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Manipulation of Glutathione and Amino Acid Biosynthesis in the
Chloroplast 总被引:19,自引:1,他引:18 下载免费PDF全文
Graham Noctor Ana-Carolina M. Arisi Lise Jouanin Christine H. Foyer 《Plant physiology》1998,118(2):471-482
Poplars (Populus tremula × Populus alba) were transformed to overexpress Escherichia coli γ-glutamylcysteine synthetase (γ-ECS) or glutathione synthetase in the chloroplast. Five independent lines of each transformant strongly expressed the introduced gene and possessed markedly enhanced activity of the gene product. Glutathione (GSH) contents were unaffected by high chloroplastic glutathione synthetase activity. Enhanced chloroplastic γ-ECS activity markedly increased γ-glutamylcysteine and GSH levels. These effects are similar to those previously observed in poplars overexpressing these enzymes in the cytosol. Similar to cytosolic γ-ECS overexpression, chloroplastic overexpression did not deplete foliar cysteine or methionine pools and did not lead to morphological changes. Light was required for maximal accumulation of GSH in poplars overexpressing γ-ECS in the chloroplast. High chloroplastic, but not cytosolic, γ-ECS activities were accompanied by increases in amino acids synthesized in the chloroplast. We conclude that (a) GSH synthesis can occur in the chloroplast and the cytosol and may be up-regulated in both compartments by increased γ-ECS activity, (b) interactions between GSH synthesis and the pathways supplying the necessary substrates are similar in both compartments, and (c) chloroplastic up-regulation of GSH synthesis is associated with an activating effect on the synthesis of specific amino acids formed in the chloroplast. 相似文献
3.
Purification and Characterization of Phosphoribulokinase from the
Marine Chromophytic Alga Heterosigma
carterae 总被引:2,自引:0,他引:2 下载免费PDF全文
In this study we characterized phosphoribulokinase (PRK, EC 2.7.1.19) from the eukaryotic marine chromophyte Heterosigma carterae. Serial column chromatography resulted in approximately 300-fold purification of the enzyme. A polypeptide of 53 kD was identified as PRK by sequencing the amino terminus of the protein. This protein represents one of the largest composite monomers identified to date for any PRK. The native holoenzyme demonstrated by flow performance liquid chromatography a molecular mass of 214 ± 12.6 kD, suggesting a tetrameric structure for this catalyst. Because H. carterae PRK activity was insensitive to NADH but was stimulated by dithiothreitol, it appears that the enzyme may require a thioredoxin/ferredoxin rather than a metabolite mode of regulation. Kinetic analysis of this enzyme demonstrated Michaelis constant values of ribulose-5-phosphate (226 μm) and ATP (208 μm), respectively. In summary, H. carterae PRK is unique with respect to holoenzyme structure and function, and thus may represent an alternative evolutionary pathway in Calvin-cycle kinase development. 相似文献
4.
Transport of Palmitic Acid Across the
Tegument of the Entomophilic Nematode
Romanomermis culicivorax
Romanomermis culicivorax juveniles, dissected out of Aedes aegypti larvae 7 days after infection, were incubated under controlled conditions in isotonic saline containing ¹⁴C-U-palmitic acid to investigate the nature of the transport mechanism(s) used by the nematode for transcuticular uptake of palmitic acid. Net uptake of the isotope by the nematode was of a logarithmic nature with respect to time. Uptake of palmitic acid was accomplished by a combination of diffusion and a mediated process which was substrate saturable and competitively inhibited by myristic and stearic acids. Both 2,4-dinitrophenol and ouabain inhibited uptake of palmitic acid and thus supported the hypothesis that the carrier system is of the active transport variety and is coupled to a Na⁺K⁺ ATPase pump. 相似文献
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Nicoletta Bodrato Luisa Franco Chiara Fresia Lucrezia Guida Cesare Usai Annalisa Salis Iliana Moreschi Chiara Ferraris Claudia Verderio Giovanna Basile Santina Bruzzone Sonia Scarf�� Antonio De Flora Elena Zocchi 《The Journal of biological chemistry》2009,284(22):14777-14787
Abscisic acid (ABA) is a phytohormone regulating important functions in
higher plants, notably responses to abiotic stress. Recently, chemical or
physical stimulation of human granulocytes was shown to induce production and
release of endogenous ABA, which activates specific cell functions. Here we
provide evidence that ABA stimulates several functional activities of the
murine microglial cell line N9 (NO and tumor necrosis factor-α
production, cell migration) through the second messenger cyclic ADP-ribose and
an increase of intracellular calcium. ABA production and release occur in N9
cells stimulated with bacterial lipopolysaccharide, phorbol myristate acetate,
the chemoattractant peptide f-MLP, or β-amyloid, the primary plaque
component in Alzheimer disease. Finally, ABA priming stimulates N9 cell
migration toward β-amyloid. These results indicate that ABA is a
pro-inflammatory hormone inducing autocrine microglial activation, potentially
representing a new target for anti-inflammatory therapies aimed at limiting
microglia-induced tissue damage in the central nervous system.Microglial cells are the monocyte/macrophage equivalent of the central
nervous system and represent the first line of defense in the brain, by
removing infectious agents and damaged cells
(1). Microglia can also release
a variety of trophic factors and cytokines able to regulate the communication
between neuronal and other glial cells and can contribute to tissue repair and
neuroprotection
(2–4).
Pathologic microglial activation, however, confers neurotoxic properties to
these cells, thereby causing neuronal degeneration
(5). Excessive activation of
microglia, under conditions of chronic inflammation, can contribute to the
pathogenesis of neurodegenerative diseases, such as multiple sclerosis and
Alzheimer and Parkinson diseases, by producing and releasing a number of
potentially cytotoxic substances, including pro-inflammatory cytokines and NO
(4,
6–8).
Therefore, identification of the molecular mechanisms underlying microglial
activation might lead to the development of new anti-inflammatory drugs for
the treatment of these diseases.Abscisic acid
(ABA)2 is a plant
hormone regulating important biological functions in higher plants, including
response to abiotic stress, control of stomatal closure, regulation of seed
dormancy, and germination (9).
Recently, ABA was shown to behave as an endogenous pro-inflammatory hormone in
human granulocytes (10),
stimulating several functional activities of these cells (migration,
phagocytosis, reactive oxygen species, and NO production) through a signaling
cascade that involves a protein kinase A-mediated ADP-ribosyl cyclase
phosphorylation and consequent overproduction of the universal Ca2+
mobilizer cyclic ADP-ribose (cADPR)
(11). This mechanism leads to
an increase of the intracellular Ca2+ concentration, which is
ultimately responsible for granulocyte activation
(10).The facts that microglial cells play a defensive role in the central
nervous system similar to that of granulocytes in other tissues and that cADPR
has been described as the second messenger involved in the activation of
microglia induced by lipopolysaccharide (LPS)
(12) prompted us to
investigate the effect of ABA in these cells.Indeed, exogenous ABA, at concentrations ranging from 250 nm to
20 μm, elicits functional activation of murine N9 cells,
stimulating TNF-α release and cell migration through activation of the
ADP-ribosyl cyclase CD38 and overproduction of cADPR. Moreover, N9 cells
produce and release ABA when stimulated with LPS, amyloid β-peptide
(βA), phorbol myristate acetate (PMA), or the chemoattractant peptide
f-MLP. These results indicate that ABA behaves as an endogenous,
pro-inflammatory hormone in murine microglia and provide a new target for
future investigations into the role of this hormone in inflammatory and
degenerative diseases of the central nervous system accompanied by microglial
activation. 相似文献
8.
Development of Multiplex PCR Assays for the Identification of the 33 Serotypes of Streptococcus
suis
Zhijie Liu Han Zheng Marcelo Gottschalk Xuemei Bai Ruiting Lan Shaobo Ji Haican Liu Jianguo Xu 《PloS one》2013,8(8)
Streptococcus
suis
is an important zoonotic agent causing severe diseases in pigs and humans. To date, 33 serotypes of
S
. suis
have been identified based on antigenic differences in the capsular polysaccharide. The capsular polysaccharide synthesis (cps) locus encodes proteins/enzymes that are responsible for capsular production and variation in the capsule structures are the basis of
S
. suis
serotyping. Multiplex and/or simplex PCR assays have been developed for 15 serotypes based on serotype-specific genes in the cps gene cluster. In this study, we developed a set of multiplex PCR (mPCR) assays to identify the 33 currently known
S
. suis
serotypes. To identify serotype-specific genes for mPCR, the entire genomes of reference strains for the 33 serotypes were sequenced using whole genome high-throughput sequencing, and the cps gene clusters from these strains were identified and compared. We developed a set of 4 mPCR assays based on the polysaccharide polymerase gene wzy, one of the serotype-specific genes. The assays can identify all serotypes except for two pairs of serotypes: 1 and 14, and 2 and 1/2, which have no serotype-specific genes between them. The first assay identifies 12 serotypes (serotypes 1 to 10, 1/2, and 14) that are the most frequently isolated from diseased pigs and patients; the second identifies 10 serotypes (serotypes 11 to 21 except 14); the third identifies the remaining 11 serotypes (serotypes 22 to 31, and 33); and the fourth identifies a new cps cluster of
S
. suis
discovered in this study in 16 isolates that agglutinated with antisera for serotypes 29 and 21. The multiplex PCR assays developed in this study provide a rapid and specific method for molecular serotyping of
S
. suis
. 相似文献
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We refer to Oswaldo Cruz''s reports dating from 1913 about the necessities of a
healthcare system for the Brazilian Amazon Region and about the journey of Carlos
Chagas to 27 locations in this region and the measures that would need to be adopted.
We discuss the risks of endemicity of Chagas disease in the Amazon Region. We
recommend that epidemiological surveillance of Chagas disease in the Brazilian Amazon
Region and Pan-Amazon region should be implemented through continuous monitoring of
the human population that lives in the area, their housing, the environment and the
presence of triatomines. The monitoring should be performed with periodic
seroepidemiological surveys, semi-annual visits to homes by health agents and the
training of malaria microscopists and healthcare technicians to identify
Trypanosoma cruzi from patients'' samples and T.
cruzi infection rates among the triatomines caught. We recommend health
promotion and control of Chagas disease through public health policies, especially
through sanitary education regarding the risk factors for Chagas disease. Finally, we
propose a healthcare system through base hospitals, intermediate-level units in the
areas of the Brazilian Amazon Region and air transportation, considering the
distances to be covered for medical care. 相似文献
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The influenza virus matrix protein 2 (M2) assembles into a tetramer in the host membrane during viral uncoating and maturation. It has been used as a model system to understand the relative contributions of protein-lipid and protein-protein interactions to membrane protein structure and association. Here we investigate the effect of lipid chain length on the association of the M2 transmembrane domain into tetramers using Förster resonance energy transfer. We observe that the interactions between the M2 helices are much stronger in 1,2-dilauroyl-sn-glycero-3-phosphocholine than in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine bilayers. Thus, lipid chain length and bilayer thickness not only modulate peptide interactions, but could also be a major determinant of the association of transmembrane helices into functional membrane protein oligomers. 相似文献
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Fluoroacetic acid is known to lead to inhibition of aconitase and block both the Krebs and glyoxylate cycles. In this study, we discovered it to be a potent and specific inhibitor of reproduction in a bioassay using the nematode Caenorhabditis elegans. Fluoroacetic acid added to the growth medium reduced reproduction in the second generation by 50% at concentrations 3,000 times lower than the concentrations that reduced 24-hour survival by 50%. Four concentrations (2, 4, 8, and 17 mM) of fluoroacetic acid were tested thoroughly. At the two lower concentrations, the survival rates were unaffected, and first-generation reproduction was greatly reduced but not completely eliminated. Survival was reduced at the higher concentrations. Malonate, which inhibits the Krebs cycle, and itaconate, which inhibits the glyoxylate cycle, were tested individually and in combination. The combination did not specifically inhibit reproduction, suggesting another mode of action for fluoroacetic acid. Fluoroacetic acid shows promise as a tool in studies requiring age synchrony. 相似文献
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We have achieved, to our knowledge,
the first high-level heterologous expression of the gene encoding
d-ribulose-5-phosphate 3-epimerase from any source, thereby
permitting isolation and characterization of the epimerase as found in
photosynthetic organisms. The extremely labile recombinant spinach
(Spinacia oleracea L.) enzyme was stabilized by
dl-α-glycerophosphate or ethanol and destabilized by
d-ribulose-5-phosphate or 2-mercaptoethanol. Despite this
lability, the unprecedentedly high specific activity of the purified
material indicates that the structural integrity of the enzyme is
maintained throughout isolation. Ethylenediaminetetraacetate and
divalent metal cations did not affect epimerase activity, thereby
excluding a requirement for the latter in catalysis. As deduced from
the sequence of the cloned spinach gene and the electrophoretic
mobility under denaturing conditions of the purified recombinant
enzyme, its 25-kD subunit size was about the same as that of the
corresponding epimerases of yeast and mammals. However, in contrast to
these other species, the recombinant spinach enzyme was octameric
rather than dimeric, as assessed by gel filtration and polyacrylamide
gel electrophoresis under nondenaturing conditions. Western-blot
analyses with antibodies to the purified recombinant enzyme confirmed
that the epimerase extracted from spinach leaves is also octameric.As a participant in the oxidative pentose phosphate pathway, Ru5P
epimerase (EC 5.1.3.1), which catalyzes the interconversion of Ru5P and
Xu5P, is widely distributed throughout nature. Beyond its catabolic
role, the epimerase is also vital anabolically to photosynthetic
organisms in the regenerative phase of the reductive pentose phosphate
pathway (the Calvin cycle). In this capacity, Ru5P epimerase directs
Xu5P, formed in two distinct transketolase reactions of the cycle, to
Ru5P. Phosphorylation of the latter regenerates
d-ribulose-1,5-bisphosphate, the substrate for net
CO2 fixation. Because both the oxidative and
reductive pentose phosphate pathways coexist in chloroplasts
(Schnarrenberger et al., 1995), Ru5P epimerase and R5P isomerase
facilitate partitioning of pentose phosphates between the two pathways,
as dictated by the metabolic needs and redox status of the cell.Scant structural and mechanistic information about Ru5P epimerase is
available despite its inherent importance and dual metabolic roles.
This neglect may in part reflect the low natural abundance of the
enzyme. For example, achievement of electrophoretic homogeneity
required a 2000-fold purification from yeast (Bär et al., 1996)
and spinach (Spinacia oleracea L.) chloroplasts (Teige et
al., 1998) and 9000-fold purification from beef liver (Terada et al.,
1985). Although low overall recoveries (<10%) further limited the
availability of pure material, molecular sieving and denaturing
electrophoresis established that the epimerases from mammals (Wood,
1979; Karmali et al., 1983; Terada et al., 1985) and yeast (Bär
et al., 1996) are homodimers of approximately 23-kD subunits, whereas
the enzyme from spinach chloroplasts may be an octamer of 23-kD
subunits (Teige et al., 1998). DNA-deduced amino acid sequences of Ru5P
epimerases from both photosynthetic and nonphotosynthetic sources,
which confirm this estimated subunit size, show greater than 50%
similarities among the most evolutionarily distant species examined
(Kusian et al., 1992; Blattner et al., 1993; Falcone and Tabita, 1993;
Lyngstadaas et al., 1995; Nowitzki et al., 1995; Teige et al.,
1995).Although Ru5P epimerase has very recently been purified from a
photosynthetic organism (spinach) for the first time (Teige et al.,
1998), the low recovery (100 μg from 3.8 g of soluble
chloroplast protein, representing an overall yield of 5%) imposes
severe constraints on the directions of future experiments.
Furthermore, despite successful cloning of cDNA fragments encoding Ru5P
epimerase of several photosynthetic organisms (Kusian et al., 1992;
Nowitzki et al., 1995; Teige et al., 1995), to our knowledge high-level
heterologous expression and purification of enzymically active
recombinant enzyme have not been achieved. Because of our interest in
the regulation of photosynthetic carbon assimilation and the requisite
need for ample supplies of the participant enzymes for use in
mechanistic studies, we have attempted to optimize the heterologous
expression of the spinach gene for Ru5P epimerase. In this paper we
report cDNA clones that encode the mature chloroplastic enzyme or its
cytoplasmic precursor. We also describe an efficient isolation
procedure for the mature spinach enzyme synthesized in
Escherichia coli and some of the properties of the purified
enzyme. Contrasting features of the plant Ru5P epimerase, relative to
the animal and yeast counterparts, include an octameric rather than a
dimeric structure (also see Teige et al., 1998) and striking
instability under routine laboratory conditions. 相似文献
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Jo?o Victor Leite Dias Dimas Ramon Mota Queiroz Helen Rodrigues Martins David Eladio Gorla Herton Helder Rocha Pires Liléia Diotaiuti 《Memórias do Instituto Oswaldo Cruz》2016,111(1):43-50
Reports of triatomine infestation in urban areas have increased. We analysed the
spatial distribution of infestation by triatomines in the urban area of Diamantina,
in the state of Minas Gerais, Brazil. Triatomines were obtained by community-based
entomological surveillance. Spatial patterns of infestation were analysed by Ripley’s
K function and Kernel density estimator. Normalised difference vegetation index
(NDVI) and land cover derived from satellite imagery were compared between infested
and uninfested areas. A total of 140 adults of four species were captured (100
Triatoma vitticeps, 25Panstrongylus geniculatus,
8 Panstrongylus megistus, and 7 Triatoma
arthurneivai specimens). In total, 87.9% were captured within domiciles.
Infection by trypanosomes was observed in 19.6% of 107 examined insects. The spatial
distributions ofT. vitticeps, P. geniculatus,
T. arthurneivai, and trypanosome-positive triatomines were
clustered, occurring mainly in peripheral areas. NDVI values were statistically
higher in areas infested by T. vitticeps and P.
geniculatus. Buildings infested by these species were located closer to
open fields, whereas infestations of P. megistus andT.
arthurneivai were closer to bare soil. Human occupation and modification
of natural areas may be involved in triatomine invasion, exposing the population to
these vectors. 相似文献
16.
Photosystem I (PSI) is a reaction center
associated with oxygenic photosynthesis. Unlike the monomeric reaction centers in
green and purple bacteria, PSI forms trimeric
complexes in most cyanobacteria with a 3-fold rotational symmetry that is primarily
stabilized via adjacent PsaL subunits; however, in plants/algae, PSI is monomeric. In this study, we discovered a
tetrameric form of PSI in the thermophilic
cyanobacterium Chroococcidiopsis sp TS-821 (TS-821). In TS-821,
PSI forms tetrameric and dimeric species.
We investigated these species by Blue Native PAGE, Suc density gradient
centrifugation, 77K fluorescence, circular dichroism, and single-particle analysis.
Transmission electron microscopy analysis of native membranes confirms the presence
of the tetrameric PSI structure prior to
detergent solubilization. To investigate why TS-821 forms tetramers instead of
trimers, we cloned and analyzed its psaL gene. Interestingly, this
gene product contains a short insert between the second and third predicted
transmembrane helices. Phylogenetic analysis based on PsaL protein sequences shows
that TS-821 is closely related to heterocyst-forming cyanobacteria, some of which
also have a tetrameric form of PSI. These
results are discussed in light of chloroplast evolution, and we propose that PSI evolved stepwise from a trimeric form to
tetrameric oligomer en route to becoming monomeric in plants/algae. 相似文献
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