Rapid Shape Change and Release of Ninhydrin-Positive Substances by Leishmania major Promastigotes in Response to Hypo-Osnjotic Stress

ABSTRACT. Leishmania major promastigotes were grown to late-log phase and washed and resuspended in an isosmotic buffer. When osmolality was suddenly decreased by 50%, the cells rapidly became shorter and increased in width. Cell volume, calculated assuming a prolate-ellipsoidal shape, increased 1.4 times after 1 min. Over the next several minutes, the average length and width returned to control values while the volume returned to baseline, indicating the ability to regulate volume. Concomitantly with the swelling, large amounts of alanine and other ninhydrin-positive substances were released. All of the alanine pool was released within 1 min after reduction of the osmolality by 66%. Cells pre-loaded with [¹⁴C]-aminoisobutyric acid also released it very rapidly upon hypo-osmotic stress. Release of ninhydrin-positive substances resulted from decreased osmolality rather than changes in ionic composition. The same results were obtained if osmolality was decreased by reducing only the NaCl content of the buffer instead of diluting it with water, and mannitol could substitute for the NaCl. Promastigotes were able to grow well over several days in media as low as 154 mOsm/kg. The nature of the signalling mechanism(s) that initiates the rapid shape change and efflux of ninhydrin-positive substances in response to hypo-osmotic stress is at present unknown.     

Osmotic and Metabolic-Induced Changes in Light Scattering of Leishmania donovani as Measured by Flow Cytometry

Leishmania donovani promastigotes were collected from cultures in log and stationary phases of growth and resuspended in Hank's Balanced Salt Solution containing 1 mM sodium acetate. Changes in the forward and side scattering of the cells were measured by flow cytometry in response to acute changes in osmolality and to the addition of several different substrates. Forward and side scattering of cells from log phase cultures decreased when the osmolality was decreased by the addition of H₂O and increased when the osmolality was increased by the addition of NaCl. Cells from stationary phase cultures had about the same forward scatter as cells from log phase cultures, but almost a four-fold lower side scatter, and their side scatter values did not change significantly in response to a reduction in osmolality. Microscopic observation showed that both log and stationary cells got longer and thinner, on average, in response to hyperosmolality. The light scattering properties of log (but not of stationary) cells changed in a reproducible manner when substrates were added to the buffer. The ratio of forward to side scatter increased in the following order: controls in balanced salt solution >aspartate >glutamate, glucose or 2-deoxyglucose >alanine >proline. Thus the light scattering properties of L. donovani promastigotes change with culture age, in response to changes in osmolality, and, in log phase cells, in response to the presence of several substrates.     

Effects of Long-Term in Vitro Cultivation on Leishmania donovani Promastigotes

Promastigotes of Leishmania donovani that had been subcultued in modified Tobie's medium for 2 to 3 years showed decreased infectivity and lack of virulence for hamsters and mice compared to newly transformed promastigotes. Amastigotes derived from these long-term promastigote cultures decreased in number rapidly in hamsters, but only slightly in mice, over a 48-day period. In cultured mouse and hamster macrophags infected in vitro, amastigotes derived from long-term cultures rapidly decreased to low numbers, which persisted. The same pattern was seen in macriphages treated with catalase, an inhibitor of the oxygen-dependent killing mechanism of the macrophage. Promastigotes from long-term cultures also differed from virulent first-passage promastigotes in size, growth patterns in Tabie's medium, and in the quantities of some of their antigens.     

Evidence for Phosphorylation of the Extracellular Acid Phosphatase of Leishmania donovani1

The presence of two phosphorylated molecular species in the culture supernatants of axenically cultivated Leishmania donovani promastigotes was demonstrated by biosynthetically labeling cultures with [³²P]phosphate. One of these species was resolved into two bands with M_r's of 149,000 and 97,000 by dissociating polyacrylamide gel electrophoresis and copurified with the extracellular acid phosphatase activity produced by the promastigotes. The site of phosphorylation of the extracellular acid phosphatase is not yet known.     

高温酸性磷酸酶acp基因的克隆、表达和功能的初步鉴定

从高温塘泥(温度在40～85℃之间)中分离到一组嗜热微生物,提取环境基因组DNA,通过随机测序,发现许多基因与Thermus属的DNA相似性很高,根据Genbank上Thermus thermophilusHB8基因组序列中推定的酸性磷酸酶基因(TTHA1616),设计引物,PCR扩增得到一段798bp的DNA序列,测序结果与Thermus thermophilusHB8基因组中酸性磷酸酶基因相似性达98%,含有完整的酸性磷酸酶保守区,于是将该基因在大肠杆菌中表达,得到一条约33KD的特异蛋白条带,酶活检测证实该推定蛋白具有酸性磷酸酶活力。     

大肠杆菌酸性磷酸酶基因克隆表达及应用

将大肠杆菌K-12的酸性磷酸酶(AphA)完整基因和去信号肽基因分别克隆到pET-28a(+)栽体上,并转化入大肠杆菌BL21( DE3)中.经诱导检测,重组菌均能表达出高活性的可溶性酶蛋白,去信号肽表达更稳定.对重组菌的活性研究表明,相对于野生菌,重组菌酶活力得到大幅度提高,同时,以pNPP、肌苷为底物进行磷酸转移催化反应,在pH4.0-6.0、反应温度37℃条件下,约有30％的肌苷可转化为IMP,但随着反应的进行所形成的IMP又被该酶降解,向反应液中加入EDTA即可明显抑制酶的水解活性,减缓IMP的降解速率.     

Effects of Osmotic Pressure on the Oxidative Metabolism of Leishmania major Promastigotes

Leishmania major promastigotes were washed and resuspended in an iso-osmotic buffer. The rate of oxidation of ¹⁴C-labeled substrates was then measured as a function of osmolality. An acute decrease in osmolality (achieved by adding H₂O to the cell suspension) caused an increase in the rates of ¹⁴CO₂ production from [6-¹⁴C]glucose and, to a lesser extent, from [1, (3)-¹⁴C]glycerol. An acute increase in osmolality (achieved by adding NaCl, KCl, or mannitol) strongly inhibited the rates of ¹⁴CO₂ production from [1-: ¹⁴C]alanine, [1-¹⁴C]glutamate, and [1, (3)-¹⁴C]glycerol. The rates of ¹⁴CO₂ formation from [1-¹⁴C]laurate, [1-¹⁴C]acetate, and [2-¹⁴C]glucose (all of which form [1-¹⁴C]acetyl CoA prior to oxidation) were also inhibited, but less strongly, by increasing osmolality. These data suggest that with increasing osmolality there is an inhibition of mitochondrial oxidative capacity, which could facilitate the increase in alanine pool size that occurs in response to hyper-osmotic stress. Similarly, an increase in oxidative capacity would help prevent a rebuild up of the alanine pool after its rapid loss to the medium in response to hypo-osmotic stress.     

An 18 kDa Acid Phosphatase from Chicken Heart Possesses Phosphotransferase Activity

A low molecular weight acid phosphatase was purified to homogeneity from chicken heart with a specific activity of 42 U/mg and a recovery of about 1%. Nearly 800 fold purification was achieved. The molecular weight was estimated to be 18 kDa by SDS-polyacrylamide gel electrophoresis. Para-nitrophenyl phosphate, phenyl phosphate and flavin mononucleotide were efficiently hydrolysed by the enzyme and found to be good substrates. Fluoride and tartrate had no inhibitory effect while phosphate, vanadate and molybdate strongly inhibited the enzyme. The acid phosphatase was stimulated in the presence of glycerol, ethylene glycol, methanol, ethanol and acetone, which reflected the phosphotransferase activity. When phosphate acceptors such as ethylene glycol concentrations were increased, the ratio of phosphate transfer to hydrolysis was also increased, demonstrating the presence of a transphosphorylation reaction where an acceptor can compete with water in the rate limiting step involving hydrolysis of a covalent phospho enzyme intermediate. Partition experiments carried out with two substrates, para-nitrophenyl phosphate and phenyl phosphate, revealed a constant product ratio of 1.7 for phosphotransfer to ethylene glycol versus hydrolysis, strongly supporting the existence of common covalent phospho enzyme intermediate. A constant ratio of K _cat/K _m, 4.3×10⁴, found at different ethylene glycol concentrations, also supported the idea that the rate limiting step was the hydrolysis of the phospho enzyme intermediate.     

磷胁迫下大豆酸性磷酸酶活性变化及磷效率基因型差异分析

在3种磷水平处理后,对23个大豆品种的叶片和根尖酸性磷酸酶活性、根冠比、干物质量、全磷含量及磷效率进行测定、比较和分析。结果表明,叶片或根尖酸性磷酸酶活性、叶片或根尖酸性磷酸酶活性相对值、根冠比,磷效率、磷效率相对值之间具有极显著差异(P相似文献   

Characterization and Purification of Acid Phosphatase from Ancient Human Bone

Abstract

In this research, acid phosphatase was purified and characterized from approximately 3000-year-old human bones from archeological excavations. Using anion exchange chromatography, two isoenzymes, TrACP and TsACP, were isolated from the bone. TrACP and TsACP were eluted separately, with a concentration gradient, from a CM-sepharose column. The resulting TrACP was further purified on a cellulose phosphate column. The activity was determined by using pNPP as substrate. Additionally, protein was determined by the Bradford and Coomassie Brilliant Blue method. The optimum pHs of TsACP and TrACP were 6 and 5, respectively. The optimum temperatures were 0 and 10°C, respectively. Molecular weights were measured by gel filtration chromatography. The isoenzyme purity was checked with SDS-PAGE. Finally, the effects of sodium molybdate and tartrate on isoenzyme activity were determined.     

Infectivity of Leishmania donovani Amastigotes and Promastigotes for Golden Hamsters*

SYNOPSIS. Intracardial injection of hamsters with from 5 to 114 million amastigotes or promastigotes of Leishmania donovani and screening of the 8th-day liver impression smears, provides a rapid and reproducible method for assaying infectivity. Amastigotes are at least 10X more infective than promastigotes, and log-phase promastigotes act as a single infective population for hamsters.     

Ultrastructural Localization of Acid Phosphatase Activity in Soybean Cotyledon Cells

In the cotyledon cells of the developing seeds (35～50 d after flowering) and the early germinating seeds (4 ～ 8 d after sowing) of soybean (Glycine max L. ), acid phosphatase (APase) activity was mainly deposited in the protein bodies (PB) and in endoplasmic reticulum (ER). In addition, in the early developing cotylendon cells, the prominent reaction product of APase activity was seen along the plasma membrane, in the cell wall and within the vesicles in the cytoplasm adjancent to the plasma membrane. And some of the vesicles seemed to be fused with the plasma membrane.     

Deprivation of L-Arginine Induces Oxidative Stress Mediated Apoptosis in Leishmania donovani Promastigotes: Contribution of the Polyamine Pathway

The growth and survival of intracellular parasites depends on the availability of extracellular nutrients. Deprivation of nutrients viz glucose or amino acid alters redox balance in mammalian cells as well as some lower organisms. To further understand the relationship, the mechanistic role of L-arginine in regulation of redox mediated survival of Leishmania donovani promastigotes was investigated. L-arginine deprivation from the culture medium was found to inhibit cell growth, reduce proliferation and increase L-arginine uptake. Relative expression of enzymes, involved in L-arginine metabolism, which leads to polyamine and trypanothione biosynthesis, were downregulated causing decreased production of polyamines in L-arginine deprived parasites and cell death. The resultant increase in reactive oxygen species (ROS), due to L-arginine deprivation, correlated with increased NADP+/NADPH ratio, decreased superoxide dismutase (SOD) level, increased lipid peroxidation and reduced thiol content. A deficiency of L-arginine triggered phosphatidyl serine externalization, a change in mitochondrial membrane potential, release of intracellular calcium and cytochrome-c. This finally led to DNA damage in Leishmania promastigotes. In summary, the growth and survival of Leishmania depends on the availability of extracellular L-arginine. In its absence the parasite undergoes ROS mediated, caspase-independent apoptosis-like cell death. Therefore, L-arginine metabolism pathway could be a probable target for controlling the growth of Leishmania parasites and disease pathogenesis.     

Effect of Hyper-Osmotic Stress On Alanine Content of Leishmania Major Promastigotes

ABSTRACT Earlier studies showed that Leishmania major promastigotes are sensitive to osmotic conditions. A reduction in osmolality caused the cells to shorten and to rapidly release most of their large internal pool of alanine. In this study some effects of hyper-osmotic stress were examined. an increase in osmolality of the culture medium from 308 to 625 mOsm/kg caused only a small decrease in growth rate. When cells grown in the usual culture medium (308 mOsm/kg) were washed, resuspended in iso-osmotic buffer, and subjected to acute hyper-osmotic stress by addition of mannitol, the alanine content increased even in the absence of exogenous substrate. Promastigotes, depleted of alanine by a 5-min exposure to hypo-osmotic conditions, also synthesized alanine when resuspended in iso-osmotic buffer. Washed cells resuspended in iso-osmotic buffer consume their internal pool of alanine under aerobic conditions, Rates of consumption decreased on addition of mannitol, becoming zero at about 440 mOsm/kg. At higher osmolalities, alanine synthesis occurred. to estimate whether proteolysis could account for alanine synthesis in the absence of exogenous substrate, cells that had been grown with [1-¹⁴C]leucine were washed and resuspended under hypo-, iso-, and hyper-osmotic conditions and the amounts of ¹⁴CO₂ and ¹⁴C-labelled peptides released in 1 h were measured. Little proteolysis occurred under these conditions, but the possibility that proteolysis was the source of the alanine increase, observed in response to hyper-osmotic stress, cannot be ruled out.     

Inhibition of Acid Phosphatase Isoforms Purified from Mature Soybean (Glyczne MAX) Seeds

The four soybean seed acid phosphatase isoforms AP1, AP2, AP3A and AP3B were competitively inhibited by phosphate, vanadate, fluoride and molybdate, using p-nitrophenylphos-phate as substrate. The four isoforms were not significantly affected by compounds that can interact with SH residues or by pyridoxal phosphate. These results indicated that cysteine and lysine residues are not present in the active site of the four soybean seed acid phosphatase isoforms. The inhibition constant values for phosphate, vanadate, fluoride and molybdate at pH 5.0 were respectively: API (250, 12.8, 1.7, 0.05 μM), AP2 (800,10, 500, 0.025 μM), AP3A (250, 24.2,250, 0.032 μM), AP3B (2400, 36.9,750, 0.05 μM).     

酵母酸性磷酸酯酶转录调控因子PHO2的功能域分析

利用定点诱变,氨基酸插入或缺失的方法对ＰＨＯ２进行结构改造,观察对其功能的影响。ＰＨＯ２蛋白的同源域中有α－螺旋２－β－转我－α螺旋３的结构,是转录因子结构ＤＮＡ的功能域。定点诱变α－螺旋３上的Ｉｌｅ１２３为Ｐｒｏ１２３或者在α－螺旋２中插入ＰＤＰＤ４个氨基酸,破坏α－螺旋的结构,可导致ＰＨＯ２功能的丧失。氨基酸片段的缺失分析表明,Ｎ端Ｇｉｎ丰富区大部缺失,对ＰＨＯ２功能没有影响;而包含酸性氨基酸     

The Ultracytochemical Localzation of Acid Phosphatase Activity in Wheat During Fertilization

No acid phosphatase activity was observed in the mature embryo sac of wheat (Triticum aestivum) except the chalazal cytoplasm Of the central cell before fertilization. During fertilization, acid phosphataseactivity was observed in the following loci: part of chromatin of the egg nucleus and most of the mitochondria in the egg cytoplasm; the perinuclear spaces of the egg and sperm nuclei at the fusion of the egg and sperm nuclei; the chalazal cytoplasm and some vacuoles of the degenerated synergid; two sperm nuclei within the cytoplasm of female cells; the cell wall of each cell of the embryo sac and that of the nucellar cells surrounding the embryo sac. No acid phosphatase was observed in the two-celled proembryo. Dense enzyme reaction product was localized in the chromatin of the free nuclei at early stage of the endosperm. The characteristic of acid phosphatase distribution during fertilization may be associated with the physiological change of the egg Cell, the reorganization of mitochondria in the egg cell cytoplasm, the degeneration of one of the two synergids, the physiological state of the sperm nuclei and the nuclear membrane fusion of the egg and sperm nuclei.     

墨兰菌根的结构及酸性磷酸酶定位研究

利用光学显微镜、电子显微镜及细胞化学方法,对墨兰菌根的结构和酸性磷酸酶定位进行了初步研究。结果表明墨兰具有典型的兰科植物根结构,发现该兰花的根的外皮层不具薄壁通道细胞,菌根真菌通过破坏部分根被和外皮层细胞而侵入根的皮层细胞并在细胞内形成菌丝结,侵入的菌丝被染菌皮层细胞质膜和电子透明物质包围,进一步被消化并聚集成衰败菌丝团块。酸性磷酸酶在染菌皮层细胞及包围菌丝的皮层细胞质膜和衰败菌丝细胞壁上有强烈的酶反应,衰败菌丝周围分布有许多单层膜的含酶小泡,它们可相互愈合形成大的含酶泡或与包围菌丝的质膜融合,类似于兰科植物共生原球茎中观察到的现象。说明皮层细胞可主动释放水解酶参与对菌丝的消化     

杜仲次生木质部分化和脱分化过程中酸性磷酸酶的超微细胞化学定位

杜仲（EucommiaulmoidesOliv．）次生木质部分化过程中,在形成层刚衍生的木薄壁细胞中,酸性磷酸酶（APase）主要分布于核膜边缘和高尔基体;在分化程度较高的木薄壁细胞中,APase散布于整个核中,进而,在各种细胞器残体上聚集;在成熟的木薄壁细胞中,APase沿细胞壁内侧分布。在未成熟导管分子中,核、质膜及纹孔上明显存在APase聚集,进而,核解体;在即将分化成熟的导管分子中,APase主要集中于初生壁;在已分化成熟的导管分子中,APase集中于次生壁。脱分化过程中,只在细胞质中可见分散的APase活性,而细胞核和细胞壁上未见此酶的分布;更深层的即将分化成熟和已分化成熟的导管分子,未见有细胞分裂,其上APase的分布与剥皮前相同。通过比较分化和脱分化过程中APase的分布,推测不同的APase同工酶可能分别参与了次生木质部细胞程序性死亡过程中原生质体的解体和次生壁的建成。APase的聚集程度可能是决定细胞能否脱分化的一个重要特征。     

Distribution of Macromolecular Methylations in Promastigotes of Leishmania donovani and Impact of Sinefungin

ABSTRACT. Sinefungin, an antifungal and antiparasitic nucleoside antibiotic, is a very potent antileishmanial agent both in vitro and in vivo. This molecule, structurally related to S-adenosylmethionine, is a good competitive inhibitor of methyltransferases in vitro. The aim of this report is to analyze the impact of sinefungin on methylation pattern and the subcellular localisation of methyl groups and various methylases in promastigotes of Leishmania donovani . We have shown the presence of various methylated macromolecules in different subcellular fractions, with somewhat higher concentration in membrane fraction. In vitro, sinefungin inhibits the three main protein methylases, but in cells cultured in its presence the protein carboxylmethylations are specifically inhibited.