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1.
L5178Y mouse lymphoma cells normally appear to possess two functional thymidine kinase alleles (TK +/+). TK-deficient (TK ?/?) clonal lines can be derived from these cells by treatment with EMS or other mutagens. Mezger-Freed [12] has argued that such stable phenotypic variants do not arise as the result of gene mutations but instead represent epigenetic events such as normally occur during differentiation without any permanent gene alteration. If this be so, then rare TK +/? revertants arising in TK ?/? cultures should possess TK enzyme identical with one of those present in the original TK +/+ cells, since only depression of the TK gene is involved. Our studies show that this is not the case.Among the mutant TK enzymes analyzed in vitro (those from parental TK +/? lines, each derived in turn from separate TK ?/? lines) differences were found in (1) solubility in saline; (2) solubility in3 M LiCl; (3) Km′s; and (4) ATP-Mg 2+ requirements. These findings were incompatible with a non-mutational model for the production of these stable variants and, in conjunction with reversion-rate data, they tended to favor either direct structural gene modifications or mutations affecting the expression of adult and fetal enzymes. 相似文献
2.
We measured the toxicity and mutagenicity induced in human diploid lymphoblasts by various radiation doses of X-rays and two internal emitters. [ 125I]iododeoxyuridine ([ 125I]dUrd) and [ 3H]thymidine ([ 3H]TdR), incorporated into cellular DNA. [ 125I]dUrd was more effective than [ 3H]TdR at killing cells and producing mutations to 6-thioguanine resistance (6TG R). No ouabain-resistant mutants were induced by any of these agents. Expressing dose as total disintegrations per cell (dpc), the D0 for cell killing for [ 125I]dUrd was 28 dpc and for [ 3H]TdR was 385 dpc. The D0 for X-rays was 48 rad at 37°C. The slopes of the mutation curves were approximately 75 × 10 −8 6TG R mutants per cell per disintegration for [ 125I]dUrd and 2 × 10 −8 for [ 3H]TdR. X-Rays induced 8 × 10 −8 6TG R mutants per cell per rad. Normalizing for survival, [ 125I]dUrd remained much more mutagenic at low doses (high survival levels) than the other two agents. Treatment of the cells at either 37°C or while frozen at −70°C yielded no difference in cytotoxicity or mutation for [ 125I]dUrd or [ 3H]TdR, whereas X-rays were 6 times less effective in killing cells at −70°C. Assuming that incorporation was random throughout the genome, the mutagenic efficiencies of the radionuclides could be calculated by dividing the mutation rate by the level of incorporation. If the effective target size of the 6TGR locus is 1000–3000 base pairs, then the mutagenic efficiency of [125I]dUrd is 1.0–3.0 and of [3H]TdR is 0.02–0.06 total genomic mutations per cell per disintegration. 125I disintegrations are known to produce localized DNA double-strand breaks. If these breaks are potentially lethal lesions, they must be repaired, since the mean lethal dose (D0) was 28 dpc. The observations that a single dpc has a high probability of producing a mutation (mutagenic efficiency 1.0–3.0) would suggest, however, that this repair is extremely error-prone. If the breaks need not be repaired to permit survival, then lethal lesions are a subset of or are completely different from mutagenic lesions. 相似文献
3.
Herpes simplex virus thymidine kinase (HSV-TK) gene and dendritic cells (DC) have been used as the pioneering in cancer therapy. HSV-TK gene can induce apoptosis and necrosis in tumor cells in the presence of the non-toxic prodrug ganciclovir (GCV). We investigated the anti-tumor effect of DC vaccination by introducing dying cells from HSV-TK gene treatment as an adjuvant. HepG 2-TK cell line was established by transfecting human hepatoma cell line HepG 2 (HLA-A 2 positive) with HSV-TK gene. Dying tumor cells were generated by culturing HepG 2-TK cells with GCV. After engulfed dying cells efficiently, immature DCs (imDC) derived from human monocytes were fully matured and elicited marked proliferation and cytotoxicity against HLA matched HepG 2 cells in autologous peripheral blood mononuclear cells (PBMC). It also implied that HepG 2 specific CTLs played an important role in the cytotoxicity which was primarily depended on Th1 responses. Given the feasibility of inducing dying cells by HSV-TK/GCV in vivo, our results suggest an effective method in clinical human hepatocellular carcinoma (HCC) treatment by an in vitro model of applying HSV-TK gene modified human tumor cells integrated with DC vaccination. 相似文献
4.
A mammalian cellular system, utilizing Syrian hamster embryo cells, was developed for the concomitant study of neoplastic transformation and somatic mutation. Chemically induced somatic mutation of the cells was assayed at two genetic loci. Mutants deficient in hypoxanthine phosphoribosyl transferase (HPRT) were detected by the production of colonies resistant to 8-azaguanine (AG r) or 6-thioguanine (TG r) and mutants with an altered Na +/K + ATPase were detected by the production of colonies resistant to ouabain (Oua r). Colonies resistant to each of the three selective agents were isolated and characterized. AG r and TG r resistant cells maintained their resistance to the selective agent after isolation and growth in the absence of the drug, displayed a low reversion frequency, and possessed less than 1% of the HPRT activity of the wild-type cells. AG r cells were also resistant to the cytotoxicity effects of 6TG. Oua r cells also maintained their resistance to ouabain and were less sensitive to the inhibition of 86Rb uptake by ouabain than the wild-type cells. The spontaneous frequency of all three types of resistant cells was <10 ?6, but the mutation frequency was significantly increased following exposure of the cells to known mutagens in a dosage-dependent manner. These properties indicate that AG r and TG r cells posess a mutation in the structural or regulatory gene for HPRT, and that Oua r cells have an altered Na +/K + ATPase.The factors involved in the quantification of the mutation frequencies of hamster embryo cells following exposure to carcinogens were determined. Cytotoxicity was assayed by a reduction in the cloning efficiency of the treated cells. The recovery efficiencies of the resistant cells were measured by reconstitution experiments and the degree of cross feeding effects of HPRT ? cells was determined. The expression time of the mutations following exposure of the cells to carcinogens was also examined, and the mutation frequencie at the two loci of hamster embryo cells following exposure to MNNG or benzo( a)-pyrene (B( a)P) were determined. Employing this system, a quantitative comparison can be made between the frequencies of somatic mutation and morphological transformation. 相似文献
5.
As a first step in the development of a multiple-marker, mammalian cell mutagenesis assay system, we have isolated a Chinese hamster ovary (CHO) cell line that is heterozygous for both the adenine phosphoribosyltransferase ( aprt) and thymidine kinase ( tk) loci. Presumptive aprt+/? heterozygotes with intermediate levels of APRT activity were selected from unmutagenized CHO cell populations on the basis of resistance to low concentrations of the adenine analog, 8-azaadenine. A functional aprt+/? heterozygote with ~50% wild-type APRT activity was subsequently used to derive sublines that were also heterozygous for the tk locus. Biochemical and genetic characterization of one such subline, CHO-AT3-2, indicated that it was indeed heterozygous at both the aprt and tk loci. CHO-AT3-2 cells permitted single-step selection of mutants resistant to 8-azaadenine or 5-fluorodeoxyuridine, allowing quantitation and direct comparison of mutation induction at the autosomal aprt or tk loci, as well as in the gene involved in ouabain resistance or at the X-linked, hypoxanthine-guanine phosphoribosyltransferase ( hgprt) locus. Significant dose-dependent increases in mutation frequency were observed for all 4 genetic markers after treatment of CHO-AT3-2 cells with ethyl methanesulfonate. 相似文献
6.
Mutants resistant to the microtubule inhibitor podophyllotoxin (Pod R), a codominant marker, can be readily selected in various mammalian cell lines such as, CHO, HeLa, mouse L cells, Syrian hamster cells (BHK 21) and a mouse teratocarcinoma cell line OC15. In CHO cells, the recovery of Pod R mutants is not affected by cell density (up to 1 × 10 6 cells per 100-mm diameter dish), and after treatment with the mutagen ethyl methanesulfonate maximum mutagenic effect is achieved after a relatively short expression time (40–48 h). The frequency of Pod R mutants in various cell lines increased in a dose-dependent manner in response to treatment with the mutagens ethyl methanesulfonate and N-methyl- N′-nitro- N-nitrosoguanidine. The Pod R selection system thus provides a new genetic marker which should prove useful in studies of quantitative mutagenesis in mammalian cells. 相似文献
7.
Induction and characterization of a morphological mutant are described for Heterorhabditis bacteriophora strain HP88. A homozygous inbred line was used as the base population for mutagenesis and genetic analysis of mutations. Mutagenesis was induced by exposing young hermaphrodites to 0.05 M ethyl methanesulfonate. A dumpy mutant (designated Hdpy-l) was isolated from the F₂ generation of the mutagenized population. Morphological studies with light and scanning electron microscopy revealed that the head region of the adult stage was compressed. The head region of the infective juvenile was distorted and the mouth open. Backcross with the original population was successful only between mutant hermaphrodites and wild type males; 50-100 percent of the progeny of this cross maintained the dumpy phenotype, indicating that the ratio between self- and external fertilization of the eggs is > 1 and that the dumpy mutation is recessive. 相似文献
8.
Ultraviolet irradiation (254 nm) of immortal diploid human lymphoblasts killed cells, caused mutation at three genetic loci studied, and transiently inhibited 3H-TdR uptake into DNA. A shoulder of about 6 J/m 2 and a D0 of 6 J/m 2 was observed for survival. Mutation rose in a monotonic non-linear fashion through 6 J/m 2; above 6 J/m 2, complex behavior approximating a plateau in induced mutation was observed. Irradiation at 4.4 J/m 2 caused a transient increase in the number of cells synthesizing DNA and a decrease in the rate of DNA synthesis relative to mock-irradiated controls. The parameter of rate of DNA synthesis per cell in DNA synthetic phase showed a rapid recovery toward control values between 2 and 4 h after irradiation and a slower recovery to control values by 22 h post-irradiation.Fractionated dose schedules were used to measure the effects of allowing a time interval between doses at nontoxic fluences (2.2 j/m 2), moderately toxic fluences (8.8 J/m 2) and toxic fluences (17.6 J/m 2). These measurements indicate that in the non-toxic range of fluences common to human exposure, mutational response is mediated by a post-irradiation process which seems to show to shkow enchanced ability to protect against mutation induced by subsequent irradiation. However, at moderately toxic fluences there was little effect of dose fractionation, and at toxic fluences, a time-dependent increase in mutation fraction was observed at separation times greater than 7 h. We suggest that these latter observations arise primarily from cell cycle heterogeneity with regard to sensitivity to UV killing and mutation. 相似文献
9.
Glycoproteins are difficult to crystallize because they have heterogeneous glycans composed of multiple monosaccharides with considerable rotational freedom about their O-glycosidic linkages. Crystallographers studying N-glycoproteins often circumvent this problem by using β1,2- N-acetylglucosaminyltransferase I (MGAT1)–deficient mammalian cell lines, which produce recombinant glycoproteins with immature N-glycans. These glycans support protein folding and quality control but can be removed using endo-β- N-acetylglucosaminidase H (Endo H). Many crystallographers also use the baculovirus-insect cell system (BICS) to produce recombinant proteins for their work but have no access to an MGAT1-deficient insect cell line to facilitate glycoprotein crystallization in this system. Thus, we used BICS-specific CRISPR–Cas9 vectors to edit the Mgat1 gene of a rhabdovirus-negative Spodoptera frugiperda cell line (Sf-RVN) and isolated a subclone with multiple Mgat1 deletions, which we named Sf-RVN Lec1. We found that Sf-RVN and Sf-RVN Lec1 cells had identical growth properties and served equally well as hosts for baculovirus-mediated recombinant glycoprotein production. N-glycan profiling showed that a total endogenous glycoprotein fraction isolated from Sf-RVN Lec1 cells had only immature and high mannose-type N-glycans. Finally, N-glycan profiling and endoglycosidase analyses showed that the vast majority of the N-glycans on three recombinant glycoproteins produced by Sf-RVN Lec1 cells were Endo H-cleavable Man 5GlcNAc 2 structures. Thus, this study yielded a new insect cell line for the BICS that can be used to produce recombinant glycoproteins with Endo H-cleavable N-glycans. This will enable researchers to combine the high productivity of the BICS with the ability to deglycosylate recombinant glycoproteins, which will facilitate efforts to determine glycoprotein structures by X-ray crystallography. 相似文献
10.
The NADPH-oxidase of phagocytic cells is a multicomponent enzyme that generates superoxide. It comprises a membrane flavocytochrome b558 and four cytosolic proteins; p67 phox, p47 phox, p40 phox and Rac. The NADPH-binding site of this complex was shown to be located on the flavocytochrome b558. However, a number of studies have suggested the presence of another site on the p67 phox subunit which is the key activating component. Using several approaches like tryptophan quenching fluorescence measurement, inhibition by 2′,3′-dialdehyde NADPH, and free/bound NADPH concentration measurements, we demonstrate that no NADPH binds on p67 phox, thus definitively solving the controversy on the number and location of the NADPH-binding sites on this complex. 相似文献
11.
Transfer RNA is highly modified. Nucleotide 37 of the anticodon loop is represented by various modified nucleotides. In Escherichia coli, the valine-specific tRNA (cmo 5UAC) contains a unique modification, N 6-methyladenosine, at position 37; however, the enzyme responsible for this modification is unknown. Here we demonstrate that the yfiC gene of E. coli encodes an enzyme responsible for the methylation of A37 in tRNA 1Val. Inactivation of yfiC gene abolishes m 6A formation in tRNA 1Val, while expression of the yfiC gene from a plasmid restores the modification. Additionally, unmodified tRNA 1Val can be methylated by recombinant YfiC protein in vitro. Although the methylation of m 6A in tRNA 1Val by YfiC has little influence on the cell growth under standard conditions, the yfiC gene confers a growth advantage under conditions of osmotic and oxidative stress. 相似文献
12.
Two hypertrophic cardiomyopathy-associated cardiac troponin I (cTnI) mutations, R146G and R21C, are located in different regions of cTnI, the inhibitory peptide and the cardiac-specific N terminus. We recently reported that these regions may interact when Ser-23/Ser-24 are phosphorylated, weakening the interaction of cTnI with cardiac TnC. Little is known about how these mutations influence the affinity of cardiac TnC for cTnI ( KC-I) or contractile kinetics during β-adrenergic stimulation. Here, we tested how cTnI R146G or cTnI R21C influences contractile activation and relaxation and their response to protein kinase A (PKA). Both mutations significantly increased Ca 2+ binding affinity to cTn ( KCa) and KC-I. PKA phosphorylation resulted in a similar reduction of KCa for all complexes, but KC-I was reduced only with cTnI WT. cTnI WT, cTnI R146G, and cTnI R21C were complexed into cardiac troponin and exchanged into rat ventricular myofibrils, and contraction/relaxation kinetics were measured ± PKA phosphorylation. Maximal tension (T max) was maintained for cTnI R146G- and cTnI R21C-exchanged myofibrils, and Ca 2+ sensitivity of tension ( pCa 50) was increased. PKA phosphorylation decreased pCa 50 for cTnI WT-exchanged myofibrils but not for either mutation. PKA phosphorylation accelerated the early slow phase relaxation for cTnI WT myofibrils, especially at Ca 2+ levels that the heart operates in vivo. Importantly, this effect was blunted for cTnI R146G- and cTnI R21C-exchanged myofibrils. Molecular dynamics simulations suggest both mutations inhibit formation of intra-subunit contacts between the N terminus and the inhibitory peptide of cTnI that is normally seen with WT-cTn upon PKA phosphorylation. Together, our results suggest that cTnI R146G and cTnI R21C blunt PKA modulation of activation and relaxation kinetics by prohibiting cardiac-specific N-terminal interaction with the cTnI inhibitory peptide. 相似文献
13.
Chinese hamster cells (line CHEF-125) were cultured for 1 or 4 h in the presence of tritiated thymidine ( 3HTdR). Immediately after the end of the treatment with 3HTdR or 4 h afterwards, some cultures were irradiated with X-rays, while others served as controls.Analysis of colchicine-C anaphase of M 1 and M 2 cells showed that: ( a) in the M 1 chromosomes the X-rays produced a significant departure from a 1:1 ratio of the number of silver grains counted on the sister chromatids; and ( b) in the M 2 chromosomes the X-rays increased significantly the frequency of sister-chromatid exchanges and of isolabelling regions.Subchromatid exchanges involving a single polynucleotide strand may be induced by X-rays. These exchanges would cause inequality of labelling over M 1 sister chromatids and isolabelling in the M 2 chromosomes. 相似文献
14.
The pentacyclic triterpene 3β,6β,16β-tri-hydroxilup-20(29)-ene is a natural product produced by the Brazilian medicinal plant Combretum leprosum. Its cytotoxicity has been previously reported against breast cancer cell lines. The low water solubility of this natural product, that hampers its bioavailability, motivated the investigation of a new nanoparticle formulation containing the triterpene in order to improve its bioactivity. The triterpene was encapsulated in polycaprolactone (PCL) polymer by nanoprecipitation, producing homogenic nanoparticles with nanometer sizes (122.7 ± 2.06 nm), which were characterized by FT-IR, SEM imaging and DSC. The cytotoxicity (MTT method) of the nanoparticle containing the triterpene 1, besides the free natural product and the nanoparticle control (without 1), was assayed against three human tumor cell lines [human colon carcinoma line (HCT116), prostate (PC3) and glioblastoma (SNB19)] and the normal epithelial embryo kidney human cell line (Hek293T). The nanocarrier produced a significative effect in the cytotoxicity of the natural product in the nanoformulation (IC 50 0.11–0.26 µg mL −1) when compared with its free form (IC 50 1.07–1.44 µg mL −1). Additionally, higher selectivity of the triterpene to the tumor cells was found when it was encapsulated (SI 1.92–4.54) than in its free form (SI 0.42–0.56). In this case, the nanoencapsulated triterpene was more selective to PC3 (SI 3.33) and SNB19 (SI 4.54) tumor cells. 相似文献
15.
Irvin A. D. and Young E. R. 1979. Further studies on the uptake of tritiated nucleic acid precursors by Babesia spp. of cattle and mice. International Journal for Parasitology9: 109–114. An in vitro culture technique developed earlier was used to study the metabolism of nucleic acid precursors by Babesia microti and B. rodhaini of mice and by B. divergens and B. major of cattle. [ 3H]Hypoxanthine was readily incorporated by all species of parasite, and the presence of leucocytes did not affect this uptake. When parasites were maintained in culture their ability to incorporate [ 3H]hypoxanthine fell rapidly after 24 h, but when B. major was maintained at 4°C its subsequent ability to incorporate [ 3H]hypoxanthine persisted for at least 3 days. This finding could be of practical value in assessing infectivity of stored blood in vitro.On autoradiography, [ 3H]hypoxanthine appeared to be incorporated into both DNA and RNA of parasites. Salvage pathways for purine metabolism appeared to be important in all species of Babesia whereas for pyrimidine metabolism salvage pathways were more important for murine babesias and the de novo pathway more important for bovine species. This difference may relate to different permeabilities of bovine and murine erythrocyte membranes or may be a more fundamental species difference. 相似文献
16.
The aim of the present study was to identify the enzymes in human liver catalyzing hydroxylations of bile acids. Fourteen recombinant expressed cytochrome P450 (CYP) enzymes, human liver microsomes from different donors, and selective cytochrome P450 inhibitors were used to study the hydroxylation of taurochenodeoxycholic acid and lithocholic acid. Recombinant expressed CYP3A4 was the only enzyme that was active towards these bile acids and the enzyme catalyzed an efficient 6α-hydroxylation of both taurochenodeoxycholic acid and lithocholic acid. The Vmax for 6α-hydroxylation of taurochenodeoxycholic acid by CYP3A4 was 18.2 nmol/nmol P450/min and the apparent Km was 90 μM. Cytochrome b5 was required for maximal activity. Human liver microsomes from 10 different donors, in which different P450 marker activities had been determined, were separately incubated with taurochenodeoxycholic acid and lithocholic acid. A strong correlation was found between 6α-hydroxylation of taurochenodeoxycholic acid, CYP3A levels ( r2=0.97) and testosterone 6β-hydroxylation ( r2=0.9). There was also a strong correlation between 6α-hydroxylation of lithocholic acid, CYP3A levels and testosterone 6β-hydroxylation ( r2=0.7). Troleandomycin, a selective inhibitor of CYP3A enzymes, inhibited 6α-hydroxylation of taurochenodeoxycholic acid almost completely at a 10 μM concentration. Other inhibitors, such as α-naphthoflavone, sulfaphenazole and tranylcypromine had very little or no effect on the activity. The apparent Km for 6α-hydroxylation of taurochenodeoxycholic by human liver microsomes was high (716 μM). This might give an explanation for the limited formation of 6α-hydroxylated bile acids in healthy humans. From the present results, it can be concluded that CYP3A4 is active in the 6α-hydroxylation of both taurochenodeoxycholic acid and lithocholic acid in human liver. 相似文献
17.
Based on the well-known k-mer model, we propose a k-mer natural vector model for representing a genetic sequence based on the numbers and distributions of k-mers in the sequence. We show that there exists a one-to-one correspondence between a genetic sequence and its associated k-mer natural vector. The k-mer natural vector method can be easily and quickly used to perform phylogenetic analysis of genetic sequences without requiring evolutionary models or human intervention. Whole or partial genomes can be handled more effective with our proposed method. It is applied to the phylogenetic analysis of genetic sequences, and the obtaining results fully demonstrate that the k-mer natural vector method is a very powerful tool for analysing and annotating genetic sequences and determining evolutionary relationships both in terms of accuracy and efficiency. 相似文献
18.
Two Expressed Sequence Tagged (EST) clones were identified from the Arabidopsis database as encoding putative cytidine deaminases.
Sequence analysis determined that the two clones overlapped and encoded a single cDNA. This cytidine deaminase corresponds
to the Arabidopsis thaliana gene, cda1. The deduced amino acid sequence was more closely related to prokaryotic cytidine deaminases than to eukaryotic enzymes.
The cDNA shares 44% amino acid identity with the Escherichia coli cytidine deaminase but only 26 and 27% identity with human and yeast enzymes. A unique zinc-binding domain of the E coli enzyme forms the active site. A similar putative zinc-binding domain was identified in the Arabidopsis enzyme based upon
primary sequence similarities. These similarities permitted us to model the active site of the Arabidopsis enzyme upon that
of the E coli enzyme. In this model, the active site zinc is coordinated by His 73, Cys 103, Cys 107, and an active site hydroxyl. Additional residues that participate in catalysis, Asn 64, Glu 66, Ala 78, Glu 79, and Pro 102, are conserved between the Arabidopsis and E coli enzymes suggesting that the Arabidopsis enzyme has a catalytic mechanism similar to the E coli enzyme. The two overlapping ESTs were used to prepare a single, full-length clone corresponding to the A thaliana cda1 cDNA. This cDNA was subcloned into pProExHtb and expressed as a fusion protein with an N-terminal His 6 tag. Following purification on a Ni-NTA-Agarose column, the protein was analyzed for its kinetic properties. The enzyme utilizes
both cytidine (K m = 226 μand 2’-deoxycytidine (K m= 49 μM) as substrates. The enzyme was unable to deaminate cytosine, CMP or dCMP.
journal Paper Number J-18324 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa Project No. 3340. 相似文献
19.
The yeast cytochrome bc1 complex, a component of the mitochondrial respiratory chain, is composed of ten distinct protein subunits. In the assembly of the bc1 complex, some ancillary proteins, such as the chaperone Bcs1p, are actively involved. The deletion of the nuclear gene encoding this chaperone caused the arrest of the bc1 assembly and the formation of a functionally inactive bc1 core structure of about 500-kDa. This immature bc1 core structure could represent, on the one hand, a true assembly intermediate or, on the other hand, a degradation product and/or an incorrect product of assembly. The experiments here reported show that the gradual expression of Bcs1p in the yeast strain lacking this protein was progressively able to rescue the bc1 core structure leading to the formation of the functional homodimeric bc1 complex. Following Bcs1p expression, the mature bc1 complex was also progressively converted into two supercomplexes with the cytochrome c oxidase complex. The capability of restoring the bc1 complex and the supercomplexes was also possessed by the mutated yeast R81C Bcsp1. Notably, in the human ortholog BCS1L, the corresponding point mutation (R45C) was instead the cause of a severe bc1 complex deficiency. Differently from the yeast R81C Bcs1p, two other mutated Bcs1p's (K192P and F401I) were unable to recover the bc1 core structure in yeast. This study identifies for the first time a productive assembly intermediate of the yeast bc1 complex and gives new insights into the molecular mechanisms involved in the last steps of bc1 assembly. 相似文献
20.
Although Giardia lamblia trophozoites are unable to carry out de novo phospholipid synthesis, they can assemble complex glycophospholipids from simple lipids and fatty acids acquired from the host. Previously, we have reported that G. lamblia synthesizes GP49, an invariant surface antigen with a glycosylphosphatidylinositol (GPI) anchor. It is therefore possible that myo-inositol (Ins), phosphatidylinositol (PI) and other GPI precursors are obtained from the dietary products of the human small intestine, where the trophozoites colonize. In this report, we have investigated the role of exogenous Ins and PI on GPI anchor synthesis by G. lamblia. The results demonstrate that [ 3H]Ins and PI internalized by trophozoites, metabolically transformed into GlcN(acyl)-PI and downstream GPI molecules. Further investigations suggest that G. lamblia expresses cytidine monophosphate (CMP)-dependent (Mg 2+-stimulated) and independent (Mn 2+-stimulated) inositol headgroup exchange enzymes, which are responsible for exchanging free Ins with cellular PI. We observed that 3-deoxy-3-fluoro-D- myo-inositol (3-F-Ins) and 1-deoxy-1-F- scyllo-Ins (1-F- scyllo-Ins), which are considered potent inhibitors of Mn 2+-stimulated headgroup exchange enzyme, inhibited the incorporation of [ 3H]Ins into PI and GPI molecules significantly, suggesting that CMP-independent (Mn 2+-stimulated) exchange enzyme may be important for these reactions. However, 3-F-Ins and 1-F- scyllo-Ins were not effective in blocking the incorporation of exogenously supplied [ 3H]PI into GPI glycolipids. Thus, it can be concluded that G. lamblia can use exogenously supplied [ 3H]PI and [ 3H]Ins to synthesize GPI glycolipids of GP49; while PI is directly incorporated into GPI molecules, free Ins is first converted into PI by headgroup exchange enzymes, and this newly formed PI participates in GPI anchor synthesis. 相似文献
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