首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献,搜索用时 15 毫秒
1.
ABSTRACT Leishmania donovani promastigotes were grown to late log phase, washed and resuspended in iso-osmotic buffer containing L-arginine, and the rate of urea formation was then measured under various conditions. Addition of glucose or mannose activated urea formation, whereas 2-deoxyglucose inhibited and 6-deoxyglucose had no effect. Addition of alanine or of α -aminoisobutyrate inhibited urea formation, alanine causing a greater inhibition than α -aminoisobutyrate. Addition of leucine, proline, glycine, or lysine had no effect on urea formation. The presence of glutamate also increased the rate of urea formation from arginine, but to a lesser extent than did glucose. The presence of both glucose and alanine caused no net change in urea formation, whereas the inhibitory effect of alanine exceeded the activating effect of glutamate, so that a small inhibition in the rate of urea formation occurred in the presence of both alanine and glutamate. Cells grown to 3-day stationary phase had a markedly reduced rate of arginine catabolism to urea, but the activating effect of glucose and the inhibitory effect of alanine were qualitatively similar to their effects on late log phase cells. Addition of water to cells suspended in buffer also inhibited urea formation, but this appeared to be due primarily to the release of alanine caused by the hypo-osmotic stress. Addition of mannitol to cells suspended in buffer caused a small inhibition of arginine catabolism. Addition of dibutyrylcyclic AMP, 3',5'-cyclic GMP, phorbol myristic acid, or A23187 had no effect on the rate of urea formation from arginine. It is suggested that the effects of glucose and 2-deoxyglucose on arginine catabolism depend largely upon the nature of their metabolites, whereas the effects of the various amino acids examined depend largely on the extent to which they interfere with or enhance arginine transport into the cells.  相似文献   

2.
The metabolism of leucine by Leishmania donovani was investigated. Washed promastigotes were incubated with [1-14C]- or [U-14C]leucine or [1-14C]alpha-ketoisocaproate (KIC) and 14CO2 release was measured. The amount of KIC-derived acetyl-CoA oxidized in the citric acid cycle was computed. Promastigotes from mid-stationary phase cultures oxidized each of these labeled substrates less rapidly than cells from late log phase cultures, and significantly less acetyl-CoA derived from KIC oxidation was oxidized in the citric acid cycle. Glucose was a stronger inhibitor than was acetate of CO2 formation in the citric acid cycle in log phase promastigotes, but the reverse was observed in cells from mid-stationary phase. Alanine also inhibited leucine catabolism, but glutamate had little effect. Acute hypo-osmotic stress did not affect leucine catabolism, but hyper-osmotic stress caused appreciable inhibition of leucine oxidation. Cells grown under hypo- or hyper-osmotic conditions showed no changes in the effects of hypo- or hyper-osmotic stress on leucine catabolism, i.e. L. donovani is not an osmoconformer with respect to leucine metabolism. Leucine utilization in L. donovani was insensitive to a number of drugs that affect leucine metabolism in mammalian cells, indicating that the leucine pathway in L. donovani is not regulated in the same manner as in mammalian cells.  相似文献   

3.
The present study concerns RNA metabolism of Leishmania donovani and its changes during exposure to higher temperatures. The incorporation of labeled precursors into RNA was found to be greatly decreased during incubation at 37 °C in comparison with that at the optimum temperature of growth (22 °C). The decreased incorporation was shown to be mainly due to augmented template RNA degradation. The RNA-polymerase activity remained unaffected at the elevated temperature, but the uptake of labeled uracil was markedly inhibited.  相似文献   

4.
ABSTRACT. Metabolism of tryptophan by promastigotes of Leishmania donovani donovani was investigated in cells suspended in a simple buffer solution supplemented with glucose. Metabolites from supernatant and lysed cell pellets were analyzed by capillary gas liquid chromatography and 13C nuclear magnetic resonance spectroscopy, with structural confirmation by gas liquid chromatography-mass spectrometry. Tryptophan does not appear to serve as a carbon energy source for L. d. donovani promastigotes since parasites could survive for only short periods in buffer containing tryptophan without glucose, levels of tricarboxylic acid cycle intermediates remained unchanged in the presence of added tryptophan and label from [13C]tryptophan was not detected in any of the intermediates. Leishmania d. donovani catabolized l -tryptophan via aminotransferase and aromatic lactate dehydrogenase reactions to form one major end product, indole-3-lactic acid. The activity of aromatic lactate dehydrogenase required manganese and was NADH-dependent in these organisms that lack lactate dehydrogenase. Promastigotes taken from the mid-log stage of growth produced higher concentrations of indole-3-lactic acid than those from the stationary stage. Conservation of a similar tryptophan catabolic pathway among four Leishmania species suggests the pathway is physiologically important to the parasites themselves.  相似文献   

5.
The protozoan parasite Leishmania donovani encounters large fluctuations in osmolality as it cycles between its insect vector and human host. The flagellated promastigote exhibits regulatory volume responses involving organic and inorganic osmolytes, but little is known about volume regulation in the clinically relevant amastigote that multiplies within the parasitophorous vacuoles of mammalian host cells. Using a combination of morphological, X-ray microanalytical, and biochemical approaches we determined that non-motile amastigotes respond to hypotonic stress with (1) an amino acid and l-alanine-mediated regulatory volume decrease, and (2) a parallel release of Na+, K+, P (presumably as negatively charged phosphates), and subsequently Cl- from cytoplasm and the cell as a whole. In addition P, Zn2+, and subsequently Ca2+ increase in acidocalcisomes as Cl- content declines in this compartment. This evidence is the first to document subcellular translocation of, and thus a potential role for, zinc in volume regulatory responses. These coordinated changes in organic and inorganic osmolytes demonstrate that amastigote subcellular compartments, particularly acidocalcisomes, function in maintaining ionic homeostasis in the response of Leishmania amastigotes to hypo-osmotic stress.  相似文献   

6.
ABSTRACT. Leishmania donovani promastigotes were collected, washed, resuspended in buffer, and assayed for sucrase activity. No activity was observed in the intact washed cells, but activity was measurable when the cells were permeabilized with Triton X-100. Intracellular sucrase activity was highest in promastigotes grown at pH 7.4, somewhat lower in promastigotes grown at pH 5.5, and significantly lower in "amastigotes" grown at pH 5.5. No trehalase, lactase, or maltase activities were observed. Assay of the medium in which the cells had grown showed that most the sucrase activity was extracellular, i.e. was secreted into the medium during growth.  相似文献   

7.
Protozoan parasites of the Leishmania genus express the metabolic machinery to synthesize pyrimidine nucleotides via both de novo and salvage pathways. To evaluate the relative contributions of pyrimidine biosynthesis and salvage to pyrimidine homeostasis in both life cycle stages of Leishmania donovani, individual mutant lines deficient in either carbamoyl phosphate synthetase (CPS), the first enzyme in pyrimidine biosynthesis, uracil phosphoribosyltransferase (UPRT), a salvage enzyme, or both CPS and UPRT were constructed. The Δcps lesion conferred pyrimidine auxotrophy and a growth requirement for medium supplementation with one of a plethora of pyrimidine nucleosides or nucleobases, although only dihydroorotate or orotate could circumvent the pyrimidine auxotrophy of the Δcps/Δuprt double knockout. The Δuprt null mutant was prototrophic for pyrimidines but could not salvage uracil or any pyrimidine nucleoside. The capability of the Δcps parasites to infect mice was somewhat diminished but still robust, indicating active pyrimidine salvage by the amastigote form of the parasite, but the Δcps/Δuprt mutant was completely attenuated with no persistent parasites detected after a 4-week infection. Complementation of the Δcps/Δuprt clone with either CPS or UPRT restored infectivity. These data establish that an intact pyrimidine biosynthesis pathway is essential for the growth of the promastigote form of L. donovani in culture, that all uracil and pyrimidine nucleoside salvage in the parasite is mediated by UPRT, and that both the biosynthetic and salvage pathways contribute to a robust infection of the mammalian host by the amastigote. These findings impact potential therapeutic design and vaccine strategies for visceral leishmaniasis.  相似文献   

8.
Metabolism of tryptophan by promastigotes of Leishmania donovani donovani was investigated in cells suspended in a simple buffer solution supplemented with glucose. Metabolites from supernatant and lysed cell pellets were analyzed by capillary gas liquid chromatography and 13C nuclear magnetic resonance spectroscopy, with structural confirmation by gas liquid chromatography-mass spectrometry. Tryptophan does not appear to serve as a carbon energy source for L. d. donovani promastigotes since parasites could survive for only short periods in buffer containing tryptophan without glucose, levels of tricarboxylic acid cycle intermediates remained unchanged in the presence of added tryptophan and label from [13C]tryptophan was not detected in any of the intermediates. Leishmania d. donovani catabolized L-tryptophan via aminotransferase and aromatic lactate dehydrogenase reactions to form one major end product, indole-3-lactic acid. The activity of aromatic lactate dehydrogenase required manganese and was NADH-dependent in these organisms that lack lactate dehydrogenase. Promastigotes taken from the mid-log stage of growth produced higher concentrations of indole-3-lactic acid than those from the stationary stage. Conservation of a similar tryptophan catabolic pathway among four Leishmania species suggests the pathway is physiologically important to the parasites themselves.  相似文献   

9.
Adenine aminohydrolase (AAH) is an enzyme that is not present in mammalian cells and is found exclusively in Leishmania among the protozoan parasites that infect humans. AAH plays a paramount role in purine metabolism in this genus by steering 6-aminopurines into 6-oxypurines. Leishmania donovani AAH is 38 and 23% identical to Saccharomyces cerevisiae AAH and human adenosine deaminase enzymes, respectively, catalyzes adenine deamination to hypoxanthine with an apparent K(m) of 15.4 μM, and does not recognize adenosine as a substrate. Western blot analysis established that AAH is expressed in both life cycle stages of L. donovani, whereas subcellular fractionation and immunofluorescence studies confirmed that AAH is localized to the parasite cytosol. Deletion of the AAH locus in intact parasites established that AAH is not an essential gene and that Δaah cells are capable of salvaging the same range of purine nucleobases and nucleosides as wild type L. donovani. The Δaah null mutant was able to infect murine macrophages in vitro and in mice, although the parasite loads in both model systems were modestly reduced compared with wild type infections. The Δaah lesion was also introduced into a conditionally lethal Δhgprt/Δxprt mutant in which viability was dependent on pharmacologic ablation of AAH by 2'-deoxycoformycin. The Δaah/Δhgprt/Δxprt triple knock-out no longer required 2'-deoxycoformycin for growth and was avirulent in mice with no persistence after a 4-week infection. These genetic studies underscore the paramount importance of AAH to purine salvage by L. donovani.  相似文献   

10.
Leishmania donovani promastigotes were grown to late log phase, washed and resuspended in iso-osmotic buffer containing L-arginine, and the rate of urea formation was then measured under various conditions. Addition of glucose or mannose activated urea formation, whereas 2-deoxyglucose inhibited and 6-deoxyglucose had no effect. Addition of alanine or of alpha-aminoisobutyrate inhibited urea formation, alanine causing a greater inhibition than alpha-aminoisobutyrate. Addition of leucine, proline, glycine, or lysine had no effect on urea formation. The presence of glutamate also increased the rate of urea formation from arginine, but to a lesser extent than did glucose. The presence of both glucose and alanine caused no net change in urea formation, whereas the inhibitory effect of alanine exceeded the activating effect of glutamate, so that a small inhibition in the rate of urea formation occurred in the presence of both alanine and glutamate. Cells grown to 3-day stationary phase had a markedly reduced rate of arginine catabolism to urea, but the activating effect of glucose and the inhibitory effect of alanine were qualitatively similar to their effects on late log phase cells. Addition of water to cells suspended in buffer also inhibited urea formation, but this appeared to be due primarily to the release of alanine caused by the hypo-osmotic stress. Addition of mannitol to cells suspended in buffer caused a small inhibition of arginine catabolism. Addition of dibutyrylcyclic AMP, 3',5'-cyclic GMP, phorbol myristic acid, or A23187 had no effect on the rate of urea formation from arginine.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

11.
12.
13.
Hemophagocytosis is a phenomenon in which macrophages phagocytose blood cells. There are reports on up-regulated hemophagocytosis in patients with infectious diseases including typhoid fever, tuberculosis, influenza and visceral leishmaniasis (VL). However, mechanisms of infection-associated hemophagocytosis remained elusive due to a lack of appropriate animal models. Here, we have established a mouse model of VL with hemophagocytosis. At 24 weeks after infection with 1 x 107 Leishmania donovani promastigotes, BALB/cA mice exhibited splenomegaly with an average tissue weight per body weight of 2.96%. In the tissues, 28.6% of macrophages contained phagocytosed erythrocytes. All of the hemophagocytosing macrophages were parasitized by L. donovani, and higher levels of hemophagocytosis was observed in heavily infected cells. Furthermore, more than half of these hemophagocytes had two or more macrophage-derived nuclei, whereas only 15.0% of splenic macrophages were bi- or multi-nuclear. These results suggest that direct infection by L. donovani causes hyper-activation of host macrophages to engulf blood cells. To our knowledge, this is the first report on hemophagocytosis in experimental Leishmania infections and may be useful for further understanding of the pathogenesis.  相似文献   

14.
Protozoan parasites of the genus Leishmania adapt to their arthropod and vertebrate hosts through the development of defined life cycle stages. Stage differentiation is triggered by environmental stress factors and has been linked to parasite chaperone activities. Using a null mutant approach we previously revealed important, nonredundant functions of the cochaperone cyclophilin 40 in L. donovani‐infected macrophages. Here, we characterized in more detail the virulence defect of cyp40?/? null mutants. In vitro viability assays, infection tests using macrophages, and mixed infection experiments ruled out a defect of cyp40?/? parasites in resistance to oxidative and hydrolytic stresses encountered inside the host cell phagolysosome. Investigation of the CyP40‐dependent proteome by quantitative 2D‐DiGE analysis revealed up regulation of various stress proteins in the null mutant, presumably a response to compensate for the lack of CyP40. Applying transmission electron microscopy we showed accumulation of vesicular structures in the flagellar pocket of cyp40?/? parasites that we related to a significant increase in exosome production, a phenomenon previously linked to the parasite stress response. Together these data suggest that cyp40?/? parasites experience important intrinsic homeostatic stress that likely abrogates parasite viability during intracellular infection.  相似文献   

15.
Leishmania are auxotrophic for purines, and consequently purine acquisition from the host is a requisite nutritional function for the parasite. Both adenylosuccinate synthetase (ADSS) and adenylosuccinate lyase (ASL) have been identified as vital components of purine salvage in Leishmania donovani, and therefore Δadss and Δasl null mutants were constructed to test this hypothesis. Unlike wild type L. donovani, Δadss and Δasl parasites in culture exhibited a profoundly restricted growth phenotype in which the only permissive growth conditions were a 6-aminopurine source in the presence of 2′-deoxycoformycin, an inhibitor of adenine aminohydrolase activity. Although both knock-outs showed a diminished capacity to infect murine peritoneal macrophages, only the Δasl null mutant was profoundly incapacitated in its ability to infect mice. The enormous discrepancy in parasite loads observed in livers and spleens from mice infected with either Δadss or Δasl parasites can be explained by selective accumulation of adenylosuccinate in the Δasl knock-out and consequent starvation for guanylate nucleotides. Genetic complementation of a Δasl lesion in Escherichia coli implied that the L. donovani ASL could also recognize 5-aminoimidazole-(N-succinylocarboxamide) ribotide as a substrate, and purified recombinant ASL displayed an apparent Km of ∼24 μm for adenylosuccinate. Unlike many components of the purine salvage pathway of L. donovani, both ASL and ADSS are cytosolic enzymes. Overall, these data underscore the paramount importance of ASL to purine salvage by both life cycle stages of L. donovani and authenticate ASL as a potential drug target in Leishmania.  相似文献   

16.
Characterization of strains of Leishmania donovani   总被引:11,自引:0,他引:11  
  相似文献   

17.
18.
Characterization of Leishmania donovani acid phosphatases   总被引:7,自引:0,他引:7  
A crude membrane fraction from promastigotes of Leishmania donovani grown in a liquid culture medium containing 20% fetal calf serum was prepared by freeze-thawing, centrifugation (200,000 X g, 30 min), and extraction with 2% (w/v) sodium cholate. After removal of the bile salt by chromatography on a Sephadex G-75 column, the solubilized membrane protein fraction, rich in acid phosphatase activity, was chromatographed on columns containing concanavalin A-Sepharose, QAE-Sephadex, and Sephadex G-150 and G-100. Three distinct acid phosphatases were resolved: the major phosphatase activity (70% of the total) was L-(+)-tartrate-resistant (designated ACP-P1) and corresponds to the acid phosphatase localized to the outer surface of the parasite's plasma membrane; the other two phosphatases (ACP-P2 and ACP-P3) account for the remaining 30% of the particulate acid phosphatase activity, and both of these enzymes are L-(+)-tartrate-sensitive. Using a combination of sucrose density gradient centrifugation, gel filtration chromatography, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, it was determined that ACP-P1 is a 128,000-dalton protein composed of two subunits of 65,000-68,000 daltons. ACP-P1 has an isoelectric point of 4.1, a pH optimum of 5.5, hydrolyzes fructose 1,6-diphosphate, but no other sugar phosphates and dephosphorylates phosphotyrosine, yeast mannan, and the phosphorylated form of rat liver pyruvate kinase. ACP-P2 (pI, 5.4) and ACP-P3 (pI, 7.1) with molecular masses of 132,000 and 108,000 daltons, respectively, are both tartrate-sensitive and are distinguished from each other on the basis of their sensitivity to inhibition by polyanionic molybdenum complexes. These two phosphatases also have their pH optima in the pH 5.0-6.0 range, but have a considerably broader substrate specificity than ACP-P1.  相似文献   

19.
SYNOPSIS. Peritoneal macrophages from hamsters were monolayered on coverslips in Leighton tubes. Twenty-four hours later these were transferred to a perfusion chamber. Leptomonads were added with fresh medium and the infection process observed with the aid of phase contrast. In the perfusion chamber free-swimming leptomonads attached to the macrophage by the tip of their flagella. Shortly after this initial attachment the macrophage extended a narrow pseudopodium around the flagellum which eventually reached and enveloped the body of the parasite. Upon complete envelopment the pseudopod containing the leptomonad was retracted into the central body of the macrophage. When first seen in the granular endoplasm of the macrophage, most of the leptomonads appeared to be surrounded by vacuoles. In most cases these vacuoles disappeared in a few minutes making it difficult to distinguish the parasite from the host cell cytoplasm.
Leptomonads also were added directly to Leighton tube cultures, and the coverslips with the adherent macrophages and parasites were removed, fixed and stained periodically during the infection process. In these preparations most of the parasites were in clumps in the vicinity of macrophages. Details of the ingestion of the clumps could not be seen, but occasionally a single organism was seen with its flagellum and part of its body enclosed by an extended pseudopod. Most of the intracellular leptomonads were in large vacuoles. Forms intermediate between elongate leptomonads and LD bodies were surrounded by smaller vacuole-like spaces. The halo-like vacuoles most frequently seen around LD bodies may have been fixation artifacts. Under favorable conditions the leptomonads transformed to LD bodies in 1–4 hours, but it was 48 hours before a population increase could be found.  相似文献   

20.
The pathogenic protozoan parasite Leishmania donovani is capable of both de novo pyrimidine biosynthesis and salvage of pyrimidines from the host milieu. Genetic analysis has authenticated L. donovani uracil phosphoribosyltransferase (LdUPRT), an enzyme not found in mammalian cells, as the focal enzyme of pyrimidine salvage because all exogenous pyrimidines that can satisfy the requirement of the parasite for pyrimidine nucleotides are funneled to uracil and then phosphoribosylated to UMP in the parasite by LdUPRT. To characterize this unique parasite enzyme, LdUPRT was expressed in Escherichia coli, and the recombinant enzyme was purified to homogeneity. Kinetic analysis revealed apparent Km values of 20 and 99 μm for the natural substrates uracil and phosphoribosylpyrophosphate, respectively, as well as apparent Km values 6 and 7 μm for the pyrimidine analogs 5-fluorouracil and 4-thiouracil, respectively. Size exclusion chromatography revealed the native LdUPRT to be tetrameric and retained partial structure and activity in high concentrations of urea. L. donovani mutants deficient in de novo pyrimidine biosynthesis, which require functional LdUPRT for growth, are hypersensitive to high concentrations of uracil, 5-fluorouracil, and 4-thiouracil in the growth medium. This hypersensitivity can be explained by the observation that LdUPRT is substrate-inhibited by uracil and 4-thiouracil, but 5-fluorouracil toxicity transpires via an alternative mechanism. This substrate inhibition of LdUPRT provides a protective mechanism for the parasite by facilitating purine and pyrimidine nucleotide pool balance and by sparing phosphoribosylpyrophosphate for consumption by the nutritionally indispensable purine salvage process.  相似文献   

设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号