SYNOPSIS Leishmania tropica promastigotes do not utilize glucose provided in the medium until late log phase. Rapid depletion of glucose from the medium, however, occurs during late log and stationary phases. At about the same time, the cells show maximal rates of glucose uptake as well as peak levels of phosphofructokinase and pyruvate kinase activities. The glucose analog, 2‐deoxy‐D‐glucose inhibits glucose transport. Incorporation of this analog in the growth medium results in inhibition of growth. The hexokinase of L. tropica phosphorylates 2‐deoxy‐D‐glucose. Pyruvate kinase is activated by fructose‐1, 6‐diphosphate and adenosine monophosphate.
SUMMARY In Indotristicha ramosissima (Wight) van Royen there is normal syngamy. However, the polar nucleus is not fertilized so that a primary endosperm nucleus is not formed. Consequently, there is no endosperm in the seed. The post‐fertilization dissolution of the nucellar cells below the embryo sac results in the formation of a pseudo‐embryo sac into which the growing pro‐embryo penetrates. The first division of the zygote is transverse and the development of the embryo conforms to the Solanad type. The embryo possesses a large, coenocytic haustorial cell which disorganizes by the time the embryo is fully differentiated. The integuments and the ovary wall are rich in starch deposits which disappear during maturation. In the mature seed, the outermost layer of the outer integument becomes mucilaginous. The cells of the inner epidermis of the ovary wall become binucleate and occasionally multinucleate.
S ummary The embryo sac in Dicraea is tetra‐nucleate and follows a bisporic development. It comprises a single synergid, an egg cell and two juxtaposed antipodal cells. This justifies the establishment of a new type under the bisporic embryo sacs which may be designated as the Dicraea type.
S ummary The plant body in Terniola zeylanica (Gardner) Tulasne, a member of the Podostemaceae, consists of a creeping thallus. The flowers are bisexual, hypogynous and trimerous. The anthers are dithecous and all the microsporangia are arranged on the inner side of the anther and parallel to each other. The anther wall consists of four layers. The tapetum is of the secretory type. Quadripartition of the mother cells is simultaneous and the tetrads are isobilateral or tetrahedral. The microspore nucleus divides by an asymmetric spindle and the pollen grains are shed at the two‐celled stage. The ovules are anatropous, tenuinucellar and bitegminal with the micropyle formed solely by the outer integument. Both the integuments differentiate simultaneously. The embryo sac is bisporic, five‐nucleate and follows the reduced Allium type. A pseudo‐embryo sac is formed during early embryogeny. Both entomophily and anemophily are prevalent. Syngamy takes place normally. Previous reports of double fertilization are not confirmed. A primary endosperm nucleus is not formed and there is no endosperm. The development of the embryo follows the Solanad type and the embryo possesses a large, coeno‐cytic, haustorial cell. The development and maturation of the seed coat and pericarp have been studied. The cells of the inner epidermis of the ovary wall become binucleate and at times even multinucleate.
Amastigotes (non-flagellated tissue forms) of Leishmania sp. reside and multiply within the phagolysosomes in tissue macrophages of vertebrate hosts. They are true obligate, intracellular parasites and do not persist as viable entities outside of host cells. This absolute dependence on host cells raises interesting questions about the metabolic capabilities of the amastigote stage. It is assumed that in general, intracellular stages of parasitic protozoa, exhibit reduced metabolic capabilities when compared with insect or culture forms. This hypothesis is largely untested in Leishmania sp. although there is evidence to indicate that several enzymes in amastigotes are less active than their counterparts in promastigotes (Coombs et al., 1982, Molecular and Biochemical Parasitology 5: 199-211; Meade et al., 1984, Journal of Protozoology 31: 156-161). Both forms also differ in their ability to utilize various substrates (Hart et al., 1981, Molecular and Biochemical Parasitology 4: 39-51; Hart and Coombs, 1982, Experimental Parasitology 54: 397-409). The presence of several enzymes of intermediary metabolism in amastigotes of L. donovani (Looker et al., 1983, Molecular and Biochemical Parasitology 9: 15-28; Meade et al., loc. cit.) and L. mexicana (Coombs et al., 1982, loc. cit.) suggests that they are indeed capable of carrying out independent metabolic activities. Since in vitro demonstration of enzyme activity in cell free extracts in and of itself does not ensure functional activity in vivo, the capacity of L. donovani amastigotes to catabolize and incorporate substrates or precursors into macromolecules was tested. Amastigotes of L. donovani (Sudan strain 1S) were isolated and purified from infected hamster spleens as described before (Meade et al., 1984, loc. cit.). Parasites were washed twice and resuspended in basal salts solution (Mukkada et al., 1974, Journal of Protozoology 21: 393-397), pH 5.5 to a density of 0.35 mg protein/ml. Incorporation of glucose and proline into macromolecules was determined by measuring the incorporation of label into cold trichloroacetic acid (TCA) insoluble materials (Mukkada and Simon, 1977, Experimental Parasitology 42: 87-96). Amastigote suspensions (4.9 ml) were incubated on a shaking waterbath at 30 C for 20 min for temperature equilibration. [U-_4C]D-glucose and [U-_4C]-L-proline were added to a final concentration of 0.1 mM and a specific radioactivity of 0.4 A,Ci/umole. Samples of 0.5 ml were withdrawn at various intervals and immediately added to 0.5 ml cold 10% TCA. After 15 min of extraction, 0.5 ml of the suspension was filtered through a millipore filter (0.8 ,tm porosity), washed with 3 ml basal salts and the filter pads with the cells thereon were dropped into scintillation vials containing 10 ml Bray's solution (Bray, 1960, Analytical Biochemistry 1: 279-285); radioactivity was determined in a Packard scintillation spectrometer Model 3003. Radioactivity in amastigotes after extraction with cold TCA represents incorporation of label into macromolecules. Synthesis of nucleic acids was followed by determining the incorporation of [3H] thymidine and [3H] uridine into TCA insoluble materials. Amastigote suspensions were incubated with [3H] thymidine and [3H] uridine at a final concentration of 0.4 mM and specific radioactivity of 0.6 ACi/,umole. Samples of 0.5 ml were withdrawn at intervals, filtered rapidly through glass microfiber filters (1.2 ,um porosity; Whatman GF/C) and immediately washed with 3 ml cold 10% TCA. They were then rinsed with 1 ml ethanol (95%) and transferred to vials containing 10 ml Biofluor scintillation fluid (NEN) and the radioactivity determined. Protein was determined by the method of Oyama and Eagle (1956, Proceedings of the Society of Experimental Biology and Medicine 91: 305-307). Since amastigotes were found to carry out a variety of metabolic activities optimally at pH 5.0-5.5 (data not shown) these studies were conducted at pH 5.5. Label from glucose and proline were readily incorporated into TCA insoluble materials in a time dependent fashion (Fig. 1). Though details are not shown, trapping in methyl benzethonium hydroxide (Weiss et al., 1967, Nature 213: 1020-1022) showed the evolution of significant amounts of labelled CO2 from both substrates which clearly indicate that amasti-
Amastigotes (tissue forms) of Leishmania donovani isolated from infected hamster spleens carried out several physiological activities (respiration, catabolism of energy substrates, and incorporation of precursors into macromolecules) optimally at pH 4.0 to 5.5. All metabolic activities that were examined decreased sharply above the optimal pH. Promastigotes (culture forms), on the other hand, carried out the same metabolic activities optimally at or near neutral pH. This adaptation to an acid environment may account in part for the unusual ability of amastigotes to survive and multiply within the acidic environment of the phagolysosomes in vivo.
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