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71.
Cells and body fluids contain numerous, different proteinases; to identify and characterize them are both important and difficult tasks. Especially difficult to identify and characterize are highly specific proteinases. Here, we present an extremely sensitive and quantitative method to characterize proteinases fractionated by SDS-PAGE that cleave specific rhodamine-based fluorogenic substrates. To test the sensitivity of the technique, we used trypsin as our model system. Filter paper impregnated with rhodamine-based fluorogenic substrates was placed on a gel, and bands of fluorescence originating from specific proteinases were visualized in real time. The method is very sensitive; picogram amounts of trypsin can be detected. The method should be very general, in that even proteinases whose substrates require amino acids C-terminal to the cleavage site may be identified and characterized. The results allow one to obtain not only information on the substrate specificity of a specific enzyme but also information about its molecular weight. 相似文献
72.
Whittock NV Eady RA McGrath JA 《Biochemical and biophysical research communications》2000,267(1):462-465
Keratin intermediate filaments are the major components of the cytoskeleton in epithelial cells. Mutations in keratin genes have been documented in many disorders of the skin, nails, hair, and mucous membranes. Although no mutations have been described in either keratin 15 or keratin 19, they are good candidates for other as yet uncharacterized genetic disorders of keratinization, particularly as the skin, nails, hair, and conjunctiva are sites of keratin 15 and 19 expression. To facilitate future mutation detection analyses, we have therefore characterized the intron-exon organization of the human keratin 15 and keratin 19 genes. The keratin 15 gene comprises 8 exons spanning approximately 5.1 kb on 17q21, and the keratin 19 gene consists of 6 exons covering approximately 4.7 kb on 17q21. We have also developed a PCR-based mutation detection strategy using primers placed on flanking introns followed by direct sequencing of the PCR products. 相似文献
73.
Cellular compartmentation of cadmium and zinc in relation to other elements in the hyperaccumulator Arabidopsis halleri 总被引:21,自引:0,他引:21
The cellular compartmentation of elements was analysed in the Zn hyperaccumulator Arabidopsis halleri (L.) O'Kane & Al-Shehbaz (=Cardaminopsis halleri) using energy-dispersive X-ray microanalysis of frozen-hydrated tissues. Quantitative data were obtained using oxygen as
an internal standard in the analyses of vacuoles, whereas a peak/background ratio method was used for quantification of elements
in pollen and dehydrated trichomes. Arabidopsis halleri was found to hyperaccumulate not only Zn but also Cd in the shoot biomass. While large concentrations of Zn and Cd were found
in the leaves and roots, flowers contained very little. In roots grown hydroponically, Zn and Cd accumulated in the cell wall
of the rhizodermis (root epidermis), mainly due to precipitation of Zn/Cd phosphates. In leaves, the trichomes had by far
the largest concentrations of Zn and Cd. Inside the trichomes there was a striking sub-cellular compartmentation, with almost
all the Zn and Cd being accumulated in a narrow ring in the trichome base. This distribution pattern was very different from
that for Ca and P. The epidermal cells other than trichomes were very small and contained lower concentrations of Zn and Cd
than mesophyll cells. In particular, the concentrations of Cd and Zn in the mesophyll cells increased markedly in response
to increasing Zn and Cd concentrations in the nutrient solution. This indicates that the mesophyll cells in the leaves of
A. halleri are the major storage site for Zn and Cd, and play an important role in their hyperaccumulation.
Received: 4 April 2000 / Accepted: 16 May 2000 相似文献
74.
Vaccination of macaques against pathogenic simian immunodeficiency virus with Venezuelan equine encephalitis virus replicon particles
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Davis NL Caley IJ Brown KW Betts MR Irlbeck DM McGrath KM Connell MJ Montefiori DC Frelinger JA Swanstrom R Johnson PR Johnston RE 《Journal of virology》2000,74(1):371-378
Vaccine vectors derived from Venezuelan equine encephalitis virus (VEE) that expressed simian immunodeficiency virus (SIV) immunogens were tested in rhesus macaques as part of the effort to design a safe and effective vaccine for human immunodeficiency virus. Immunization with VEE replicon particles induced both humoral and cellular immune responses. Four of four vaccinated animals were protected against disease for at least 16 months following intravenous challenge with a pathogenic SIV swarm, while two of four controls required euthanasia at 10 and 11 weeks. Vaccination reduced the mean peak viral load 100-fold. The plasma viral load was reduced to below the limit of detection (1,500 genome copies/ml) in one vaccinated animal between 6 and 16 weeks postchallenge and in another from week 6 through the last sampling time (40 weeks postchallenge). The extent of reduction in challenge virus replication was directly correlated with the strength of the immune response induced by the vectors, which suggests that vaccination was effective. 相似文献
75.
The biodegradation by Rhizobium huakuii PMY1 of up to 10 mM phosphonomycin as a carbon, energy, and phosphorus source with accompanying Pi release is described. This biodegradation represents a further mechanism of resistance to this antibiotic and a novel, phosphate-deregulated route for organophosphonate metabolism by Rhizobium spp. 相似文献
76.
Nigel G. Ternan John W. McGrath John P. Quinn 《Applied and environmental microbiology》1998,64(6):2291-2294
A strain of Burkholderia cepacia isolated by enrichment culture utilized l-2-amino-3-phosphonopropionic acid (phosphonoalanine) at concentrations up to 20 mM as a carbon, nitrogen, and phosphorus source in a phosphate-insensitive manner. Cells contained phosphoenolpyruvate phosphomutase activity, presumed to be responsible for cleavage of the C—P bond of phosphonopyruvate, the transamination product of l-phosphonoalanine; this was inducible in the presence of phosphonoalanine.Organophosphonates are characterized by the presence of a stable, covalent carbon-to-phosphorus (C—P) bond. In the majority of previous studies they have been utilized only under phosphate-limited conditions and only as sole sources of phosphorus for microbial growth (3, 4, 21, 22). The C—P bond may be cleaved by at least three distinct bacterial enzymes: the C—P lyase enzyme complex(es) (17, 24, 25, 27, 28), phosphonoacetaldehyde hydrolase (5, 6, 9, 12), and phosphonoacetate hydrolase (14–16). The latter enzyme is unique in that its expression is independent of the phosphate status of the cell and is inducible solely by phosphonoacetate. It is likely that organophosphonate biodegradation in the environment is mediated largely by a C—P lyase(s), with organisms capable of mineralizing organophosphonates as sources of carbon and energy being rare (2, 13).Phosphonoalanine (2-amino-3-phosphonopropionic acid) is one of the naturally occurring C—P compounds synthesized by lower organisms, such as the sea anemone Zoanthus sociatus (10) and the protozoan Tetrahymena pyriformis (8, 23, 29). In this paper, we report the isolation of a bacterium capable of mineralizing l-phosphonoalanine as a carbon, energy, nitrogen, and phosphorus source independently of the phosphate status of the cell.Enrichment was carried out with a basal mineral salts medium which contained the following (per liter): KCl, 0.2 g; MgSO4 · 7H2O, 0.2 g; CaCl2 · 2H2O, 0.01 g; ferric ammonium citrate, 1.0 mg; trace element solution (11), 1 ml; and vitamin solution (14), 1 ml. Filter-sterilized (0.22-μm pore size) dl-phosphonoalanine (8 mM) was routinely added as a carbon, energy, nitrogen, and phosphorus source. The pH of the medium was initially adjusted to 7.2, and where required, filter-sterilized solutions of sodium pyruvate as a carbon source (final concentration, 10 g liter−1), NH4Cl as an inorganic nitrogen source (final concentration, 5 g liter−1), and/or phosphate buffer (final concentration, 1 mM) were added to the medium. Enrichment cultures (25 ml in 250-ml Erlenmeyer flasks) were inoculated with a 0.5% (vol/vol) composite inoculum from an activated sludge plant (Dunmurry, Northern Ireland), a laundry waste disposal lagoon (Summit Lake, Wis.), and a sheep dip disposal site (County Antrim, Northern Ireland). All sites were known to have a history of exposure to organophosphonates. Cultures were incubated at 28°C on an orbital shaker at 100 rpm. Microbial growth was measured by the increase in optical density at 650 nm (OD650) using a Pye-Unicam 8265 UV-visible light spectrophotometer (Pye-Unicam Ltd., Cambridge, United Kingdom). Release of inorganic phosphate and ammonium into culture supernatants was monitored by the methods of Fiske and SubbaRow (7) and Weatherburn (30), respectively.Three gram-negative isolates, each capable of growth on 8 mM dl-phosphonoalanine as a carbon, nitrogen, and phosphorus source were obtained following five rounds of serial enrichment. Of these, isolate Pal6 grew most quickly on phosphonoalanine and was chosen for further investigation. It was identified by the National Collection of Industrial and Marine Bacteria Ltd., Aberdeen, Scotland, as a strain of Burkholderia cepacia.When dl-phosphonoalanine (8 mM) was supplied as a carbon, nitrogen, and phosphorus source for growth of B. cepacia Pal6, some 47% of substrate phosphorus and 44% of substrate nitrogen was released concomitantly with growth as Pi and ammonium (results not shown). When the compound was supplied as the sole phosphorus source (Fig. (Fig.1),1), transient release of approximately 30% of substrate phosphorus to the medium as Pi was observed; this phenomenon has not previously been reported for the utilization of any organophosphorus compound as a phosphorus source. When B. cepacia Pal6 was grown on dl-phosphonoalanine as a nitrogen and phosphorus (Fig. (Fig.2)2) or nitrogen source, removal of 50% of phosphonoalanine from the medium was demonstrated by the method of Moore and Stein (18), along with release of just less than 50% of substrate phosphorus as Pi. A subsequent experiment in which the d- and l-enantiomers were separately supplied as sole sources of phosphorus indicated that only l-phosphonoalanine supported growth of B. cepacia Pal6. It is therefore clear that the catabolism of l-phosphonoalanine by this isolate is independent of the phosphate status of the cell, a marked departure from the many examples of classical pho regulon-controlled biodegradation of organophosphonates reported in the literature (26, 27). Open in a separate windowFIG. 1Growth of B. cepacia Pal6 on phosphonoalanine (1 mM) as the sole phosphorus source, with NH4Cl as a nitrogen source (5 g liter−1) and pyruvate as a carbon source (10 g liter−1). Symbols: •, OD650; ▴, phosphate release.Open in a separate windowFIG. 2Growth of B. cepacia Pal6 on phosphonoalanine (5 mM) as a nitrogen and phosphorus source, with pyruvate as a carbon source (10 g liter−1). Symbols: •, OD650; ▴, phosphate release (mM); □, phosphonoalanine remaining in medium (mM).B. cepacia Pal6 was grown on a range of dl-phosphonoalanine concentrations as carbon and nitrogen source in the presence of 1 mM inorganic phosphate. The cell yield was proportional to the concentration of phosphonoalanine supplied up to 20 mM, the highest concentration tested, again with release of less than 50% substrate phosphorus and nitrogen to the medium (results not shown), indicating no toxicity on the part of either the substrate or its breakdown products at these concentrations.In addition to phosphonoalanine, B. cepacia Pal6 was able to utilize 6 of 14 organophosphonate substrates supplied as the sole phosphorus source (Table (Table1);1); however, with the exception of 2-aminoethylphosphonic acid (2AEP), no phosphate release was observed during growth on these compounds, suggesting classical pho regulon control of their biodegradation and the involvement of a C—P lyase(s) or similar enzymes. B. cepacia Pal6 was also capable of growing on 2AEP as a carbon, energy, nitrogen, and phosphorus source, with concomitant release of excess phosphorus and nitrogen to the medium as inorganic phosphate and ammonium, respectively. It did not utilize any of the other phosphonates tested as the carbon and/or nitrogen and phosphorus source. The metabolism by B. cepacia Pal6 of 2AEP as a carbon, nitrogen and phosphorus source suggests that a phosphate-deregulated pathway is also responsible for the mineralization of this compound.
Open in a separate windowaResults were scored negative if the protein yield, as measured by the method of Binks et al. (1), was less than 20% of that of the positive control containing 1 mM inorganic phosphate. Results are means of duplicates which on no occasion varied by more than 5%. b2AEP was also metabolized as the sole carbon, nitrogen, and phosphorus source. No in vitro cleavage of the C—P bond of phosphonoalanine was detected in cell extracts of B. cepacia Pal6 grown on the compound, nor did such extracts contain detectable phosphonatase or phosphonoacetate hydrolase activities when assayed by the methods of La Nauze et al. (12) and McMullan and Quinn (16), respectively. The only other documented enzyme capable of in vitro-detectable C—P bond cleavage is phosphoenolpyruvate phosphomutase, which catalyses the reversible intramolecular rearrangement of phosphonopyruvate to phosphoenolpyruvate (PEP); it has been implicated in the utilization of phosphonoalanine as the sole phosphorus source by Pseudomonas gladioli B-1 (19, 20). The initial step in this catabolic pathway is the transamination of phosphonoalanine to phosphonopyruvate (20); no such activity was detected in cells of B. cepacia Pal6 grown on phosphonoalanine. However, extracts prepared from d,l-phosphonoalanine-grown cells did indeed contain PEP phosphomutase activity when assayed by the method of Nakashita et al. (19); this was inducible above a basal level (some 17% of the maximum) in the presence of dl-phosphonoalanine. The induction of PEP phosphomutase activity in resting cells of B. cepacia Pal6 pregrown on complete mineral salts medium and resuspended (1 g of cells/50 ml) with dl-phosphonoalanine as a sole carbon, nitrogen, and phosphorus source is shown in Fig. Fig.3.3. Open in a separate windowFIG. 3Induction of PEP phosphomutase activity in resting cells of B. cepacia Pal6 pregrown on complete medium and resuspended in mineral salts containing 8 mM phosphonoalanine as a carbon, nitrogen, and phosphorus source. Symbol: •, PEP phosphomutase activity.PEP phosphomutase activity in cell extracts was obtained only when phosphonopyruvate was supplied as a substrate, with no activity being observed in the presence of phosphonoalanine, 2AEP, phosphonoacetaldehyde, or phosphonoacetate. No activity was obtained in the control assays lacking either cell extract or phosphonopyruvate. That this activity is responsible for cleavage of the C—P bond of phosphonoalanine cannot be definitely confirmed, however, in the absence of a mutant strain of B. cepacia Pal6 deficient in PEP phosphomutase activity. It is unlikely, given the previously demonstrated involvement of PEP phosphomutase in the utilization of phosphonoalanine by P. gladioli B-1 as the sole phosphorus source (19, 20), that the enzyme is merely gratuitously induced by phosphonoalanine in B. cepacia Pal6. Moreover, the hypothesis that PEP phosphomutase is responsible for the cleavage of the C—P bond of phosphonoalanine via a phosphonopyruvate intermediate is also strengthened by the fact that activity of none of the existing known C—P bond-cleaving enzymes was obtained in cell extracts of B. cepacia Pal6.As cells of B. cepacia Pal6 grown on mineral salts supplemented with carbon, nitrogen, and phosphorus sources in the absence of phosphonoalanine were observed to have relatively high levels of constitutive PEP phosphomutase activity (Fig. (Fig.3),3), it was considered likely that the organism, like P. gladioli B-1, would also be capable of producing a C—P bond-containing compound. A sample of broth was taken prior to inoculation and again following 24-h growth of B. cepacia Pal6 on complete medium containing 5 mM inorganic phosphate as the sole source of phosphorus. 31P-labeled nuclear magnetic resonance spectra were obtained for both samples (19), and a new signal, with a shift relative to inorganic phosphate of 13.20 ppm, was observed in the 24-h sample. The experiment was repeated, with identical results. The shift obtained for the unknown compound was similar, but did not correspond, to those shifts obtained for 2-phosphonoacetaldehyde (5.55 ppm), phosphonopyruvate (6.40 ppm), 2-aminoethylphosphonate (15.90 ppm), or phosphonoalanine (14.03 ppm). The appearance of this additional resonance thus suggests the production of a C—P bond-containing compound and is further confirmation of the presence of PEP phosphomutase activity in B. cepacia Pal6.The phosphonoalanine biodegradation pathway in B. cepacia Pal6 would appear to be different from that described for both rats and Tetrahymena (8). In cell-free preparations from these organisms, phosphonoalanine biodegradation was shown to involve a deamination to phosphonopyruvate, which is converted by decarboxylation to 2-phosphonoacetaldehyde, followed by either dephosphonylation or amination of the aldehyde to give acetaldehyde or 2AEP, respectively (8). In B. cepacia Pal6, PEP produced by the intramolecular rearrangement of phosphonopyruvate by PEP phosphomutase would readily enter intermediary metabolism, serving as a carbon and phosphorus source with excess phosphorus being excreted as Pi.The isolation of three different phosphonoalanine-degrading microorganisms by enrichment culture suggests that this ability may be relatively common in the natural environment. Phosphonoalanine is a biogenic organophosphonate; it is therefore unsurprising that microbial systems for its effective utilization exist. In addition to being capable of producing a C—P bond-containing compound, B. cepacia Pal6 is the first microorganism reported to mineralize the l-enantiomer of phosphonoalanine and joins a growing number of reports of microorganisms capable of deregulated scission of the C—P bond of organophosphonates. 相似文献
TABLE 1
Range of organophosphonate substrates utilized by B. cepacia Pal6 as the sole phosphorus sourceSubstrate (1 mM) | Growth (μg of protein/ml)a |
---|---|
Inorganic phosphate | 200 |
2-Phosphonopropionic acid | 200 |
2AEPb | 200 |
Phenylphosphonic acid | 160 |
Hydroxymethylphosphonic acid | 160 |
Methylphosphonic acid | 120 |
Phosphonoacetic acid | 120 |
1-Aminobutylphosphonic acid | 30 |
Aminomethylphosphonic acid | 30 |
3-Aminopropylphosphonic acid | 20 |
Ethylphosphonic acid | 10 |
2-Amino-4-phosphonobutyric acid | 10 |
Phosphonoformic acid | 10 |
4-Aminobutylphosphonic acid | 10 |
1-Aminoethylphosphonic acid | 10 |
Phosphate-free medium | 0 |
77.
Somatic hybrids between Solanum bulbocastanum and potato: a new source of resistance to late blight 总被引:4,自引:0,他引:4
J. P. Helgeson J. D. Pohlman S. Austin G. T. Haberlach S. M. Wielgus D. Ronis L. Zambolim P. Tooley J. M. McGrath R. V. James W. R. Stevenson 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1998,96(6-7):738-742
Solanum bulbocastanum, a wild, diploid (2n=2x=24) Mexican species, is highly resistant to Phytophthora infestans, the fungus that causes late blight of potato. However this 1 EBN species is virtually impossible to cross directly with
potato. PEG-mediated fusion of leaf cells of S. bulbocastanum PI 245310 and the tetraploid potato line S. tuberosum PI 203900 (2n=4x=48) yielded hexaploid (2n= 6x=72) somatic hybrids that retained the high resistance of the S. bulbocastanum parent. RFLP and RAPD analyses confirmed the hybridity of the materials. Four of the somatic hybrids were crossed with potato
cultivars Katahdin or Atlantic. The BC1 progeny segregated for resistance to the US8 genotype (A-2 mating type) of P. Infestans. Resistant BC1 lines crossed with susceptible cultivars again yielded populations that segregated for resistance to the fungus. In a 1996
field-plot in Wisconsin, to which no fungicide was applied, two of the BC1 lines, from two different somatic hybrids, yielded 1.36 and 1.32 kg/plant under a severe late-blight epidemic. In contrast,
under these same conditions the cultivar Russet Burbank yielded only 0.86 kg/plant. These results indicate that effective
resistance to the late-blight fungus in a sexually incompatible Solanum species can be transferred into potato breeding lines by somatic hybridization and that this resistance can then be further
transmitted into potato breeding lines by sexual crossing.
Received: 27 October 1997 / Accepted: 11 November 1997 相似文献
78.
Objectives The aim of this study was to was to determine whether older adults perceive oral health as affecting their life quality and to identify variations in impacts in relation to socio-demographic factors, dental service utilisation and method of payment. Design This study formed part of the Office for National Statistics Omnibus Survey, which utilised a random probability sample of addresses from the British Postcode Address File (PAF). Setting 3,000 homes were selected from one hundred post sectors across Britain. Respondents were interviewed in their homes about how oral health affects their quality of life. Subjects 454 older people (aged 65 and over) took part in the survey. Main outcome measures The study group perceived oral health as impacting on their quality of life in general (negative and/or positive impact) (70%, 318), enhancing (53%, 241) and detracting (44%, 199) from their life quality. The most frequently perceived way in which oral health affects life quality was its effect on eating and comfort. Older people from higher socio-economic groups reported that oral health had a greater impact on their quality of life in general (positive and/or negative impacts), (OR=1.77,95% CI= 1.22,2.78) and specifically, enhancing their quality of life (OR=1.52, 95% CI=1.01,2.30). Those who reported attending the dentist within the last year perceived that their oral health enhanced their life quality (OR=1.55, 95% CI=1.01,2.38). Conclusions Socio-economic background and dental attendance pattern are associated with how older people perceived oral health as affecting quality of life. These findings may have implications for promoting regular dental attendance and auditing dental services for older people. 相似文献
79.
Phylogenetic relationships were determined for 76 partial P-element
sequences from 14 species of the melanogaster species group within the
Drosophila subgenus Sophophora. These results are examined in the context
of the phylogeny of the species from which the sequences were isolated.
Sequences from the P-element family fall into distinct subfamilies, or
clades, which are often characteristic for particular species subgroups.
When examined locally among closely related species, the evolution of P
elements is characterized by vertical transmission, whereby the P-element
phylogeny traces the species phylogeny. On a broader scale, however, the
P-element phylogeny is not congruent with the species phylogeny. One
feature of P-element evolution in the melanogaster group is the presence of
more than one P-element subfamily, differing by as much as 36%, in the
genomes of some species. Thus, P elements from several individual species
are not monophyletic, and a likely explanation for the incongruence between
P-element and species phylogenies is provided by the comparison of
paralogous sequences. In certain instances, horizontal transfer seems to be
a valid alternative explanation for lack of congruence between species and
P-element phylogenies. The canonical P-element subfamily, which represents
the active, autonomous transposable element, is restricted to D.
melanogaster. Thus, its origin clearly lies outside of the melanogaster
species group, consistent with the earlier conclusion of recent horizontal
transfer.
相似文献
80.