Phosphoenolpyruvate carboxykinase from Fasciola hepatica

Behm C. A. and Bryant C. 1982. Phosphoenolpyruvate carboxykinase from Fasciola hepatica. International Journal for Parasitology12: 271–278. The kinetic properties of a partially purified preparation of phosphoenolpyruvate carboxykinase (PEPCK) from F. hepatica were examined. The pH optimum for the carboxylation reaction is 5.8–6.2. The enzyme is more active with Mn²⁺ than Mg²⁺ and the Mn²⁺ saturation curve was sigmoid. Apparent K_m values for the substrates GDP, IDP, PEP and HCO₃^? were determined and found to be in the same range as those reported for other helminths except that the enzyme is less sensitive to low PEP concentrations. GTP and ATP at 0.5 and 1.0 mM inhibit the enzyme; the GTP inhibition was greater in the presence of Mg²⁺ than Mn²⁺ and was competitive with GDP. It was concluded that the activity of PEPCK from F. hepatica is controlled by the concentration of reactants and the ambient pH, that the accumulation of GTP is a sensitive mechanism for inhibiting the carboxylation reaction and that PEPCK activity in the cytosol is likely to be favoured over that of pyruvate kinase except when pH is high and PEP concentration low.     

Sexual dimorphism and resource allocation in male and female shrubs of Simmondsia chinensis

Summary Desert populations of the evergreen dioecious shrub Simmondsia chinensis exhibit sex-related leaf and canopy dimorphisms not present in populations from more mesic coastal environments. Leaves on female shrubs have characteristically larger sizes, greater specific weights, and greater water-holding capacity than male leaves in desert habitats. In coastal scrub environments no significant difference is present, with leaf characteristics of both sexes similar to those of desert male shrubs. Desert female shrub canopies are typically relatively open with little mutual branch shading. In male shrubs canopies are more densely branched with considerable mutual shading of branches. Female plants allocate a greater proportion of their vegetative resources to leaves than do male plants. Considering total biomass, male plants allocate 10–15% of their resources (biomass, calories, glucose-equivalents, nitrogen, phosphorus) to reproductive tissues. Female allocation is dependent on seed set. At 100% seed set females would allocate 30–40% of their resources to reproduction, while female reproductive investment would equal that of males at approximately 30% seed set. Sexual dimorphism and the associated physiological characteristics in Simmondsia act as an alternative to differential habitat selection by male and female plants. Female plants respond to limited water resources in desert areas by increasing their efficiency in allocating limited resources to reproductive structures.     

The combining site of the dinitrophenyl-binding immunoglobulin A myeloma protein MOPC 315

Magnetic-resonance techniques are used to refine the model of the combining site of the Fv fragment of the dinitrophenyl-binding mouse myeloma protein MOPC 315 constructed by Padlan, Davies, Pecht, Givol & Wright (1976) (Cold Spring Harbor Symp. Quant. Biol. 41, in the press). Light-absorption studies indicate a dinitrophenyl–tryptophan interaction in the Fv fragment of the type occurring in free solution. The Dnp-aspartate–tryptophan complex is therefore used as a starting point for the n.m.r. (nuclear-magnetic-resonance) analysis of the dinitrophenyl–Fv fragment interaction. Ring-current calculations are used to determine the geometry of the complex. The specificity of complex-formation between dinitrophenyl and tryptophan is confirmed by the lack of ring-current shifts of the dinitrophenyl resonances when tryptophan is replaced by any other aromatic amino acid. Proton n.m.r. difference spectra (at 270MHz), resulting from the addition of a variety of haptens to the Fv fragment, show that the combining site is highly aromatic in nature. Calculations on the basis of ring-current shifts define the geometry of the combining site, which involves a dinitrophenyl ring in van der Waals contact with four aromatic amino acid residues on the protein. The observation of a nuclear Overhauser effect on the H₍₃₎ resonance of the dinitrophenyl ring provides additional constraints on the relative geometry of the H₍₃₎ proton and an aromatic amino acid residue on the Fv fragment. The specificity of the Fv fragment for dinitrophenyl ligands arises from a stacking interaction of the dinitrophenyl ring with tryptophan-93_L, in an `aromatic box' of essentially tryptophan-93_L, phenylalanine-34_H and tyrosine-34_L; asparagine-36_L and tyrosine-34_L also contribute by forming hydrogen bonds with the nitro groups on the dinitrophenyl ring. The n.m.r. results also confirm that the antibody–hapten reaction may be visualized as a single encounter step. An Appendix shows the method of calculation of ring currents for the four aromatic amino acids and their use in calculating structures.     

Histochemical analysis of rat testicular glycoconjugates. 1. Subsets of N-linked saccharides in seminiferous tubules

Summary The distribution of N-linked glycans in rat testis has been probed using a panel of lectins derived fromGalanthus nivalis (snowdrop, GNA),Canavalia ensiformis (jack bean, Con A),Lens culinaris (lentil, LCA),Pisum sativum (garden pea, PSA) andPhaseolus vulgaris, erythro- and leucoagglutinins (kidney bean, ePHA and IPHA). Several classes of N-linked glycan were identified in the spermatogenic series, and during differentiation into spermatozoa they altered in both their pattern of distribution and relative abundance. A population of tetra-antennary, non-bisected, complex glycans, detected by IPHA, was lost during the transition from spermatogonia to spermatocytes, while high-mannose structures were acquired; these were most abundant in spermatocytes, as were bi- and tri-antennary complex, non-bisected glycans, the latter becoming increasingly abundant on acrosomes and spermatozoa. Their bisected counterparts were more generally expressed throughout spermatogenic cells, although marked localization onto acrosomes and nuclear caps was again seen. Transition from spermatocytes to spermatids involved mainly changes of the acrosomal granule and nuclear cap, which were carried through to the final stages of differentiation. Sertoli cell surfaces and cytoplasmic granules showed a high level of N-glycan expression.     

Histochemical analysis of rat testicular glycoconjugates. 2. Beta-galactosyl residues in O- and N-linked glycans in seminiferous tubules.

Summary Rat testes have been examined with a panel of lectins that bind specifically to oligosaccharide sequences having terminal or subterminal -galactosyl residues in O-linked glycans, or in the outer chains of complex N-linked glycans:Arachis hypogaea (peanut, AHA),Erythrina cristagalli (coral tree, ECA),Ricinus communis (castor bean, RCA120) andAbrus precatorius (jequirity bean, APA) agglutinins. Pretreatment of sections with neuraminidase, -galactosidase and removal of alkali-labile O-linked sequences by -elimination allowed the structure of these glycans to be further explored. In spermatogonia and spermatocytes there was little evidence of glycans terminating in -galactosyl residues, although these were present at non-reducing terminals as sialylgalactosides. The acrosome contained two subsets of O-linked glycans terminating in sialylgalactosides, while the nuclear cap showed at least two subsets of N-linked sialylgalactosyl as well as O-linked glycans. Spermatozoa exhibited minor changes in the pattern of glycosylation, although the overall pattern of -galactosyl expression was similar. Binding to Sertoli cells showed the presence of some unsubstituted -galactosyl terminals on O-linked glycans but few such N-linked residues, while terminal -galactosides were scanty in tubular basement membranes.     

Pneumocystis carinii: Freeze-fracture study of stages of the organism

The morphology and life cycle of Pneumocystis carinii were studied in cortico-steroid-treated rats by ultrathin section and freeze-fracture electron microscopy. The following stages of P. carinii were noted: trophic, precyst, and cyst. The crescent-shaped cysts appeared to be intermediate forms between precyst and cyst. The cell wall of the trophic stage showed membrane structures suggestive of protozoan endocytosis, whereas the surface of the precyst stage was smooth. The cell wall of the cyst lacked the specialized structural differentiation of yeasts and resembled that of Plasmodium spp. We conclude that P. carinii belongs to the Protozoa, and is presumably Rhizopoda.     

Arrangement of Integrated Avian Sarcoma Virus DNA Sequences Within the Cellular Genomes of Transformed and Revertant Mammalian Cells

We have examined the arrangement of integrated avian sarcoma virus (ASV) DNA sequences in several different avian sarcoma virus transformed mammalian cell lines, in independently isolated clones of avian sarcoma virus transformed rat liver cells, and in morphologically normal revertants of avian sarcoma virus transformed rat embryo cells. By using restriction endonuclease digestion, agarose gel electrophoresis, Southern blotting, and hybridization with labeled avian sarcoma virus complementary DNA probes, we have compared the restriction enzyme cleavage maps of integrated viral DNA and adjacent cellular DNA sequences in four different mouse and rat cell lines transformed with either Bratislava 77 or Schmidt-Ruppin strains of avian sarcoma virus. The results of these experiments indicated that the integrated viral DNA resided at a different site within the host cell genome in each transformed cell line. A similar analysis of several independently derived clones of Schmidt-Ruppin transformed rat liver cells also revealed that each clone contained a unique cellular site for the integration of proviral DNA. Examination of several morphologically normal revertants and spontaneous retransformants of Schmidt-Ruppin transformed rat embryo cells revealed that the internal arrangement and cellular integration site of viral DNA sequences was identical with that of the transformed parent cell line. The loss of the transformed phenotype in these revertant cell lines, therefore, does not appear to be the result of rearrangement or deletions either within the viral genome or in adjacent cellular DNA sequences. The data presented support a model for ASV proviral DNA integration in which recombination can occur at multiple sites within the mammalian cell genome. The integration and maintenance of at least one complete copy of the viral genome appear to be required for continuous expression of the transformed phenotype in mammalian cells.     

Schistosoma mansoni: Ultrastructure of early transformation of skin- and shear-pressure-derived schistosomules

Against the background of cercarial fine structure, ultrastructural changes were compared in schistosomules of Schistosoma mansoni 30 min and 1 hr after their production in vivo by skin penetration and in vitro by shear pressure. The same developmental pattern was observed in schistosomules of both derivations. In vitro schistosomules, however, developed more slowly, resembled cercariae more closely, and varied less among organisms than did in vivo schistosomules. The greatest morphological changes were observed in the 1-hr in vivo schistosomules. These were as follows: (1) in tegument, formation of transient microvilli, a hepatalaminate outer membrane and accented surface invaginations, loss of glycocalyx, movement outward of cyton vesicles via bridges, accumulation of multilaminate bodies around bridge openings; (2) in the anterior organ (oral sucker), movement of head gland vesicles via the ducts into tegument followed by collapse of the gland fundus, disappearance of the circumfundal cells and two large support cells, and the appearance in these areas of membranes and parenchymal cells; (3) secretion of the acetabular gland contents, collapse of the glands and replacement by membranes and parenchymal cells; (4) peristaltic activity of the digestive tract as shown by alternate areas of lumen constriction and dilation; (5) loss of bladder and contraction of the small aboral collecting tubules; and (6) conversion of heterochromatic parenchymal cell nuclei to euchromatic. In contrast, the 1-hr in vitro shear schistosomules resembled 30-min in vivo schistosomules, retaining many cercarial features.     

Regulatory properties of a partially purified preparation of pyruvate kinase from Fasciola hepatica

It possesses sigmoid kinetics with PEP; FBP activation changes the relationship to a rectangular hyperbola. The enzyme is inhibited by malate, which competes with PEP; FBP relieves the inhibition slightly. ATP and bicarbonate ions are also inhibitory at high concentrations. ATP inhibition is mixed-competitive with PEP; bicarbonate inhibition is non-competitive. It is suggested that pyruvate kinase may regulate both lactate and acetate production by moderating the size of the cytosolic pyruvate pool.     

Kinetics and morphology of glucose-limited cultures of moulds grown in a chemostat and on solid media

The affinity (K _s value) of Geotrichum candidum for glucose determined from chemostat cultures was ca. 1 mg/l. K _s values for glucose were also estimated from the radial growth rates of colonies of G. candidum and Neurospora crassa grown on media solidified with agar or silica gel. An assessment is made of the use of colony radial growth rate to determine substrate affinities. The length of apical and intercalary hyphal comparte ments, internode length and the diameter of leading hyphaat the margin of colonies grown on solid media were all reduced at low glucose concentrations.