An airtight approach to the inebriometer

The mechanism by which inhaled anesthetics work is not fully understood, although they have been extensively used. Much research has been done showing the likelihood that there is more than one pathway or mechanism of action. A long-term goal of our laboratory is to decipher these mechanisms using Drosophila melanogaster, an excellent model organism for this purpose. In order to do this, we have modified and constructed an apparatus called the inebriometer to quantitatively analyze the response of flies to inhaled anesthetics. While the inebriometer is not new to the fly community, our updated design provides a relatively low-labor, high-throughput means for performing screens in search of genes involved in the anesthetic mechanism. Here we describe our construction of an airtight inebriometer that we have designed for this purpose. We also provide data that validates this apparatus and establishes a procedure for its use.     

Autophagy across the eukaryotes

Autophagy is conserved throughout the eukaryotes and for many years, work in Saccharomyces cerevisiae has been at the forefront of autophagy research. However as our knowledge of the autophagic machinery has increased, differences between S. cerevisiae and mammalian cells have become apparent. Recent work in other organisms, such as the amoeba Dictyostelium discoideum, indicate an autophagic pathway much more similar to mammalian cells than S. cerevisiae, despite its earlier evolutionary divergence. S. cerevisiae therefore appear to have significantly specialized, and the autophagic pathway in mammals is much more ancient than previously appreciated, which has implications for how we interpret data from organisms throughout the eukaryotic tree.     

Xanthomonas citri ssp. citri requires the outer membrane porin OprB for maximal virulence and biofilm formation

Xanthomonas citri ssp. citri (Xcc) causes canker disease in citrus, and biofilm formation is critical for the disease cycle. OprB (Outer membrane protein B) has been shown previously to be more abundant in Xcc biofilms compared with the planktonic state. In this work, we showed that the loss of OprB in an oprB mutant abolishes bacterial biofilm formation and adherence to the host, and also compromises virulence and efficient epiphytic survival of the bacteria. Moreover, the oprB mutant is impaired in bacterial stress resistance. OprB belongs to a family of carbohydrate transport proteins, and the uptake of glucose is decreased in the mutant strain, indicating that OprB transports glucose. Loss of OprB leads to increased production of xanthan exopolysaccharide, and the carbohydrate intermediates of xanthan biosynthesis are also elevated in the mutant. The xanthan produced by the mutant has a higher viscosity and, unlike wild‐type xanthan, completely lacks pyruvylation. Overall, these results suggest that Xcc reprogrammes its carbon metabolism when it senses a shortage of glucose input. The participation of OprB in the process of biofilm formation and virulence, as well as in metabolic changes to redirect the carbon flux, is discussed. Our results demonstrate the importance of environmental nutrient supply and glucose uptake via OprB for Xcc virulence.     

CHRYSOCHROMULINA BREVITURRITA SP. NOV., A NEW FRESHWATER MEMBER OF THE PRYMNESIOPHYCEAE1

Chrysochromulina breviturrita sp. nov. is described as a new haptonema-bearing member of the freshwater Prymnesiophyceae. It is ca. three times larger than the only other known freshwater member of the genus (C. parva Lackey)and possesses a haptonema only ca. 0.2–0.3 as long as the haptonema of C. parva. Two equal flagella (1.5–2 as long as the haptonema), a large contractile vacuole, two parietal plasties, a single pyremid, nucleus, Chrysolaminarin-like droplets and a food vacuole are features readily observed with the light microscope. Dried and shadoweast whole mounts examined with the electron microscope reveal the cell surface covered with two types of small, delicate scales: outer spined scales with a clubshaped terminus and with the spine anchored to a circular to oval base-plate by 2–6 (7) branching arms; and, spineles oval plate scales with concentric lines and radiating ridges on one face and parallel ridges on the other. C. breviturrita has been found in nine lakes in Ontario (Canada). Pereliminary observations suggest that it is an important component of lake plankton and has probably not been delected in previous work, more because of its fragility than because of its scarcity. Living specimens are a prerequisite for initial identification, since cells frequently rupture and haptonemata are often lost when the organisms are killed with the commonly used fixatives.     

Iron homeostasis in <Emphasis Type="Italic">Brucella abortus</Emphasis>: the role of bacterioferritin

Brucella abortus is the etiological agent of bovine brucellosis, an infectious disease of humans and cattle. Its pathogenesis is mainly based on its ability to survive and multiply inside macrophages. It has been demonstrated that if B. abortus ferrochelatase cannot incorporate iron into protoporphyrin IX to synthesize heme, the intracellular replication and virulence in mice is highly attenuated. Therefore, it can be hypothesized that the unavailability of iron could lead to the same attenuation in B. abortus pathogenicity. Thus, the purpose of this work was to obtain a B. abortus derivative unable to keep an internal iron pool and test its ability to replicate under iron limitation. To achieve this, we searched for iron-storage proteins in the genome of brucellae and found bacterioferritin (Bfr) as the sole ferritin encoded. Then, a B. abortus bfr mutant was built up and its capacity to store iron and replicate under iron limitation was investigated. Results indicated that B. abortus Bfr accounts for 70% of the intracellular iron content. Under iron limitation, the bfr mutant suffered from enhanced iron restriction with respect to wild type according to its growth retardation pattern, enhanced sensitivity to oxidative stress, accelerated production of siderophores, and altered expression of membrane proteins. Nonetheless, the bfr mutant was able to adapt and replicate even inside eukaryotic cells, indicating that B. abortus responds to internal iron starvation before sensing external iron availability. This suggests an active role of Bfr in controlling iron homeostasis through the availability of Bfr-bound iron.     

ATP-citrate lyase activity and carotenoid production in batch cultures of Phaffia rhodozyma under nitrogen-limited and nonlimited conditions

ATP-citrate lyase (ACL) is the key cytoplasmic enzyme which supplies acetyl-CoA for fatty acids in oleaginous yeast. Although it has been suggested that fatty acid and carotenoid biosynthesis may have a common source of acetyl-CoA in Phaffia rhodozyma, the source for carotenoids is currently unknown. The purpose of this work was to analyze the development of ACL activity during batch cultures of P. rhodozyma under ammonium-limited and nonammonium-limited conditions and study its possible relationship with carotenoid synthesis. Every experiment showed carotenoid accumulation linked to an increasing ACL activity. Moreover, the ACL activity increased with dissolved oxygen (DO), i.e., ACL responded to DO in a similar way as carotenoid synthesis. Additionally, in the ammonium-limited culture, ACL activity increased upon ammonium depletion. However, the contribution to carotenoid accumulation in that case was negligible. This suggests that P. rhodozyma has developed two components of ACL, each one responsive to a different environmental stimulus, i.e., DO and ammonium depletion. The role of each component is still unknown; however, considering that the former responds to DO and the known role of carotenoids as antioxidants, it may be a provider of acetyl-CoA for carotenoid synthesis.     

The Pinus pinea L. woodlands along the coast of South-western Spain: data for a new geobotanical interpretation

The origin and natural range of the Stone pine (Pinus pinea L.) has been questioned for more than a century. In this work, we focus the investigation on one of the most important and controversial regions, viz., the Iberian Peninsula and, specifically, the Huelva and Cadiz populations in Andalusia, one of the most representative population cores. Although some authors maintain that it is an autochthonous Iberian species, most of them consider it to be exotic. From this idea, many works have been done and a sintaxonomic scheme has been created, which is accepted by the majority of the scientific community, not including Pinus pinea, nor its formations, since they are considered as man-induced forest crops. However, Stone has been present for several thousand years in the Iberian Peninsula and in the territory studied, as several paleobotanic and historical data show, proving that Pinus pinea is an autochthonous species of this region. This is a clear consequence to the field of geobotany, since – at least – the Stone pine woodlands from the Iberian Southeast must be considered as communities predominated by an autochthonous species that must be included in the sintaxonomichal schemes. This revised version was published online in June 2006 with corrections to the Cover Date.     

The Role of Teleological Thinking in Learning the Darwinian Model of Evolution

Human beings are predisposed to think of evolution as teleological—i.e., having a purpose or directive principle—and the ways scientists talk about natural selection can feed this predisposition. This work examines the suggestion that students’ teleological thinking operates as an obstacle when the natural selection evolution model is taught. What we mean by obstacle is an established way of thinking that resists change due to its explanatory power. In light of this approach, the challenges of teaching evolution in biology education have been revised, and improved methodological strategies aimed at a better comprehension of the Darwinian evolution model are suggested.     

Solenoid: a new aquiferous system to Porifera

The aquiferous system is an essential character of poriferans and supports their monophyly. Within the Calcarea, this system displays its greatest variety and traditionally is classified as: asconoid, syconoid, sylleibid, and leuconoid. Species of Leucascus, however, present a different type of aquiferous system composed of anastomosed (interconnected) choanocyte tubes and have an atrium lacking choanoderm. There is such confusion about the classification of the aquiferous system of Leucascus that, depending on the author, it has been classified as asconoid, syconoid, or leuconoid. Therefore, in the present work, we describe a new type of aquiferous system for Leucascus: the solenoid aquiferous system. This new aquiferous system is defined by the presence of anastomosed tubes internally lined by choanocytes and atrium without choanoderm. Although no deep phylogenetic significance has been attributed to the aquiferous system, the solenoid system raises important evolutionary questions about the variety of systems found among the poriferans.     

Application of Two‐Dimensional NMR Spectroscopy to Metabotyping Laboratory Escherichia coli Strains

NMR Spectroscopy has been established as a major tool for identification and quantification of metabolites in a living system. Since the metabolomics era began, one‐dimensional NMR spectroscopy has been intensively employed due to its simplicity and quickness. However, it has suffered from an inevitable overlap of signals, thus leading to inaccuracy in identification and quantification of metabolites. Two‐dimensional (2D) NMR has emerged as a viable alternative because it can offer higher accuracy in a reasonable amount of time. We employed ¹H,¹³C‐HSQC to profile metabolites of six different laboratory E. coli strains. We identified 18 metabolites and observed clustering of six strains according to their metabolites. We compared the metabolites among the strains, and found that a) the strains specialized for protein production were segregated; b) XL1‐Blue separated itself from others by accumulating amino acids such as alanine, aspartate, glutamate, methionine, proline, and lysine; c) the strains specialized for cloning purpose were spread out from one another; and d) the strains originating from B strain were characterized by succinate accumulation. This work shows that 2D‐NMR can be applied to identify a strain from metabolite analysis, offering a possible alternative to genetic analysis to identify E. coli strains.     

A molecular marker confirms that the rate of adult maturation is largely independent of the rate of pre-adult development in Drosophila melanogaster

The separation of adult from pre-adult life seen with animals such as Drosophila melanogaster, which are holometabolous and undergo complete metamorphosis, provides the opportunity to examine the contribution of pre-adult rate of development on the rate of maturation and aging of the adult. Recent work has shown that when ambient temperature is used to alter the rate of development there is little effect on adult life span. From this work it has been concluded that the rate of aging is largely independent of the rate of pre-adult development. However, the techniques used to examine life span did not allow for the examination of the earliest events of adult life. Our experimental design used a molecular marker linked to life span as a sensitive measure of determining physiological age. In this way, we were able to evaluate the effect of pre-adult rate of development on the earliest events of adult life. Using ambient temperature to alter both the rate of development in the pre-adult and the rate of aging in the adult independently, we were able to show that it is the ambient temperature at which the adults are living that is the principle determinant of the rate of maturation and aging of the adult. Little effect was seen on the rate of adult maturation in response to an acceleration or a slowing down of the rate of pre-adult development as measured by our molecular marker. These data support the conclusions drawn by others who examined the effect of the rate of development on adult life expectancy. The timing mechanisms at work during pre-adult and adult life appear to be largely regulated separately. If there is such a thing as a physiological clock, it appears to be reset upon eclosion. © 1996 Wiley-Liss, Inc.     

A Novel Geometry Mass Spectrometer, the Q-TOF, for Low-Femtomole/Attomole-Range Biopolymer Sequencing

Ultra-high-sensitivity, biopolymer sequencing is a goal in many fields of molecular biology, and collisionally activated decomposition electrospray mass spectrometry (CAD ES MS/MS) using a triple quadrupole mass spectrometer has become a method of choice for work in the high- to mid-femtomole range. However, when the detection of ions becomes statistical, as it may in that range, the mass assignment of fragment ions is inaccurate and either sequencing becomes impossible or ambiguities result due, for example, to the closeness in amino acid residue masses (I/L, N or K/Q, E). Some ambiguities may be resolved by synthesizing possible sequences, but this is unsatisfactory. In considering the limitations of triple quadrupole MS/MS with respect to scanning ion detection, resolution, transmission, and mass accuracy, we reasoned that a novel geometry quadrupole orthogonal acceleration time-of-flight (Q-TOF) instrument would have special merit for ultra-high-sensitivity MS/MS sequencing, and suggested its construction for this purpose some three years ago. A prototype Q-TOF has now been built by Micromass [Morris et al. (1996), Rapid Commun. Mass Spectrom. 10, 889–896], and in the first research on the instrument, including MHC antigen and filarial nematode glycoprotein studies, we demonstrate low-femtomole- and attomole-range sequencing with mass accuracy of better than 0.1 Da throughout the daughter-ion spectrum, thus removing sequencing ambiguities in some of the most challenging work demanding the highest sensitivity.     

Green fluorescent protein is superior to blue fluorescent protein as a quantitative reporter of promoter activity in <Emphasis Type="Italic">E. coli</Emphasis>

Fluorescent proteins related to and derived from green fluorescent protein (GFP) are widely used as tools for investigating a wide range of biological processes. In particular, GFP and its relatives have been used extensively as qualitative reporters of gene expression in many different organisms, but relatively few studies have investigated fluorescent proteins as quantitative reporters of gene expression. GFP has some limitations as a reporter gene, including possible toxicity when expressed at high levels. Therefore, it would be useful if other fluorescent proteins could be identified for use as quantitative reporters. Toward this end, we investigated BFP as a quantitative reporter of promoter activity in E. coli and directly compared it with GFPuv using a set of well-characterized synthetic constitutive promoters. The fluorescence produced in E. coli strains expressing GFPuv or BFP grown on solid medium was quantified using a CCD camera and fluorimetry. GFPuv consistently gave more reliable and statistically significant results than did BFP in all assays. Correspondingly, we found that the signal-to-noise ratio for GFPuv fluorescence is substantially higher than for BFP. We conclude that, under the conditions assessed in this study, GFPuv is superior to BFP as a quantitative reporter of promoter activity in E. coli. J. Bayes, M. Calvey, L. Reineke, A. Colagiavanni, and M. Tscheiner made equivalent contributions to this work.     

Adaptation and Natural Selection revisited

In Adaptation and Natural Selection, George C. Williams linked the distinction between group and individual adaptation with the distinction between group and individual selection. Williams’ Principle, as we will call it, says that adaptation at a level requires selection at that level. This is a necessary but not a sufficient condition; for example, group adaptation requires group selection, but the fact that group selection influences a trait’s evolution does not suffice for the resulting trait frequency to be a group adaptation. What more is required? In this paper, we describe an answer to this question that has been developed in multilevel selection theory. We also discuss an alternative framework for defining units of adaptation that violates Williams’ Principle.     

Identification and characterization of AtCASP,a plant transmembrane Golgi matrix protein

Golgins are a family of coiled-coil proteins that are associated with the Golgi apparatus. They are necessary for tethering events in membrane fusion and may act as structural support for Golgi cisternae. Here we report on the identification of an Arabidopsis golgin which is a homologue of CASP, a known transmembrane mammalian and yeast golgin. Similar to its homologues, the plant CASP contains a long N-terminal coiled-coil region protruding into the cytosol and a C-terminal transmembrane domain with amino acid residues which are highly conserved across species. Through fluorescent protein tagging experiments, we show that plant CASP localizes at the plant Golgi apparatus and that the C-terminus of this protein is sufficient for its localization, as has been shown for its mammalian counterpart. In addition, we demonstrate that the plant CASP is able to localize at the mammalian Golgi apparatus. However, mutagenesis of a conserved tyrosine in the transmembrane domain revealed that it is necessary for ER export and Golgi localization of the Arabidopsis CASP in mammalian cells, but is not required for its correct localization in plant cells. These data suggest that mammalian and plant cells have different mechanisms for concentrating CASP in the Golgi apparatus.†These authors have contributed equally to the work     

Dof基因家族调节植物生长发育功能的研究进展

Dof（DNA binding with one finger）蛋白是一类植物特异性转录因子,通常含有200～400个氨基酸和2个主要结构域。该家族成员的N 末端为高度保守的单锌指Dof结构域,具有与DNA和蛋白质相互作用的双重功能,其C末端的氨基酸序列则较为多变,是Dof蛋白重要的特异转录调控结构域。研究表明,Dof蛋白作为转录激活物或阻遏物参与了多方面的植物生长发育过程。随着基因组测序技术的发展,已有大量的Dof基因从植物基因组数据库中鉴定出来。该文对近年来国内外有关Dof基因家族的结构特点、全基因组鉴定、蛋白互作以及生物学功能等方面的研究进展进行综述,以期为Dof转录因子的深入研究提供参考。     

Identification of efflux proteins using efficient radial basis function networks with position‐specific scoring matrices and biochemical properties

Efflux proteins are membrane proteins, which are involved in the transportation of multidrugs. The annotation of efflux proteins in genomic sequences would aid to understand the function. Although the percentage of membrane proteins in genomes is estimated to be 25–30%, there is no information about the content of efflux proteins. For annotating such class of proteins it is necessary to develop a reliable method to identify efflux proteins from amino acid sequence information. In this work, we have developed a method based on radial basis function networks using position specific scoring matrices (PSSM) and amino acid properties. We noticed that the C‐terminal domain of efflux proteins contain vital information for discrimination. Our method showed an accuracy of 78 and 92% in discriminating efflux proteins from transporters and membrane proteins, respectively using fivefold cross‐validation. We utilized our method for annotating the genomes E. coli and P. aeruginosa and it predicted 8.7 and 9.2% of proteins as efflux proteins in these genomes, respectively. The predicted efflux proteins have been compared with available experimental data and we observed a very good agreement between them. Further, we developed a web server for classifying efflux proteins and it is freely available at http://rbf.bioinfo.tw/～sachen/EFFLUXpredict/Efflux‐RBF.php . We suggest that our method could be an effective tool for annotating efflux proteins in genomic sequences.Proteins 2013. © 2013 Wiley Periodicals, Inc.     

The Challenge of Pneumocystis carinii Culture1

ABSTRACT. Published and unpublished data on the cultivation of P. carinii were reviewed by a panel of investigators convened by the National Institutes of Health. Although several cell culture systems allow propagation of P. carinii for a limited time with modest rates of replication, these have not proved adequate for isolation of P. carinii in sufficient quantity to explore important basic biological investigation. Attempts at cell-free culture have yielded only transient proliferation. Because much of the unsuccessful work on cultivation of the organism has been unpublished, the panel agreed that these data may be useful to other investigators in designing experimental strategies for cultivation. Therefore, the purpose of this report is to make available this information to researchers, lest others unknowingly repeat unsuccessful methods. It is hoped that by documenting the history and the complexities of Pneumocystis culture, renewed interest and efforts will be directed toward this fundamental scientific challenge.