During protein import into chloroplasts, one of the Hsp70 proteins in pea (Hsp70-IAP), previously... more During protein import into chloroplasts, one of the Hsp70 proteins in pea (Hsp70-IAP), previously reported to localize in the intermembrane space of chloroplasts, was found to interact with the translocating precursor protein but the gene for Hsp70-IAP has not been identified yet. In an attempt to identify the Arabidopsis homolog of Hsp70-IAP, we employed an in vitro protein import assay to determine the localization of three Arabidopsis Hsp70 homologs (AtHsp70-6 through 8), predicted for chloroplast targeting. AtHsp70-6 and AtHsp70-7 were imported into chloroplasts and processed into similar-sized mature forms. In addition, a smaller-sized processed form of AtHsp70-6 was observed. All the processed forms of both AtHsp70 proteins were localized in the stroma. Organelle-free processing assays revealed that the larger processed forms of both AtHsp70-6 and AtHsp70-7 were cleaved by stromal processing peptidase, whereas the smaller processed form of AtHsp70-6 was produced by an unspecified peptidase.
Sulfated glycosphingolipids are present on the surface of a variety of cells. They are active par... more Sulfated glycosphingolipids are present on the surface of a variety of cells. They are active participants in adhesion processes in many systems and appear to be involved in the regulation of cell proliferation, differentiation and other developmental cellular events. However, the body of knowledge about synthesis, structure, and function of glycolipids in parasitic protozoa is very limited so far. In this work, we show by metabolic incorporation of [14C]palmitic acid, [14C]glucose and Na235SO4 that sulfoglycosphingolipids are biosynthesized in the three intraerythrocytic stages of Plasmodium falciparum. After saponification, purification of the labelled acidic components was achieved and two components named SPf1 and SPf2 were characterized. Chemical degradations and TLC analysis pointed out to sulfolipidic structures. Analysis by UV-MALDI-TOF mass spectrometry in the negative ion mode using nor-harmane as matrix showed for SPf2 a structure consisting in a disulfated hexose linked to a 20:1 sphingosine acylated with C18:0 fatty acid. Interestingly, parasite treatment with low concentrations of d,l-threo-phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP) caused an arrest on parasite development associated to the inhibition of sulfoglycolipid biosynthesis. Taking into account that sulfoglycolipidic structures are currently involved in adhesion processes, our findings open the possibility to study the participation of this type of structures in the described aggregation phenomena in severe malaria and may contribute to clarify the pathogenesis of the disease. This report shows for the first time the synthesis of sulfoglycolipids in Apicomplexa.
The in vitro protein import experiment is one of the most important techniques for determining pr... more The in vitro protein import experiment is one of the most important techniques for determining protein localization. For chloroplastic proteins, proteins of interest are incubated with isolated chloroplasts in the presence of energy sources. Radio-labeled proteins synthesized either in vitro or in vivo have been widely used as substrate proteins. Here we report our development of the protein import assay system in which non-radio-labeled proteins, overexpressed in Escherichia coli, were applied. In this system, substrate proteins were designed to carry epitope-tags, thus allowing analysis of imported proteins by SDS-PAGE, followed by immunoblotting to detect these tags. Furthermore, the imported proteins were found to be incorporated into their native form. These observations indicated that recombinant proteins were imported into chloroplasts and folded correctly. Therefore, this assay system could represent another valuable tool for determining protein localization.
Matrix-assisted ultraviolet laser-desorption ionization time-of-flight mass spectrometry (UV-MALD... more Matrix-assisted ultraviolet laser-desorption ionization time-of-flight mass spectrometry (UV-MALDI-TOF-MS) was applied to sulfated xylo-mannan fractions from Nothogenia fastigiata in order to determine their molecular weights and distribution profiles. The number-average molecular weight calculated from the spectra was similar to that determined by chemical end-group analysis for the lower molecular weight fractions. For the other fractions, the number-average molecular weight was lower than that chemically determined; the increased difference may be attributed to higher desorption difficulties and, consequently, mass-dependent discrimination. A reconstructed spectrum, using the peaks obtained from all the fractions, suggested an unimodal distribution. The best results were obtained by using 2,5-dihydroxybenzoic acid as matrix doped with 1-hydroxyisoquinoline and with harmane and nor-harmane.
Journal of The American Society for Mass Spectrometry, 2008
Single-cell cytoplasm sap (1–10 pL) was extracted by using a pressure probe glass microcapillary ... more Single-cell cytoplasm sap (1–10 pL) was extracted by using a pressure probe glass microcapillary tip from tulip leaf and bulb and analyzed by UV-MALDI-TOF MS for free underivatized carbohydrate content. Three matrices including 2,5-dihydroxybenzoic acid (DHB), 2,4,6-trihydroxyacetophenone (THAP), and carbon nanotubes (CNTs) in positive ion mode were selected for analysis because of acceptable carbohydrate-related signal reproducibility. Disaccharide and oligosaccharide (up to 15 Hex when THAP was used, 11 Hex with DHB, and 7 Hex with CNTs) were detected in tulip bulb cell cytoplasm sample. When DHB was used as matrix, neutral carbohydrates were more abundantly detected as sodiated cations; the sugar-related signals, however, appeared as dominant potassiated cations when THAP and CNTs were used. Small amount of monosaccharide was also detected in bulb cell cytoplasm with CNTs as matrix. UV-MALDI-TOF MS of leaf cell extract resulted in high-resolution detection of hexose and disaccharide with DHB, THAP, and CNTs.
Journal of The American Society for Mass Spectrometry, 2009
Probe electrospray ionization (PESI) is a recently developed ESI-based ionization technique which... more Probe electrospray ionization (PESI) is a recently developed ESI-based ionization technique which generates electrospray from the tip of a solid needle. In this study, we have applied PESI interfaced with a time of flight mass spectrometer (TOF-MS) for direct profiling of phytochemicals in a section of a tulip bulb in different regions, including basal plate, outer and inner rims of scale, flower bud and foliage leaves. Different parts of tulip petals and leaves have also been investigated. Carbohydrates, amino acids and other phytochemicals were detected. A series of in vivo PESI-MS experiments were carried out on the second outermost scales of four living tulip bulbs to monitoring the change of carbohydrate content during the first week of initial growth. The breakdown of carbohydrates was observed which was in accordance with previous reports achieved by other techniques. This study has indicated that PESI-MS can be used for rapid and direct analysis of phytochemicals in living biological systems with advantages of low sample consumption and little sample preparation. Therefore, PESI-MS can be a new choice for direct analysis/profiling of bioactive compounds or monitoring metabolic changes in living biological systems.
Underivatized carbohydrates of tulip bulb and leaf tissues were characterized in situ by matrix-a... more Underivatized carbohydrates of tulip bulb and leaf tissues were characterized in situ by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI–TOF MS) by using carbon nanotubes (CNTs) as matrix. Two sample preparation methods—(i) depositing CNTs on the fresh tissue slices placed on the probe and (ii) locating semitransparent tissues on a dried layer of CNTs on the probe—were examined. Furthermore, practicability of in situ starch analysis by MALDI–TOF MS was examined by detection of glucose originated from on-probe amyloglucosidase-catalyzed degradation of starch on the tissue surface. Besides, CNTs could efficiently desorb/ionize natural mono-, di-, and oligosaccharides extracted from tulip bulb tissues as well as glucose resulting from starch enzymatic degradation in vitro. These results were compared with those obtained by in situ MALDI–TOF MS analysis of similar tissues. Positive ion mode showed superior signal reproducibility. CNTs deposited under semitransparent tissue could also desorb/ionize neutral carbohydrates, leading to nearly complete elimination of matrix cluster signals but with an increase in tissue-originated signals. Furthermore, several experiments were carried out to compare the efficiency of 2,5-dihydroxybenzoic acid, nor-harmane, α-cyano-4-hydroxycinnamic acid, and CNTs as matrices for MALDI of neutral carbohydrates from the intact plant tissue surface and for enzymatic tissue starch degradation; these results are discussed in brief. Among matrices studied, the lowest laser power was needed to acquire carbohydrate signals with high signal-to-noise ratio and resolution when CNTs were used.
... Hernan A. Orgueira, † Rosa Erra-Balsells, † Hiroshi Nonami, ‡ and Oscar Varela* †. ... Sci. 1... more ... Hernan A. Orgueira, † Rosa Erra-Balsells, † Hiroshi Nonami, ‡ and Oscar Varela* †. ... Sci. 1996, 4, 25−31. [CAS]. (2) Selegny, E.; Merle-Aubry, L. In Optically Active Polymers; Selegny, E., Ed.; Reidel: Dordrecht, The Netherlands, 1979; pp 15−110. ...
This work was undertaken to determine the growth parameters of Lockhart’s equation for finding wh... more This work was undertaken to determine the growth parameters of Lockhart’s equation for finding which component was predominantly contributing to the cell expansion rates of plants subjected to environmental stresses under tissue-culture conditions. Embryos isolated from soybean (Glycine max [L.] Merr.) and kidney bean (Phaseolus vulgaris L.) seeds were grown under tissue-culture conditions. The water potential of culture media ranged from − 0·02 to − 0·94 MPa so that nutrient deficiency and salt stress conditions could be applied. Additionally, the temperature of culture conditions was set from 10 to 40 °C to apply low-temperature and high-temperature stresses on plants grown at the optimum concentration of culture medium. Cell expansion could be inhibited completely by adding 2,4-dichlorophenoxyacetic acid and benzylaminopurine to culture media to form callus tissue. The sizes of the water potential gradient between the water source and elongating cells correlated with the speed of growth rates under nutrient deficiency, salt stress, growth retardation induced by plant hormones, low-temperature and high-temperature conditions in the present study, indicating that cell expansion rates were mainly associated with how much water could be absorbed by elongating cells regardless of the kinds of environmental stress conditions applied.
Abstract Silsesquioxanes obtained by the hydrolytic condensation of (3-glycidoxypropyl) trimethox... more Abstract Silsesquioxanes obtained by the hydrolytic condensation of (3-glycidoxypropyl) trimethoxysilane (GPMS) in diglycidyl ether of bisphenol A (DGEBA) were characterized by electrospray ionization time-of-flight mass spectrometry (ESI-TOF MS) and matrix-assisted ...
Trypanosoma cruzi, the parasitic protozoan that causes Chagas disease, contains a major cysteine ... more Trypanosoma cruzi, the parasitic protozoan that causes Chagas disease, contains a major cysteine proteinase, cruzipain. This lysosomal enzyme bears an unusual C-terminal extension that contains a number of post-translational modifications, and most antibodies in natural and experimental infections are directed against it. In this report we took advantage of UV-MALDI-TOF mass spectrometry in conjunction with peptide N-glycosidase F deglycosylation and high performance anion exchange chromatography analysis to address the structure of the N-linked oligosaccharides present in this domain. The UV-MALDI-TOF MS analysis in the negative-ion mode, using nor-harmane as matrix, allowed us to determine a new striking feature in cruzipain: sulfated high-mannose type oligosaccharides. Sulfated GlcNAc2Man3 to GlcNAc2Man9 species were identified. In accordance, after chemical or enzymatic desulfation, the corresponding signals disappeared. In addition, by UV-MALDI-TOF MS analysis (a) a main population of high-mannose type oligosaccharides was shown in the positive-ion mode, (b) lactosaminic glycans were also identified, among them, structures corresponding to monosialylated species were detected, and (c) as an interesting fact a fucosylated oligosaccharide was also detected. The presence of the deoxy sugar was further confirmed by high performance anion exchange chromatography. In conclusion, the total number of oligosaccharides occurring in cruzipain was shown to be much higher than previous estimates. This constitutes the first report on the presence of sulfated glycoproteins in Trypanosomatids.
Malaria remains a major health problem especially in tropical and subtropical regions of the worl... more Malaria remains a major health problem especially in tropical and subtropical regions of the world, and therefore developing new antimalarial drugs constitutes an urgent challenge. Lipid metabolism has been attracting a lot of attention as an application for malarial chemotherapeutic purposes in recent years. However, little is known about glycosphingolipid biosynthesis in Plasmodium falciparum. In this report we describe for the first time the presence of an active glucosylceramide synthase in the intraerythrocytic stages of the parasite. Two different experiments, using UDP-[14C]glucose as donor with ceramides as acceptors, or UDP-glucose as donor and fluorescent ceramides as acceptors, were performed. In both cases, we found that the parasitic enzyme was able to glycosylate only dihydroceramide. The enzyme activity could be inhibited in vitro with low concentrations of d,l-threo-phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP). In addition, de novo biosynthesis of glycosphingolipids was shown by metabolic incorporation of [14C]palmitic acid and [14C]glucose in the three intraerythrocytic stages of the parasite. The structure of the ceramide, monohexosylceramide, trihexosylceramide and tetrahexosylceramide fractions was analysed by UV-MALDI-TOF mass spectrometry.When PPMP was added to parasite cultures, a correlation between arrest of parasite growth and inhibition of glycosphingolipid biosynthesis was observed. The particular substrate specificity of the malarial glucosylceramide synthase must be added to the already known unique and amazing features of P. falciparum lipid metabolism; therefore this enzyme might represent a new attractive target for malarial chemotherapy.
Organotrialkoxysilanes containing secondary hydroxyl groups were synthesized by reacting 1 mole o... more Organotrialkoxysilanes containing secondary hydroxyl groups were synthesized by reacting 1 mole of (3-aminopropyl)triethoxysilane (APS) with 1 or 2 mole of phenylglycidylether (PGE). Resulting products, APS–PGE and APS–PGE2, respectively, were subjected to hydrolytic condensation at 50 °C during 24 h. For APS–PGE, the reaction was performed using a molar ratio [H2O]/Si=3, without addition of an external catalyst. For APS–PGE2, the reaction was catalyzed by HCOOH or NaOH. Resulting poly(silsesquioxanes) (PSSO) were characterized by size exclusion chromatography, Fourier-transformed infrared spectroscopy and matrix-assisted ultraviolet laser desorption/ionization time-of-flight mass spectrometry (UV-MALDI-TOF MS). PSSO derived from APS–PGE and APS–PGE2, catalyzed by HCOOH, exhibited a relatively narrow distribution of polyhedral structures. This constitutes a simple one-step synthesis of polyhedral oligomeric silsesquioxanes (POSS) functionalized with amine and/or hydroxyl groups. The directionality of the reaction pathway towards the formation of polyhedral structures was ascribed to the formation of intramolecular SiOC bonds through the reaction of SiOEt or SiOH groups with secondary hydroxyl groups. Intramolecular SiOC bonds were found in the structures of APS–PGE and APS–PGE2, and in most of the species of the PSSO obtained from the NaOH-catalyzed reaction of APS–PGE2. A small fraction of surviving SiOC bonds was also found in the polyhedral structures of the PSSO derived from the hydrolytic condensation of APS–PGE and APS–PGE2 catalyzed by formic acid. By usual organic reactions transforming hydroxyl groups into other functional groups, it is possible to generate narrow distribution of multi-functionalized POSS starting from an OH-functionalized organotrialkoxysilane.The hydrolytic condensation of organotrialkoxysilanes containing secondary hydroxyl groups led to a narrow distribution of OH-functionalized polyhedral oligomeric silsesquioxanes. The generation of closed structures was ascribed to the reversible formation of intramolecular cycles through SiOC bonds. Under appropriate conditions, structures containing these cycles were present in high concentrations.
Several commercial sulfated neocarrabiose oligosaccharides were analyzed by matrix-assisted ultra... more Several commercial sulfated neocarrabiose oligosaccharides were analyzed by matrix-assisted ultraviolet laser-desorption ionization time-of-flight mass spectrometry (UV-MALDI-TOF-MS). UV-MALDI-TOF-MS was carried out in the linear and reflectron modes and, as routine, in both the positive- and negative-ion modes. 2,5-Dihydroxybenzoic acid and nor-harmane were used as matrices. In the positive- and negative-ion modes, with both matrices, peaks corresponding to (M+Na)+ and (M−Na)− ions, respectively, were obtained, with only some signals due to glycosidic linkage cleavages (prompt fragmentation). With 2,5-dihydroxybenzoic acid abundant matrix signals were observed; nor-harmane afforded very few matrix signals in both ion modes, but more desulfation (prompt fragmentation) of the compounds occurred. When the desorption/ionization process was highly efficient, the post-source decay (PSD) fragmentation patterns were also investigated; most of the fragments detected derived from glycosidic linkage cleavages. Electrospray-ionization time-of-flight mass spectrometry (ESI-TOF-MS) in the negative-ion mode confirmed, with the observation of the (M−Na)− and the multiply charged anions, the identity and the purity of the samples.Several commercial sulfated neocarrabiose oligosaccharides were analyzed by UV-MALDI-TOF-MS in the positive- and negative-ion modes, using 2,5-dihydroxybenzoic acid and nor-harmane as matrices; PSD patterns were investigated. ESI-TOF-MS of these analytes was also performed.
During protein import into chloroplasts, one of the Hsp70 proteins in pea (Hsp70-IAP), previously... more During protein import into chloroplasts, one of the Hsp70 proteins in pea (Hsp70-IAP), previously reported to localize in the intermembrane space of chloroplasts, was found to interact with the translocating precursor protein but the gene for Hsp70-IAP has not been identified yet. In an attempt to identify the Arabidopsis homolog of Hsp70-IAP, we employed an in vitro protein import assay to determine the localization of three Arabidopsis Hsp70 homologs (AtHsp70-6 through 8), predicted for chloroplast targeting. AtHsp70-6 and AtHsp70-7 were imported into chloroplasts and processed into similar-sized mature forms. In addition, a smaller-sized processed form of AtHsp70-6 was observed. All the processed forms of both AtHsp70 proteins were localized in the stroma. Organelle-free processing assays revealed that the larger processed forms of both AtHsp70-6 and AtHsp70-7 were cleaved by stromal processing peptidase, whereas the smaller processed form of AtHsp70-6 was produced by an unspecified peptidase.
Sulfated glycosphingolipids are present on the surface of a variety of cells. They are active par... more Sulfated glycosphingolipids are present on the surface of a variety of cells. They are active participants in adhesion processes in many systems and appear to be involved in the regulation of cell proliferation, differentiation and other developmental cellular events. However, the body of knowledge about synthesis, structure, and function of glycolipids in parasitic protozoa is very limited so far. In this work, we show by metabolic incorporation of [14C]palmitic acid, [14C]glucose and Na235SO4 that sulfoglycosphingolipids are biosynthesized in the three intraerythrocytic stages of Plasmodium falciparum. After saponification, purification of the labelled acidic components was achieved and two components named SPf1 and SPf2 were characterized. Chemical degradations and TLC analysis pointed out to sulfolipidic structures. Analysis by UV-MALDI-TOF mass spectrometry in the negative ion mode using nor-harmane as matrix showed for SPf2 a structure consisting in a disulfated hexose linked to a 20:1 sphingosine acylated with C18:0 fatty acid. Interestingly, parasite treatment with low concentrations of d,l-threo-phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP) caused an arrest on parasite development associated to the inhibition of sulfoglycolipid biosynthesis. Taking into account that sulfoglycolipidic structures are currently involved in adhesion processes, our findings open the possibility to study the participation of this type of structures in the described aggregation phenomena in severe malaria and may contribute to clarify the pathogenesis of the disease. This report shows for the first time the synthesis of sulfoglycolipids in Apicomplexa.
The in vitro protein import experiment is one of the most important techniques for determining pr... more The in vitro protein import experiment is one of the most important techniques for determining protein localization. For chloroplastic proteins, proteins of interest are incubated with isolated chloroplasts in the presence of energy sources. Radio-labeled proteins synthesized either in vitro or in vivo have been widely used as substrate proteins. Here we report our development of the protein import assay system in which non-radio-labeled proteins, overexpressed in Escherichia coli, were applied. In this system, substrate proteins were designed to carry epitope-tags, thus allowing analysis of imported proteins by SDS-PAGE, followed by immunoblotting to detect these tags. Furthermore, the imported proteins were found to be incorporated into their native form. These observations indicated that recombinant proteins were imported into chloroplasts and folded correctly. Therefore, this assay system could represent another valuable tool for determining protein localization.
Matrix-assisted ultraviolet laser-desorption ionization time-of-flight mass spectrometry (UV-MALD... more Matrix-assisted ultraviolet laser-desorption ionization time-of-flight mass spectrometry (UV-MALDI-TOF-MS) was applied to sulfated xylo-mannan fractions from Nothogenia fastigiata in order to determine their molecular weights and distribution profiles. The number-average molecular weight calculated from the spectra was similar to that determined by chemical end-group analysis for the lower molecular weight fractions. For the other fractions, the number-average molecular weight was lower than that chemically determined; the increased difference may be attributed to higher desorption difficulties and, consequently, mass-dependent discrimination. A reconstructed spectrum, using the peaks obtained from all the fractions, suggested an unimodal distribution. The best results were obtained by using 2,5-dihydroxybenzoic acid as matrix doped with 1-hydroxyisoquinoline and with harmane and nor-harmane.
Journal of The American Society for Mass Spectrometry, 2008
Single-cell cytoplasm sap (1–10 pL) was extracted by using a pressure probe glass microcapillary ... more Single-cell cytoplasm sap (1–10 pL) was extracted by using a pressure probe glass microcapillary tip from tulip leaf and bulb and analyzed by UV-MALDI-TOF MS for free underivatized carbohydrate content. Three matrices including 2,5-dihydroxybenzoic acid (DHB), 2,4,6-trihydroxyacetophenone (THAP), and carbon nanotubes (CNTs) in positive ion mode were selected for analysis because of acceptable carbohydrate-related signal reproducibility. Disaccharide and oligosaccharide (up to 15 Hex when THAP was used, 11 Hex with DHB, and 7 Hex with CNTs) were detected in tulip bulb cell cytoplasm sample. When DHB was used as matrix, neutral carbohydrates were more abundantly detected as sodiated cations; the sugar-related signals, however, appeared as dominant potassiated cations when THAP and CNTs were used. Small amount of monosaccharide was also detected in bulb cell cytoplasm with CNTs as matrix. UV-MALDI-TOF MS of leaf cell extract resulted in high-resolution detection of hexose and disaccharide with DHB, THAP, and CNTs.
Journal of The American Society for Mass Spectrometry, 2009
Probe electrospray ionization (PESI) is a recently developed ESI-based ionization technique which... more Probe electrospray ionization (PESI) is a recently developed ESI-based ionization technique which generates electrospray from the tip of a solid needle. In this study, we have applied PESI interfaced with a time of flight mass spectrometer (TOF-MS) for direct profiling of phytochemicals in a section of a tulip bulb in different regions, including basal plate, outer and inner rims of scale, flower bud and foliage leaves. Different parts of tulip petals and leaves have also been investigated. Carbohydrates, amino acids and other phytochemicals were detected. A series of in vivo PESI-MS experiments were carried out on the second outermost scales of four living tulip bulbs to monitoring the change of carbohydrate content during the first week of initial growth. The breakdown of carbohydrates was observed which was in accordance with previous reports achieved by other techniques. This study has indicated that PESI-MS can be used for rapid and direct analysis of phytochemicals in living biological systems with advantages of low sample consumption and little sample preparation. Therefore, PESI-MS can be a new choice for direct analysis/profiling of bioactive compounds or monitoring metabolic changes in living biological systems.
Underivatized carbohydrates of tulip bulb and leaf tissues were characterized in situ by matrix-a... more Underivatized carbohydrates of tulip bulb and leaf tissues were characterized in situ by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI–TOF MS) by using carbon nanotubes (CNTs) as matrix. Two sample preparation methods—(i) depositing CNTs on the fresh tissue slices placed on the probe and (ii) locating semitransparent tissues on a dried layer of CNTs on the probe—were examined. Furthermore, practicability of in situ starch analysis by MALDI–TOF MS was examined by detection of glucose originated from on-probe amyloglucosidase-catalyzed degradation of starch on the tissue surface. Besides, CNTs could efficiently desorb/ionize natural mono-, di-, and oligosaccharides extracted from tulip bulb tissues as well as glucose resulting from starch enzymatic degradation in vitro. These results were compared with those obtained by in situ MALDI–TOF MS analysis of similar tissues. Positive ion mode showed superior signal reproducibility. CNTs deposited under semitransparent tissue could also desorb/ionize neutral carbohydrates, leading to nearly complete elimination of matrix cluster signals but with an increase in tissue-originated signals. Furthermore, several experiments were carried out to compare the efficiency of 2,5-dihydroxybenzoic acid, nor-harmane, α-cyano-4-hydroxycinnamic acid, and CNTs as matrices for MALDI of neutral carbohydrates from the intact plant tissue surface and for enzymatic tissue starch degradation; these results are discussed in brief. Among matrices studied, the lowest laser power was needed to acquire carbohydrate signals with high signal-to-noise ratio and resolution when CNTs were used.
... Hernan A. Orgueira, † Rosa Erra-Balsells, † Hiroshi Nonami, ‡ and Oscar Varela* †. ... Sci. 1... more ... Hernan A. Orgueira, † Rosa Erra-Balsells, † Hiroshi Nonami, ‡ and Oscar Varela* †. ... Sci. 1996, 4, 25−31. [CAS]. (2) Selegny, E.; Merle-Aubry, L. In Optically Active Polymers; Selegny, E., Ed.; Reidel: Dordrecht, The Netherlands, 1979; pp 15−110. ...
This work was undertaken to determine the growth parameters of Lockhart’s equation for finding wh... more This work was undertaken to determine the growth parameters of Lockhart’s equation for finding which component was predominantly contributing to the cell expansion rates of plants subjected to environmental stresses under tissue-culture conditions. Embryos isolated from soybean (Glycine max [L.] Merr.) and kidney bean (Phaseolus vulgaris L.) seeds were grown under tissue-culture conditions. The water potential of culture media ranged from − 0·02 to − 0·94 MPa so that nutrient deficiency and salt stress conditions could be applied. Additionally, the temperature of culture conditions was set from 10 to 40 °C to apply low-temperature and high-temperature stresses on plants grown at the optimum concentration of culture medium. Cell expansion could be inhibited completely by adding 2,4-dichlorophenoxyacetic acid and benzylaminopurine to culture media to form callus tissue. The sizes of the water potential gradient between the water source and elongating cells correlated with the speed of growth rates under nutrient deficiency, salt stress, growth retardation induced by plant hormones, low-temperature and high-temperature conditions in the present study, indicating that cell expansion rates were mainly associated with how much water could be absorbed by elongating cells regardless of the kinds of environmental stress conditions applied.
Abstract Silsesquioxanes obtained by the hydrolytic condensation of (3-glycidoxypropyl) trimethox... more Abstract Silsesquioxanes obtained by the hydrolytic condensation of (3-glycidoxypropyl) trimethoxysilane (GPMS) in diglycidyl ether of bisphenol A (DGEBA) were characterized by electrospray ionization time-of-flight mass spectrometry (ESI-TOF MS) and matrix-assisted ...
Trypanosoma cruzi, the parasitic protozoan that causes Chagas disease, contains a major cysteine ... more Trypanosoma cruzi, the parasitic protozoan that causes Chagas disease, contains a major cysteine proteinase, cruzipain. This lysosomal enzyme bears an unusual C-terminal extension that contains a number of post-translational modifications, and most antibodies in natural and experimental infections are directed against it. In this report we took advantage of UV-MALDI-TOF mass spectrometry in conjunction with peptide N-glycosidase F deglycosylation and high performance anion exchange chromatography analysis to address the structure of the N-linked oligosaccharides present in this domain. The UV-MALDI-TOF MS analysis in the negative-ion mode, using nor-harmane as matrix, allowed us to determine a new striking feature in cruzipain: sulfated high-mannose type oligosaccharides. Sulfated GlcNAc2Man3 to GlcNAc2Man9 species were identified. In accordance, after chemical or enzymatic desulfation, the corresponding signals disappeared. In addition, by UV-MALDI-TOF MS analysis (a) a main population of high-mannose type oligosaccharides was shown in the positive-ion mode, (b) lactosaminic glycans were also identified, among them, structures corresponding to monosialylated species were detected, and (c) as an interesting fact a fucosylated oligosaccharide was also detected. The presence of the deoxy sugar was further confirmed by high performance anion exchange chromatography. In conclusion, the total number of oligosaccharides occurring in cruzipain was shown to be much higher than previous estimates. This constitutes the first report on the presence of sulfated glycoproteins in Trypanosomatids.
Malaria remains a major health problem especially in tropical and subtropical regions of the worl... more Malaria remains a major health problem especially in tropical and subtropical regions of the world, and therefore developing new antimalarial drugs constitutes an urgent challenge. Lipid metabolism has been attracting a lot of attention as an application for malarial chemotherapeutic purposes in recent years. However, little is known about glycosphingolipid biosynthesis in Plasmodium falciparum. In this report we describe for the first time the presence of an active glucosylceramide synthase in the intraerythrocytic stages of the parasite. Two different experiments, using UDP-[14C]glucose as donor with ceramides as acceptors, or UDP-glucose as donor and fluorescent ceramides as acceptors, were performed. In both cases, we found that the parasitic enzyme was able to glycosylate only dihydroceramide. The enzyme activity could be inhibited in vitro with low concentrations of d,l-threo-phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP). In addition, de novo biosynthesis of glycosphingolipids was shown by metabolic incorporation of [14C]palmitic acid and [14C]glucose in the three intraerythrocytic stages of the parasite. The structure of the ceramide, monohexosylceramide, trihexosylceramide and tetrahexosylceramide fractions was analysed by UV-MALDI-TOF mass spectrometry.When PPMP was added to parasite cultures, a correlation between arrest of parasite growth and inhibition of glycosphingolipid biosynthesis was observed. The particular substrate specificity of the malarial glucosylceramide synthase must be added to the already known unique and amazing features of P. falciparum lipid metabolism; therefore this enzyme might represent a new attractive target for malarial chemotherapy.
Organotrialkoxysilanes containing secondary hydroxyl groups were synthesized by reacting 1 mole o... more Organotrialkoxysilanes containing secondary hydroxyl groups were synthesized by reacting 1 mole of (3-aminopropyl)triethoxysilane (APS) with 1 or 2 mole of phenylglycidylether (PGE). Resulting products, APS–PGE and APS–PGE2, respectively, were subjected to hydrolytic condensation at 50 °C during 24 h. For APS–PGE, the reaction was performed using a molar ratio [H2O]/Si=3, without addition of an external catalyst. For APS–PGE2, the reaction was catalyzed by HCOOH or NaOH. Resulting poly(silsesquioxanes) (PSSO) were characterized by size exclusion chromatography, Fourier-transformed infrared spectroscopy and matrix-assisted ultraviolet laser desorption/ionization time-of-flight mass spectrometry (UV-MALDI-TOF MS). PSSO derived from APS–PGE and APS–PGE2, catalyzed by HCOOH, exhibited a relatively narrow distribution of polyhedral structures. This constitutes a simple one-step synthesis of polyhedral oligomeric silsesquioxanes (POSS) functionalized with amine and/or hydroxyl groups. The directionality of the reaction pathway towards the formation of polyhedral structures was ascribed to the formation of intramolecular SiOC bonds through the reaction of SiOEt or SiOH groups with secondary hydroxyl groups. Intramolecular SiOC bonds were found in the structures of APS–PGE and APS–PGE2, and in most of the species of the PSSO obtained from the NaOH-catalyzed reaction of APS–PGE2. A small fraction of surviving SiOC bonds was also found in the polyhedral structures of the PSSO derived from the hydrolytic condensation of APS–PGE and APS–PGE2 catalyzed by formic acid. By usual organic reactions transforming hydroxyl groups into other functional groups, it is possible to generate narrow distribution of multi-functionalized POSS starting from an OH-functionalized organotrialkoxysilane.The hydrolytic condensation of organotrialkoxysilanes containing secondary hydroxyl groups led to a narrow distribution of OH-functionalized polyhedral oligomeric silsesquioxanes. The generation of closed structures was ascribed to the reversible formation of intramolecular cycles through SiOC bonds. Under appropriate conditions, structures containing these cycles were present in high concentrations.
Several commercial sulfated neocarrabiose oligosaccharides were analyzed by matrix-assisted ultra... more Several commercial sulfated neocarrabiose oligosaccharides were analyzed by matrix-assisted ultraviolet laser-desorption ionization time-of-flight mass spectrometry (UV-MALDI-TOF-MS). UV-MALDI-TOF-MS was carried out in the linear and reflectron modes and, as routine, in both the positive- and negative-ion modes. 2,5-Dihydroxybenzoic acid and nor-harmane were used as matrices. In the positive- and negative-ion modes, with both matrices, peaks corresponding to (M+Na)+ and (M−Na)− ions, respectively, were obtained, with only some signals due to glycosidic linkage cleavages (prompt fragmentation). With 2,5-dihydroxybenzoic acid abundant matrix signals were observed; nor-harmane afforded very few matrix signals in both ion modes, but more desulfation (prompt fragmentation) of the compounds occurred. When the desorption/ionization process was highly efficient, the post-source decay (PSD) fragmentation patterns were also investigated; most of the fragments detected derived from glycosidic linkage cleavages. Electrospray-ionization time-of-flight mass spectrometry (ESI-TOF-MS) in the negative-ion mode confirmed, with the observation of the (M−Na)− and the multiply charged anions, the identity and the purity of the samples.Several commercial sulfated neocarrabiose oligosaccharides were analyzed by UV-MALDI-TOF-MS in the positive- and negative-ion modes, using 2,5-dihydroxybenzoic acid and nor-harmane as matrices; PSD patterns were investigated. ESI-TOF-MS of these analytes was also performed.
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Papers by Hiroshi Nonami