric point of 8.11. EhPGDH showed high homology to PGDH from bacteroides and another enteric protozoan ciliate, Entodinium caudatum. EhPGDH lacks both the carboxyl-terminal serine binding domain and the 13-14 amino acid regions containing the conserved Trp139 (of Escherichia coli PGDH) in the nucleotide binding domain shown to be crucial for tetramerization, which are present in other organisms including higher eukaryotes. EhPGDH catalyzed reduction of phosphohydroxypyruvate to phosphoglycerate utilizing NADH and, less efficiently, NADPH; EhPGDH did not utilize 2-oxoglutarate. Kinetic parameters of EhPGDH were similar to those of mammalian PGDH, for example the preference of NADH cofactor, substrate specificities and salt-reversible substrate inhibition. In contrast to PGDH from bacteria, plants and mammals, the EhPGDH protein is present as a homodimer as demonstrated by gel filtration chromatography. The E. histolytica lysate contained PGDH activity of 26 nmol NADH utilized per min per mg of lysate protein in the reverse direction, which consisted 0.2-0.4% of a total soluble protein. Altogether, this parasite represents a unique unicellular protist that possesses both phosphorylated and nonphosphorylated serine metabolic pathways, reinforcing the biological importance of serine metabolism in this organism. Amino acid sequence comparison and phylogenetic analysis of various PGDH sequences showed that E. histolytica forms a highly supported monophyletic group with another enteric protozoa, cilliate E. caudatum, and bacteroides.     

Effect of colchicine on polarization of cells on narrow strips of an adherent substrate

Effect of antitubulin colcemide on polarization of mouse fibroblast-like cells on anisotropic substrate was studied. Such a substrate was obtained by scratching narrow strips in lipid films, adsorbed on the glass. The control cells were seen spread only along the strips, and in 4-6 hours they approached the length of120-150 mcm. In colcemide-containing media, the cells remain in an unspread state for a long time; they extrude their outgrowths both along the strip and perpendicularly to it. Due to frequent refractions of outgrowths, two thirds of colcemide-treated cells were detached from the substrate. Possible mechanisms of these effects of antitubulins are discussed.     

Plasmodium subtilisin-like protease 1 (SUB1): insights into the active-site structure, specificity and function of a pan-malaria drug target

Release of the malaria merozoite from its host erythrocyte (egress) and invasion of a fresh cell are crucial steps in the life cycle of the malaria pathogen. Subtilisin-like protease 1 (SUB1) is a parasite serine protease implicated in both processes. In the most dangerous human malarial species, Plasmodium falciparum, SUB1 has previously been shown to have several parasite-derived substrates, proteolytic cleavage of which is important both for egress and maturation of the merozoite surface to enable invasion. Here we have used molecular modelling, existing knowledge of SUB1 substrates, and recombinant expression and characterisation of additional Plasmodium SUB1 orthologues, to examine the active site architecture and substrate specificity of P. falciparum SUB1 and its orthologues from the two other major human malaria pathogens Plasmodium vivax and Plasmodium knowlesi, as well as from the rodent malaria species, Plasmodium berghei. Our results reveal a number of unusual features of the SUB1 substrate binding cleft, including a requirement to interact with both prime and non-prime side residues of the substrate recognition motif. Cleavage of conserved parasite substrates is mediated by SUB1 in all parasite species examined, and the importance of this is supported by evidence for species-specific co-evolution of protease and substrates. Two peptidyl alpha-ketoamides based on an authentic PfSUB1 substrate inhibit all SUB1 orthologues examined, with inhibitory potency enhanced by the presence of a carboxyl moiety designed to introduce prime side interactions with the protease. Our findings demonstrate that it should be possible to develop 'pan-reactive' drug-like compounds that inhibit SUB1 in all three major human malaria pathogens, enabling production of broad-spectrum antimalarial drugs targeting SUB1.     

Axonal shortening and the mechanisms of axonal motility

Axons in tissue culture retract and shorten if their tips are detached from the substrate. The shortening reaction of the axon involves contractile forces that also arise during normal axonal motility, elongation, and retraction. We studied shortening in axonal segments isolated from their parent axons by transecting the axon between the growth cone and the most distal point of adhesion to the substrate. Within 15-20 minutes after transection, an isolated axonal segment shortened and pulled its tail end toward the growth cone. During the shortening process, long sinusoidal bends arose along the axon. The identical shortening reaction occurs without transection, when the axon tip is detached from the substrate. Pharmacological studies with inhibitors of glycolysis indicate that the shortening mechanisms utilize metabolic energy, presumably ATP. The rate of sinusoidal shortening is similar to both the rate of polymer translocation in the axon by slow axonal transport and the rate of normal axonal elongation. Taxol inhibits the shortening reaction with a similar dose dependence to its inhibition of axonal growth. Together, all these observations suggest that the same basic intracellular motility mechanisms are involved in normal axonal growth, in slow axonal transport, and in the shortening reaction: the intracellular dynamic system that utilizes ATP to generate longitudinal movements of polymers within the axon may be the same mechanism underlying both the retraction and the elongation of the axon.     

Uridine phosphorylase from Schistosoma mansoni

Uridine phosphorylase is the only pyrimidine nucleoside cleaving activity that can be detected in extracts of Schistosoma mansoni. The enzyme is distinct from the two purine nucleoside phosphorylases contained in this parasite. Although Urd is the preferred substrate, uridine phosphorylase can also catalyze the reversible phosphorolysis of dUrd and dThd, but not Cyd, dCyd, or orotidine. The enzyme was purified 170-fold to a specific activity of 2.76 nmol/min/mg of protein with a 16% yield. It has a Mr of 56,000 as determined by molecular sieving on Sephadex G-100. The mechanism of uridine phosphorylase is sequential. When Urd was the substrate, the KUrd = 13 microM and the KPi = 533 +/- 78 microM. When dThd was used as a substrate, the KdThd = 54 microM and the KPi = 762 +/- 297 microM. The Vmax with dThd was 53 +/- 9.8% that of Urd. dThd was a competitive inhibitor when Urd was used as a substrate. The enzyme showed substrate inhibition by Urd, dThd (greater than 0.125 mM) and phosphate (greater than 10 mM). 5-(Benzyloxybenzyloxybenzyl)acyclouridine was identified as a potent and specific inhibitor of parasite (Ki = 0.98 microM) but not host uridine phosphorylase. Structure-activity relationship studies suggest that uridine phosphorylase from S. mansoni has a hydrophobic pocket adjacent to the 5-position of the pyrimidine ring and indicate differences between the binding sites of the mammalian and parasite enzymes. These differences may be useful in designing specific inhibitors for schistosomal uridine phosphorylase which will interfere selectively with nucleic acids synthesis in this parasite.