Spatial-temporal studies of membrane dynamics: scanning fluorescence correlation spectroscopy (SFCS)

Giant unilamellar vesicles (GUVs) have been widely used as a model membrane system to study membrane organization, dynamics, and protein-membrane interactions. Most recent studies have relied on imaging methods, which require good contrast for image resolution. Multiple sequential image processing only detects slow components of membrane dynamics. We have developed a new fluorescence correlation spectroscopy (FCS) technique, termed scanning FCS (i.e., SFCS), which performs multiple FCS measurements simultaneously by rapidly directing the excitation laser beam in a uniform (circular) scan across the bilayer of the GUVs in a repetitive fashion. The scan rate is fast compared to the diffusion of the membrane proteins and even small molecules in the GUVs. Scanning FCS outputs a "carpet" of timed fluorescence intensity fluctuations at specific points along the scan. In this study, GUVs were assembled from rat kidney brush border membranes, which included the integral membrane proteins. Scanning FCS measurements on GUVs allowed for a straightforward detection of spatial-temporal interactions between the protein and the membrane based on the diffusion rate of the protein. To test for protein incorporation into the bilayers of the GUVs, antibodies against one specific membrane protein (NaPi II cotransporter) were labeled with ALEXA-488. Fluorescence images of the GUVs in the presence of the labeled antibody showed marginal fluorescence enhancement on the GUV membrane bilayers (poor image contrast and resolution). With the application of scanning FCS, the binding of the antibody to the GUVs was detected directly from the analysis of diffusion rates of the fluorescent antibody. The diffusion coefficient of the antibody bound to NaPi II in the GUVs was approximately 200-fold smaller than that in solution. Scanning FCS provided a simple, quantitative, yet highly sensitive method to study protein-membrane interactions.     

pH-induced conformational changes in the soluble manganese-stabilizing protein of photosystem II

In this paper, we analyzed the pH-induced changes in the conformational states of the manganese-stabilizing protein (MSP) of photosystem II. Distinct conformational states of MSP were identified using fluorescence spectra, far-UV circular dichroism, and pressure-induced unfolding at varying suspension pH values, and four different conformational states of MSP were clearly distinguished using the center of fluorescence spectra mass when suspension pH was altered from 2 to 12. MSP was completely unfolded at a suspension pH above 11 and partly unfolded below a pH of 3. Analysis of the center of fluorescence spectral mass showed that the MSP structure appears stably folded around pH 6 and 4. The conformational state of MSP at pH 4 seems more stable than that at pH 6. Studies of peak positions of tryptophan fluorescence and MSP-bound 1-anilinonaphthalene-8-sulfonic acid fluorescence spectra supported this observation. A decrease in the suspension pH to 2 resulted in significant alterations in the MSP structure possibly because of protonation of unprotonated residues at lower pH, suggesting the existence of a large number of unprotonated amino acid residues at neutral pH possibly useful for proton transport in oxygen evolution. The acidic pH-induced conformational changes of MSP were reversible upon increase of pH to neutral pH; however, N-bromosuccinimide modification of tryptophan (Trp241) blocks the recovery of pH-induced conformational changes in MSP, implying that Trp241 is a key residue for the unfolded protein to form a functional structure. Thus, pH-induced structural changes of stable MSP (pH 6-4) may be utilized to analyze its functionality as a cofactor for oxygen evolution.     

Cysteinyl-tRNA(Cys) formation in Methanocaldococcus jannaschii: the mechanism is still unknown

Most organisms form Cys-tRNA(Cys), an essential component for protein synthesis, through the action of cysteinyl-tRNA synthetase (CysRS). However, the genomes of Methanocaldococcus jannaschii, Methanothermobacter thermautotrophicus, and Methanopyrus kandleri do not contain a recognizable cysS gene encoding CysRS. It was reported that M. jannaschii prolyl-tRNA synthetase (C. Stathopoulos, T. Li, R. Longman, U. C. Vothknecht, H. D. Becker, M. Ibba, and D. S?ll, Science 287:479-482, 2000; R. S. Lipman, K. R. Sowers, and Y. M. Hou, Biochemistry 39:7792-7798, 2000) or the M. jannaschii MJ1477 protein (C. Fabrega, M. A. Farrow, B. Mukhopadhyay, V. de Crécy-Lagard, A. R. Ortiz, and P. Schimmel, Nature 411:110-114, 2001) provides the "missing" CysRS activity for in vivo Cys-tRNA(Cys) formation. These conclusions were supported by complementation of temperature-sensitive Escherichia coli cysS(Ts) strain UQ818 with archaeal proS genes (encoding prolyl-tRNA synthetase) or with the Deinococcus radiodurans DR0705 gene, the ortholog of the MJ1477 gene. Here we show that E. coli UQ818 harbors a mutation (V27E) in CysRS; the largest differences compared to the wild-type enzyme are a fourfold increase in the K(m) for cysteine and a ninefold reduction in the k(cat) for ATP. While transformants of E. coli UQ818 with archaeal and bacterial cysS genes grew at a nonpermissive temperature, growth was also supported by elevated intracellular cysteine levels, e.g., by transformation with an E. coli cysE allele (encoding serine acetyltransferase) or by the addition of cysteine to the culture medium. An E. coli cysS deletion strain permitted a stringent complementation test; growth could be supported only by archaeal or bacterial cysS genes and not by archaeal proS genes or the D. radiodurans DR0705 gene. Construction of a D. radiodurans DR0705 deletion strain showed this gene to be dispensable. However, attempts to delete D. radiodurans cysS failed, suggesting that this is an essential Deinococcus gene. These results imply that it is not established that proS or MJ1477 gene products catalyze Cys-tRNA(Cys) synthesis in M. jannaschii. Thus, the mechanism of Cys-tRNA(Cys) formation in M. jannaschii still remains to be discovered.     

Characterization of the 3a protein of SARS-associated coronavirus in infected vero E6 cells and SARS patients

Proteomics was used to identify a protein encoded by ORF 3a in a SARS-associated coronavirus (SARS-CoV). Immuno-blotting revealed that interchain disulfide bonds might be formed between this protein and the spike protein. ELISA indicated that sera from SARS patients have significant positive reactions with synthesized peptides derived from the 3a protein. These results are concordant with that of a spike protein-derived peptide. A tendency exists for co-mutation between the 3a protein and the spike protein of SARS-CoV isolates, suggesting that the function of the 3a protein correlates with the spike protein. Taken together, the 3a protein might be tightly correlated to the spike protein in the SARS-CoV functions. The 3a protein may serve as a new clinical marker or drug target for SARS treatment.     

盐地碱蓬幼嫩花序的组织培养及植株再生

选用盐地碱蓬(Suaeda salsa)幼嫩花序为外植体, 建立了快速而高效的离体培养体系。在附加1.0mg.L-16-BA和0.4 mg.L-1 IAA的MS培养基上培养25天可诱导出不定芽, 诱导频率达到82.1%; 不定芽在此培养基上可快速扩增和长期继代培养。不定芽转至MS培养基中培养2~3周生根形成完整植株。     

Home range and habitat use of male Reeves's Pheasant(Symaticus reevesii)during winter in Dongzhai National Nature Reserve,Henan Province,China

Home range and habitat use of male Reeves's Pheasant(syrmaticus reevesii)were studied during winter of 2001～2002 and 2002～2003 in the Dongzhai National Nature Reserve,Henan Province.Results from five individuals of Reeves's Pheasant with over 30 relocations,indicated that the average size of home range was 10.03±1.17 hm2 by Minimum Convex Polygon method.8.60±0.35 hm2 by 90% Harmonic Mean Transformation method,and 9.50±1.90 hm2 by 95% Fixed Kernel method.It was observed that the winter range is smaller than that in the breeding season.The mean core area of the home range was found to be 1.88±0.37 hm2.Although the habitat composition of the core area varied greatly for individuals,a large part of the habitats used were composed of conifer and broadleaf mixed forests,masson pine forests,fir forests,and shrubs.Habitat use within the study area was non-random,while habitats within home ranges were randomly used.Habitat use was dictated by tree diameter at breast height,shrub height and coverage at 2.0 m.The proximity between forests and shrubs were also found to be important in providing refuge for the birds during winter.Recommendations for conservation management include protecting the existing habitats in Dongzhai National Nature Reserve,increasing suitable habitat for Reeves's Pheasant through artificial plantations(e.g.firs),and restoring some parts of the large shrub area into forests.     

A cellular automata model for simulating fed-batch penicillin fermentation process

A cellular automata model to simulate penicillin fed-batch fermentation process(CAPFM)was established in this study,based on a morphologically structured dynamic penicillin production model,that is in turn based on the growth mechanism of penicillin producing microorganisms and the characteristics of penicillin fed-batch fermentation.CAPFM uses the three-dimensional cellular automata as a growth space,and a Moore-type neighborhood as the cellular neighborhood.The transition roles of CAPFM are designed based on mechanical and structural kinetic models of penicillin batch-fed fermentation processes.Every cell of CAPFM represents a single or specific number of penicillin producing microorganisms,and has various state.The simulation experimental results show that CAPFM replicates the evolutionary behavior of penicillin batch-fed fermentation processes described by the structured penicillin production kinetic model accordingly.     

Home range and habitat use of male Reeves’s pheasant (Syrmaticus reevesii) during winter in Dongzhai National Nature Reserve, Henan Province, China

Home range and habitat use of male Reeves’s pheasant (Syrmaticus reevesii) were studied during winter of 2001∼2002 and 2002∼2003 in the Dongzhai National Nature Reserve, Henan Province. Results from five individuals of Reeves’s pheasant with over 30 relocations, indicated that the average size of home range was 10.03 ± 1.17 hm² by Minimum Convex Polygon method, 8.60 ± 0.35 hm² by 90% Harmonic Mean Transformation method, and 9.50 ± 1.90 hm² by 95% Fixed Kernel method. It was observed that the winter range is smaller than that in the breeding season. The mean core area of the home range was found to be 1.88 ± 0.37 hm². Although the habitat composition of the core area varied greatly for individuals, a large part of the habitats used were composed of confier and broadleaf mixed forests, masson pine forests, fir forests, and shrubs. Habitat use within the study area was non-random, while habitats within home ranges were randomly used. Habitat use was dictated by tree diameter at breast height, shrub height and coverage at 2.0 m. The proximity between forests and shrubs were also found to be important in providing refuge for the birds during winter. Recommendations for conservation management include protecting the existing habitats in Dongzhai National Nature Reserve, increasing suitable habitat for Reeves’s Pheasant through artificial plantations (e.g. firs), and restoring some parts of the large shrub area into forests. __________ Translated from Biodiversity Science, 2005, 13 (5) [译自: 生物多样性, 2005,13(5)]     

Simultaneous silencing of FAD2 and FAE1 genes affects both oleic acid and erucic acid contents in Brassica napus seeds

The fatty acid composition in the seed oil was significantly modified following the introduction of transgenes. To further enhance the desirable characteristics of rapeseed oil, it would be beneficial to develop a new approach for the simultaneous silencing of two or more target genes. Our goals in the current study were to (1) increase oleic acid to more than 75%, (2) reduce polyunsaturated fatty acids (PUFA) to about 10% and erucic acid to zero, and (3) accomplish these changes in a single-transformation event. In a single transformation, two fragments amplified from the fatty acid ^Δ12-desaturase 2 (BnaFAD2) and fatty acid elongase 1 (BnaFAE1) genes of Brassica napus were linked together to form a fusion fragment. The fusion fragment was then used to assemble unique intron-spliced hairpin interfering constructs. In the transgenic plant FFRP4-4, the expression of BnaFAD2 and BnaFAE1 genes was completely inhibited. The composition of oleic acid in FFRP4-4 rose to 85%, PUFA dropped to 10% and erucic acid was undetectable. All hybrid F₁ seeds obtained from the reciprocal crossing of FFRP4-4 and GX-parents (with different genetic backgrounds) contained more than 80% oleic acid, about 10% PUFA and very low, or undetectable, erucic acid. The results confirmed that the fusion fragment silencing construct can simultaneously and effectively silence the target genes on a consistent basis. The strategy provides a useful tool for detecting gene function and advancing genetic engineering techniques for the improvement of agricultural crops.