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1.
Acidophiles are typically isolated from sulfate-rich ecological niches yet the role of sulfur metabolism in their growth and
survival is poorly defined. Studies of heterotrophically grown “ Ferroplasma acidarmanus” showed that its growth requires a minimum of 100 mM of a sulfate-containing salt. Headspace gas analyses by GC/MS determined
that the volatile sulfur compound emitted by active “ F. acidarmanus” cultures is methanethiol. In “ F. acidarmanus” cultures grown either heterotrophically or chemolithotrophically, methanethiol was produced constitutively. Radiotracer
studies with 35S-labeled methionine, cysteine, and sulfate showed that all three were used in methanethiol production. Additionally, 3H-labeled methionine was incorporated into methanethiol and was probably used as a methyl-group donor. Methanethiol production
in whole cell lysates supplied with SO 32− indicated that NADPH-dependant sulfite reductase and methyltransferase activities were present. Cell lysates also contained
enzymatic activity for methionine-γ-lyase that cleaved the side chain of either methionine to form methanethiol or cysteine
to produce H 2S. Since methanethiol was detected from the degradation of cysteine, it is likely that sulfide was methylated by a thiol methyltransferase.
Collectively, these data demonstrate that “ F. acidarmanus” produces methanethiol through the metabolism of methionine, cysteine, or sulfate. This is the first report of a methanethiol-producing
acidophile, thus identifying a new contributor to the global sulfur cycle. 相似文献
2.
A dual Hg–Au amalgam electrode is used to detect S-sulfocysteine (SSC) in this study. There exist two main components in the acetonitrile (ACN) rat brain extracts, namely, Cl − and GSSG (oxidized glutathione), that are active in our detection system (GSH is not extracted in ACN). Two strong anion-exchange columns from different companies were used to separate the samples under different conditions, but SSC and Cl − were not separated at the optimum detection pH of 5.2. The signal from Cl − was greatly decreased by lowering the potential at the downstream electrode, though it cannot be completely eliminated. While a silver cartridge removed Cl − from micromoles to several millimoles without any negative effect on the SSC signal in aqueous standards, a large negative peak which interferes with SSC detection was unfortunately introduced when a silver cartridge was applied to brain tissue samples. However, SSC and Cl − in the samples are successfully separated by ion-modified reversed-phase LC in acetate buffer at the optimum detection pH (5.2). The separation conditions are 20 m M acetic acid, 2% methanol, 0.5 m M cetyltrimethylammonium p-toluene sulfonate (CTMA) (pH 5.2). Most importantly, the sensitivity of SSC under the optimum separation conditions is not sacrificed. The detection limit is 8 n M (20 μl injected). 相似文献
3.
In this study, sclerotial differentiation in filamentous phytopathogenic fungi, representing the four main types of sclerotia,
was studied in relation to thiol redox state (TRS)-related enzymes and their substrates/products. TRS was altered by the general
TRS modulator Ν-acetylcysteine (AcCSH) and by the glutathione (GSH) biosynthesis modulators l-oxo-thiazolidine-4-carboxylate (OTC), and l-buthionine-S,R-sulfoximine (BSO). This study showed that the four studied types of sclerotial differentiation are directly
related with the antioxidant –SH groups of GSH and/or CSH, since the decrease of sclerotial differentiation concurred with
an increase of these thiols by the GSH biosynthesis modulators AcCSH, OTC, and BSO. Supportive to that conclusion is the fact
that, in general, the activities of the TRS-related enzymes GR/GPDH and Ttase decrease in the end of the undifferentiated
stage due to the substitution of their antioxidant function by the antioxidant potential of the –SH group providers AcCSH
and OTC. Moreover, it was found that BSO expectedly suppressed GSH biosynthesis in the tested fungi, and unexpectedly decreased
their sclerotial differentiation by a dose-dependent manner typical for antioxidants. The possible antioxidant role of BSO
was supported by the decrease it caused in the antioxidant enzymes GR/GPDH and Ttase. The results of this study are in accordance
with our hypothesis that sclerotial differentiation in phytopathogenic fungi is induced by oxidative stress. 相似文献
5.
Acrylamide is a neurotoxin inhibiting neurotransmission in peripheral nerves. Less is known about acrylamide influence on the central nervous system. Here we measured acrylamide influence on the acetylcholinesterase activity in brain stem, hemispheres, and cerebellum of mice (males, Swiss strain) in relation to the thiol groups and malondialdehyde concentration. Acrylamide was injected intraperitoneally (20 and 40 mg/kg, i.e. 0.52 and 1.04 mg per animal). The brain structures were taken 24, 48, and 192 h after the injection. Acetylcholinesterase activity was significantly lower ( p < 0.001 to p < 0.05) in all the structures. It was accompanied by the statistically significant ( p < 0.001 to p < 0.05) increase in malondialdehyde concentrations in most of the studied structures time periods and ACR doses. –SH groups concentrations were significantly depleted in the right hemisphere ( p < 0.01) after 24 h and in brain stem ( p < 0.05) after 48 h. We suggest that neurotoxicity of acrylamide in brain is related to acetylcholinesterase inhibition and redox imbalance. 相似文献
6.
Succinic semialdehyde dehydrogenase (EC 1.2.1.16) was purified 74-fold from wheat grain ( Triticum durum Desf.). The enzyme appears quite specific for succinic semialdehyde (SSA). Both NAD and NADP support the oxidation of the substrate, but the former is 7-fold more active than the latter. The optimum pH for activity is around 9; the enzyme is stable in the pH range 6–9 and retains its whole activity up to 40°C. The enzyme activity is strongly dependent on the presence of mercaptoethanol, other thiol compounds being much less effective. Kinetic data support the formation of a ternary complex between enzyme, substrate and coenzyme. The K
m for SSA and for NAD are 7.4x10 -6 M and 2x10 -4 M, respectively. The molecular weight of the enzyme protein was estimated by gel-filtration to be about 130,000.Abbreviations GABA
-aminobutyric acid
- GABA-T
-aminobutyric acid transaminase
- ME
mercaptoethanol
- SSA
succinic semialdehyde
- SSA-DH
succinic semialdehyde dehydrogenase 相似文献
7.
Amyloid diseases, including Alzheimer's disease, are characterized by aggregation of normally functioning proteins or peptides into ordered, β-sheet rich fibrils. Most of the theories on amyloid toxicity focus on the nuclei or oligomers in the fibril formation process. The nuclei and oligomers are transient species, making their full characterization difficult. We have isolated toxic protein species that act like an oligomer and may provide the first evidence of a stable reactive species created by disaggregation of amyloid fibrils. This reactive species was isolated by dissolving amyloid fibrils at high pH and it has a mass >100 kDa and a diameter of 48 ± 15 nm. It seeds the formation of fibrils in a dose dependent manner, but using circular dichroism and deep ultraviolet resonance Raman spectroscopy, the reactive species was found to not have a β-sheet rich structure. We hypothesize that the reactive species does not decompose at high pH and maintains its structure in solution. The remaining disaggregated insulin, excluding the toxic reactive species that elongated the fibrils, returned to native structured insulin. This is the first time, to our knowledge, that a stable reactive species of an amyloid reaction has been separated and characterized by disaggregation of amyloid fibrils. 相似文献
8.
This study showed that sclerotial differentiation in the filamentous phytopathogenic fungus Rhizoctonia solani is directly related to oxidative stress and thiol redox state (TRS). Sclerotial differentiation is modulated by the availability
of non-cytotoxic −SH groups as was shown by the inhibition of sclerorial differentiation by the TRS modulator N-acetyl cysteine
(AcCSH), and not necessarily with those of the TRS reduced components glutathione (GSH) and its precursor cysteine (CSH) as
indicated by the GSH-biosynthesis inducer and inhibitor l-2–oxo-thiazolidine-4-carboxylate and l-buthionine-S,R-sulfoximine, respectively. Moreover, inhibition of sclerotial differentiation was accompanied by decrease
of the high oxidative stress indicators, lipid peroxidation and DNA damage in the mycelial substrate where sclerotia initials
are formed, which suggests that this phenomenon is related to oxidative stress as it is predicted by our theory on sclerotial
differentiation. 相似文献
9.
A novel method for isolation and de novo sequencing of N-terminal peptides from proteins is described. The method presented here combines selective chemical tagging using succinimidyloxycarbonylmethyl tris(2,4,6-trimethoxyphenyl)phosphonium bromide (TMPP-Ac-OSu) at the Nα-amino group of peptides after digestion by metalloendopeptidase (from Grifola frondosa) and selective capture procedures using p-phenylenediisothiocyanate resin, by which the N-terminal peptide can be isolated, whether or not it is N-terminally blocked. The isolated N-terminal peptide modified N-terminally with TMPP-Ac-OSu reagent produces a simple fragmentation pattern under tandem mass spectrometric analysis to significantly facilitate sequencing. 相似文献
10.
Chitosan (Ch) was chemically modified with ethylenesulfide (Es) under solvent-free conditions to give (ChEs), displaying a high content of thiol groups due to opening of the three member cyclic reagent. Elemental analysis showed a decrease in nitrogen content. This result indicated the incorporation of two ethylenesulfide molecules for each unit of the polymeric structure of the precursor biopolymer. Infrared spectroscopy, thermogravimetry, and 13C NMR in the solid state demonstrated the effectiveness of the reaction, with signals at 30 ppm for ChEs due to the change in the methylene group environment. Divalent metal uptake by chemically modified biopolymer gave the order Cu > Ni > Co > Zn, reflecting the corresponding acidity of these cations in bonding to the sulfur and the basic nitrogen atoms available on the pendant chains. The equilibrium data were fitted to Freundlich, Temkin, and Langmuir models. The maximum monolayer adsorption capacity for the cations was found to be 1.54 ± 0.02, 1.25 ± 0.03, 1.13 ± 0.01, and 0.83 ± 0.03 mmol g −1, respectively. The Langmuir model best explained the cation–sulfur bond interactions at the solid–liquid interface. The thermodynamics for these interactions gave exothermic enthalpic values of −43.02 ± 0.03, −28.72 ± 0.02, −26.27 ± 0.04, and −17.32 ± 0.02 kJ mol −1, respectively. The spontaneity of the systems is given by negative Gibbs free energies of −31.2 ± 0 .1, −32.7 ± 0 .1, −31.7 ± 0.1, and −32.2 ± 0 .1 kJ mol −1, respectively, in spite of the unfavorable negative entropic values of −39 ± 1, −13 ± 1, −18 ± 1, and −49 ± 1 J K −1 mol −1 due to solvent ordering in the course of complexation. This newly synthesized biopolymer is presented as a chemically useful material for cation removal from aqueous solution. 相似文献
11.
Spectral reflectance and transmittance of leaves to ultraviolet irradiation were determined under laboratory conditions for seven species of hardwood trees, namely red oak ( Quercus rubra, L), black oak ( Q. velutina, Lamarch), white oak ( Q. alba, L.), sugar maple ( Acer saccharum), Norway maple ( A. plantanoides), hickory ( Carya tomemtosa), sweetgum ( Liquidambar styraciflua), and black oak litter. The experimental system consisted of a solar simulator, an integrating sphere, and a spectroradiometer. Experiments were repeated three to five times for both adaxial and abaxial surfaces of fresh leaves chosen at randomly. The spectral distributions and simple averages of the radiative properties in the wavelength ranges of ultraviolet-B (UV-B, 280–320 nm) and ultraviolet-A (UV-A, 320–400 nm) were determined. The spectral distributions of reflectance were similar between adaxial and abaxial surfaces, although the magnitude varied among tree species. Leaf reflectance was very low for the ultraviolet spectrum in general and varied among species and between adaxial and abaxial surfaces. It was generally higher over the UV-A waveband compared to UV-B, and higher on the abaxial than adaxial surface. The broadband reflectance in the UV-A range (over all species) was 5.0 and 3.9% for abaxial and adaxial surface, respectively, compared to 3.5 and 2.8% in UV-B. The transmittance through leaves was extremely small in the UV-B (<0.1%) and nearly zero in the UV-A spectral range. Consequently, the absorptance of ultraviolet radiation by leaves, as determined from the measured reflectance and transmittance, was quite high, being more than 90% for all the combinations of species and wavebands examined. The reported results are useful for studies requiring spectral radiative properties of the examined leaves with respect to ultraviolet irradiation. 相似文献
12.
Heat shock induces resistance to killing by ultraviolet light in Escherichia coli JE1011. When cells growing at 30°C were transferred to 42°C, maximum resistance to ultraviolet radition was reached after 30–45 min, but no change in heat resistance occurred. The effect was dependent on growth or protien synthesis. In contrast, E. coli B becomes more sensitive to the radiation and more heat resistant after a similar treatment. Thus, ultraviolet resistance and thermal resistance are not induced together in these two strains and may arise by independent mechanisms. It is also possible that the lon gene is involved in the effect of heat shock on ultraviolet resistance. 相似文献
13.
An improved bacterial preculture protocol for Agrobacterium-mediated genetic transformation was developed for an economic tomato cultivar ( Solanum lycopersicum L. cv. Zhongshu No. 4). Frequencies of transient gene expression and stable transformation were influenced by the density
of Agrobacterium preculture and not the density of Agrobacterium used for infection. The improved protocol presented in this study depends on the use of an overnight-grown Agrobacterium preculture density of OD 600 nm = 1.0, diluted 1/10th with Luria-Bertani (LB) liquid medium, and grown for an additional 4 h. Cultures are collected and
resuspended in a liquid cocultivation medium-I, adjusted to OD 600 nm = 0.1. Using this modified Agrobacterium preparation, transient β-glucuronidase expression was higher than 90%, and transformation efficiency reached 44.7%. This
improved transformation is simple, repeatable, does not require a feeder layer, and most notably, the transformation frequency
is stable and highly efficient. 相似文献
14.
Traditional protocols for preparing Escherichia coli for electroporation are laborious and often deliver highly variable transformation efficiencies. Many laboratories resort to purchasing expensive commercially prepared cells. This article describes a simple method for producing electrocompetent E. coli by centrifuging bacteria through a glycerol/mannitol density cushion. The method is rapid and replaces tedious multistep procedures with two 15-min centrifugations. Standard cloning strains consistently produce more than 8 × 10 9 transformants/μg pUC18, whereas the strains TG1 and LE392 display efficiencies of more than 3 × 10 10/μg DNA. 相似文献
15.
Typical physico-chemical studies of metal binding proteins are usually aimed at determination of the metal binding constant
K for a native protein ( K
n), while the significance of the K value for the thermally denatured protein ( K
u) is usually underestimated. Meanwhile, metal binding induced shift of thermal denaturation transition of a single site metal
binding protein is defined by K
n to K
u ratio, implying that knowledge of both K values is required for full characterization of the system. In the present work, the most universal approach to the studies
of single site metal binding proteins, namely construction of a protein “phase diagram” in coordinates of free metal ion concentration
– temperature, is considered in detail. The detailed algorithm of construction of the phase diagrams along with underlying
mathematic procedures developed here may be of use for studies of other simple protein-target type systems, where target represents
low molecular weight ligand. Analysis of the simplest protein-ligand system reveals that thermodynamic properties of apo-protein
dictate the maximal possible increase of its affinity to any simple ligand upon thermal denaturation of the protein. Experimental
and general problems coupled with the use of the phase diagrams are discussed. 相似文献
16.
Summary The striking similarity between the treatments that induce SOS functions and those that result in stable DNA replication (continuous DNA replication in the absence of protein synthesis) prompted us to examine the possibility of stable DNA replication being a recA
+
lexA
+-dependent SOS function. In addition to the treatments previously reported, ultraviolet (UV) irradiation or treatment with mitomycin C was also found to induce stable DNA replication.The thermal treatment of tif-1 strains did not result in detectable levels of stable DNA replication, but nalidixic acid readily induced the activity in these strains. The induction of stable DNA replication with nalidixic acid was severely suppressed in tif-1 lexA mutant strains. The inhibitory activity of lexA3 was negated by the presence of the spr-51 mutation, an intragenic suppressor of lexA3.Induced stable DNA replication was found to be considerably more resistant to UV irradiation than nromal replication both in a uvrA6 strain and a uvr
+ strain. The UV-resistant replication occurred mostly in the semiconservative manner. The possible roles of stable DNA replication in repair of damaged DNA are discussed. 相似文献
17.
Objectives: The etiology of multiple myeloma (MM) is not exactly known. This study investigated the role of thiol/disulfide homeostasis in the etiopathogenesis of MM. Methods: Some 50 patients with MM (aged 39–84 years) and 50 sex-matched healthy volunteer controls (aged 50–91 years) participated in this study. Venous blood samples were collected, and levels of native thiols, total thiols, and disulfide were measured. Results: Native and total thiol levels in the control group were determined to be higher than in the study and patient groups (P<0.001). Disulfide levels were found to be higher in the control group than in the study group and higher in newly diagnosed patients than in outpatients who were undergoing treatment (P=0.002). The ratios of thiol levels were found to be similar in both the study and control groups (P>0.05). Discussion: The results of the study show that although there was a decrease in the levels of disulfide, native thiol, and total thiol, the balance of thiol/disulfide was maintained. This is the first study to research the homeostasis of dynamic thiol/disulfide from the perspective of the new method that was used. We hope that this study will encourage and facilitate further studies in this area. 相似文献
18.
With the completion of the poplar tree genome database, Populus species have become one of the most useful model systems for the study of woody plant biology. Populus tremuloides (quaking aspen) is the most wide-spread tree species in North America, and its rapid growth generates the most abundant wood-based
biomass out of any other plant species. To study such beneficial traits, there is a need for easier and more efficient transformation
procedures that will allow the study of large numbers of tree genes. We have developed transformation procedures that are
suitable for high-throughput format transformations using either Agrobacterium tumefaciens to produce transformed trees or Agrobacterium rhizogenes to generate hairy roots. Our method uses Agrobacterium inoculated aspen seedling hypocotyls followed by direct thidiazuron (TDZ)-mediated shoot regeneration on selective media.
Transformation was verified through β-glucuronidase (GUS) reporter gene expression in all tree tissues, PCR amplification
of appropriate vector products from isolated genomic DNA, and northern hybridization of incorporated and expressed transgenes.
The hairy root protocol follows the same inoculation procedures and was tested using GUS reporter gene integration and antibiotic
selection. The benefit of these procedures is that they are simple and efficient, requiring no maintenance of starting materials
and allowing fully formed transgenic trees (or hairy roots) to be generated in only 3–4 months, rather than the 6–12 months
required by more traditional methods. Likewise, the fact that the protocols are amenable to high-throughput formats makes
them better suited for large-scale functional genomics studies in poplars. 相似文献
19.
Cells of Bacillus subtilis
168 with deletions in the citD locus were found to be sensitive to irradiation with ultraviolet light and to mitomycin C but were able to repair DNA damage
induced by methyl methanesulfonate. The recombination abilities of these cells, as determined by transformation and PBS1-mediated
transduction experiments, were unaffected by the deletion. These phenotypic characteristics do not result from a metabolic
imbalance caused by the deficiency of a functional α-ketoglutarate dehydrogenase complex, but most likely are a consequence
of a genetic locus involved in ultraviolet repair, which is deleted together with the citK gene when the deletion is formed. 相似文献
20.
The dynamics of a general in-host model with intracellular delay is studied. The model can describe in vivo infections of HIV-I, HCV, and HBV. It can also be considered as a model for HTLV-I infection. We derive the basic reproduction
number R
0 for the viral infection, and establish that the global dynamics are completely determined by the values of R
0. If R
0≤1, the infection-free equilibrium is globally asymptotically stable, and the virus are cleared. If R
0>1, then the infection persists and the chronic-infection equilibrium is locally asymptotically stable. Furthermore, using
the method of Lyapunov functional, we prove that the chronic-infection equilibrium is globally asymptotically stable when
R
0>1. Our results shows that for intercellular delays to generate sustained oscillations in in-host models it is necessary have
a logistic mitosis term in target-cell compartments. 相似文献
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