pH regulation in apoplastic and cytoplasmic cell compartments of leaves

 The regulation of pH in the apoplast, cytosol and chloroplasts of intact leaves was studied by means of fluorescent pH indicators and as a response of photosynthesis to acid stress. The apoplastic pH increased under anaerobiosis. Aeration reversed this effect. Apoplastic responses to CO₂, HCl or NH₃ differed considerably. Whereas HCl and ammonia caused rapid acidification or alkalinization, the return to initial pH values was slow after cessation of fumigation. Addition of CO₂ either did not produce the acidification expected on the basis of known apoplastic buffering or even caused some alkalinization. Removal of CO₂ shifted the apoplastic pH into the alkaline range before the pH returned to initial steady-state levels. In the presence of vanadate, the alkaline shift was absent and the apoplastic pH returned slowly to the initial level when CO₂ was removed from the atmosphere. In contrast to the response of the apoplast, anaerobiosis acidified the cytosol or, in some species, had little effect on its pH. Acidification was rapidly reversed upon re-admission of oxygen. The CO₂-dependent pH changes were very fast in the cytosol. Considerable alkalinization was observed after removal of CO₂ under aerobic, but not under anaerobic conditions. Rates of the re-entry of protons into the cytosol during recovery from CO₂ stress increased in the presence of oxygen with the length of previous exposure to high CO₂. Effective pH regulation in the chloroplasts was indicated by the recovery of photosynthesis after the transient inhibition of photosynthetic electron flow when CO₂ was increased from 0.038% to 16% in air. As photosynthesis became inhibited under high CO₂, reduction of the electron transport chain increased transiently. The time required for recovery of photosynthesis from inhibition during persistent CO₂ stress was similar to the time required for establishing steady-state pH values in the cytosol under acid stress. The high capacity of leaf cells for the rapid re-attainment of pH homeostasis in the apoplast and the cytoplasm under acid or alkaline stress suggested the rapid activation or deactivation of membrane-localised proton-transporting enzymes and corresponding ion channel regulation for co-transport of anions or counter-transport of cations together with proton fluxes. Acidification of the cytoplasm appeared to activate energy-dependent proton export primarily into the vacuoles whereas apoplastic alkalinization resulted in the pumping of protons into the apoplast. Proton export rates from the cytosol into the apoplast after anaerobiosis were about 100 nmol (m² leaf area)⁻¹ s⁻¹ or less. Proton export under acid stress into the vacuole was about 1200 nmol m⁻² s⁻¹. The kinetics of pH responses to the addition or withdrawal of CO₂ indicated the presence of carbonic anhydrase in the cytosol, but not in the apoplast. Received: 19 July 1999 / Accepted: 29 December 1999     

Misexpression screen in Drosophila melanogaster aiming to reveal novel factors involved in formation of body parts

To identify novel factors that lead a fly imaginal disc to adopt its developmental fate, we carried out a modular dominant misexpression screen in imaginal discs. We have identified two factors that appear to change the fate of the respective body structure and appear to lead to the transformation of a body part. In one mutant line, notum tissue, normally derived from wing imaginal tissue, formed close to the site of the sternopleural bristles, which are leg disc derivatives. In the other line, the arista is transformed into a tubular structure, resembling an abnormal leg. We found that ectopic expression of abrupt was responsible for this potential transformation of the arista.     

评论：塑料结合模块促进聚对苯二甲酸乙二醇酯的酶法降解

塑料的大量生产和无节制的使用已造成严重的环境污染。为了减少塑料废物对环境的影响,近年来塑料酶法降解已成为国内外研究者关注的热点。例如,通过蛋白质工程策略提高塑料降解酶催化活性和热稳定性,进一步提高酶法降解的效率。另外,通过融合酶策略将塑料结合模块与塑料降解酶融合,也可以促进塑料降解。近期发表在期刊Chem Catalysis的一项研究表明,采用碳水化合物结合模块融合策略可以在低浓度(<10 wt%)的底物聚对苯二甲酸乙二醇酯[poly(ethylene terephthalate),PET]中提高塑料降解酶的活性。但是在高浓度底物(10 wt%−20 wt%)中,该策略无法提高PET的酶法降解。该项研究对于采用塑料结合模块促进酶法降解塑料具有重要的指导意义。     

One-pot synthesis of pegylated ultrasmall iron-oxide nanoparticles and their in vivo evaluation as magnetic resonance imaging contrast agents

A well-defined copolymer poly(oligo(ethylene glycol) methacrylate-co-methacrylic acid) P(OEGMA-co-MAA) was studied as a novel water-soluble biocompatible coating for superparamagnetic iron oxide nanoparticles. This copolymer was prepared via a two-step procedure: a well-defined precursor poly(oligo(ethylene glycol) methacrylate-co-tert-butyl methacrylate), P(OEGMA-co-tBMA) (M(n) = 17300 g mol(-1); M(w)/M(n) = 1.22), was first synthesized by atom-transfer radical polymerization in the presence of the catalyst system copper(I) chloride/2,2'-bipyridyl and subsequently selectively hydrolyzed in acidic conditions. The resulting P(OEGMA-co-MAA) was directly utilized as a polymeric stabilizer in the nanoparticle synthesis. Four batches of ultrasmall PEGylated magnetite nanoparticles (i.e., with an average diameter below 30 nm) were prepared via aqueous coprecipitation of iron salts in the presence of variable amounts of P(OEGMA-co-MAA). The diameter of the nanoparticles could be easily tuned in the range 10-25 nm by varying the initial copolymer concentration. Moreover, the formed PEGylated ferrofluids exhibited a long-term colloidal stability in physiological buffer and could therefore be studied in vivo by magnetic resonance (MR) imaging. Intravenous injection into rats showed no detectable signal in the liver within the first 2 h. Maximum liver accumulation was found after 6 h, suggesting a prolongated circulation of the nanoparticles in the bloodstream as compared to conventional MR imaging contrast agents.     

Paleodistributions and comparative molecular phylogeography of leafcutter ants (Atta spp.) provide new insight into the origins of Amazonian diversity

The evolutionary basis for high species diversity in tropical regions of the world remains unresolved. Much research has focused on the biogeography of speciation in the Amazon Basin, which harbors the greatest diversity of terrestrial life. The leading hypotheses on allopatric diversification of Amazonian taxa are the Pleistocene refugia, marine incursion, and riverine barrier hypotheses. Recent advances in the fields of phylogeography and species-distribution modeling permit a modern re-evaluation of these hypotheses. Our approach combines comparative, molecular phylogeographic analyses using mitochondrial DNA sequence data with paleodistribution modeling of species ranges at the last glacial maximum (LGM) to test these hypotheses for three co-distributed species of leafcutter ants (Atta spp.). The cumulative results of all tests reject every prediction of the riverine barrier hypothesis, but are unable to reject several predictions of the Pleistocene refugia and marine incursion hypotheses. Coalescent dating analyses suggest that population structure formed recently (Pleistocene-Pliocene), but are unable to reject the possibility that Miocene events may be responsible for structuring populations in two of the three species examined. The available data therefore suggest that either marine incursions in the Miocene or climate changes during the Pleistocene--or both--have shaped the population structure of the three species examined. Our results also reconceptualize the traditional Pleistocene refugia hypothesis, and offer a novel framework for future research into the area.     

In Sup35p filaments (the [PSI+] prion), the globular C-terminal domains are widely offset from the amyloid fibril backbone

In yeast cells infected with the [PSI+] prion, Sup35p forms aggregates and its activity in translation termination is downregulated. Transfection experiments have shown that Sup35p filaments assembled in vitro are infectious, suggesting that they reproduce or closely resemble the prion. We have used several EM techniques to study the molecular architecture of filaments, seeking clues as to the mechanism of downregulation. Sup35p has an N-terminal 'prion' domain; a highly charged middle (M-)domain; and a C-terminal domain with the translation termination activity. By negative staining, cryo-EM and scanning transmission EM (STEM), filaments of full-length Sup35p show a thin backbone fibril surrounded by a diffuse 65-nm-wide cloud of globular C-domains. In diameter (～8 nm) and appearance, the backbones resemble amyloid fibrils of N-domains alone. STEM mass-per-unit-length data yield ～1 subunit per 0.47 nm for N-fibrils, NM-filaments and Sup35p filaments, further supporting the fibril backbone model. The 30 nm radial span of decorating C-domains indicates that the M-domains assume highly extended conformations, offering an explanation for the residual Sup35p activity in infected cells, whereby the C-domains remain free enough to interact with ribosomes.     

Deleterious mutations in LRBA are associated with a syndrome of immune deficiency and autoimmunity

Most autosomal genetic causes of childhood-onset hypogammaglobulinemia are currently not well understood. Most affected individuals are simplex cases, but both autosomal-dominant and autosomal-recessive inheritance have been described. We performed genetic linkage analysis in consanguineous families affected by hypogammaglobulinemia. Four consanguineous families with childhood-onset humoral immune deficiency and features of autoimmunity shared genotype evidence for a linkage interval on chromosome 4q. Sequencing of positional candidate genes revealed that in each family, affected individuals had a distinct homozygous mutation in LRBA (lipopolysaccharide responsive beige-like anchor protein). All LRBA mutations segregated with the disease because homozygous individuals showed hypogammaglobulinemia and autoimmunity, whereas heterozygous individuals were healthy. These mutations were absent in healthy controls. Individuals with homozygous LRBA mutations had no LRBA, had disturbed B cell development, defective in vitro B cell activation, plasmablast formation, and immunoglobulin secretion, and had low proliferative responses. We conclude that mutations in LRBA cause an immune deficiency characterized by defects in B cell activation and autophagy and by susceptibility to apoptosis, all of which are associated with a clinical phenotype of hypogammaglobulinemia and autoimmunity.     

Antagonistic roles of ESCRT and Vps class C/HOPS complexes in the recycling of yeast membrane proteins

In Saccharomyces cerevisiae, deficiencies in the ESCRT machinery trigger the mistargeting of endocytic and biosynthetic ubiquitinated cargoes to the limiting membrane of the vacuole. Surprisingly, impairment of this machinery also leads to the accumulation of various receptors and transporters at the plasma membrane in both yeast and higher eukaryotes. Using the well-characterized yeast endocytic cargo uracil permease (Fur4p), we show here that the apparent stabilization of the permease at the plasma membrane in ESCRT mutants results from an efficient recycling of the protein. Whereas several proteins as well as internalized dyes are known to be recycled in yeast, little is known about the machinery and molecular mechanisms involved. The SNARE protein Snc1p is the only cargo for which the recycling pathway is well characterized. Unlike Snc1p, endocytosed Fur4p did not pass through the Golgi apparatus en route to the plasma membrane. Although ubiquitination of Fur4p is required for its internalization, deubiquitination is not required for its recycling. In an attempt to identify actors in this new recycling pathway, we found an unexpected phenotype associated with loss of function of the Vps class C complex: cells defective for this complex are impaired for recycling of Fur4p, Snc1p, and the lipophilic dye FM4-64. Genetic analyses indicated that these phenotypes were due to the functioning of the Vps class C complex in trafficking both to and from the late endosomal compartment.     

Presence and Characterization of a Mosaic Genomic Island Which Distinguishes Sorbitol-Fermenting Enterohemorrhagic Escherichia coli O157:H? from E. coli O157:H7

A mosaic genomic island comprising Shigella resistance locus (SRL) sequences flanked by segments of Escherichia coli O157:H7 strain EDL933 O islands 43, 81, and 82 was identified in sorbitol-fermenting (SF) enterohemorrhagic Escherichia coli (EHEC) O157:H⁻ strain 493/89. This mosaic island is absent from strain EDL933. PCR targeting the SRL-related sequence is a useful tool to distinguish SF EHEC O157:H⁻ from EHEC O157:H7.     

Osmotic responses of preimplantation mouse and bovine embryos and their cryobiological implications

Cells subjected to the events occurring before, during, and after freezing and thawing are exposed to major changes in the osmotic pressure of the surrounding medium; i.e., the osmolalities can exceed 30. An important question in understanding the mechanisms of injury is whether cells respond as ideal osmometers to these strongly anisotonic solutions. Mouse and bovine embryos from eight-cell to blastocyst stage were used to investigate the question. They were found to behave as ideal osmometers at room temperature over a wide range of tonicities; i.e., from four times isotonic to almost 1/3 times isotonic, ideality being defined by a Boyle-van't Hoff equation. Embryo volumes increased from 40 to 200% of isotonic over this range and survivals of mouse embryos were unaffected. However, outside this range the membrane apparently becomes leaky and the survival of mouse embryos drops sharply. Osmolalities rise to high values during freezing and the paper develops the thermodynamic equations to show how computed cell volumes as a function of subzero temperature can be translated into the Boyle-van't Hoff format of cell volume as a function of the reciprocal of osmolality.