bluuebunny/crossref_metadata_2025 huggingface dataset:
The Arabidopsis pxa1 Mutant Is Defective in an ATP-Binding Cassette Transporter-Like Protein Required for Peroxisomal Fatty Acid β-Oxidation
Authors: Zolman Bethany K. , Silva Illeana D. , Bartel Bonnie
Published: November 2008

Peroxisomes are important organelles in plant metabolism, containing all the enzymes required for fatty acid β-oxidation. More than 20 proteins are required for peroxisomal biogenesis and maintenance. The Arabidopsis pxa1 mutant, originally isolated because it is resistant to the auxin indole-3-butyric acid (IBA), developmentally arrests when germinated without supplemental sucrose, suggesting defects in fatty acid β-oxidation. Because IBA is converted to the more abundant auxin, indole-3-acetic acid (IAA), in a mechanism that parallels β-oxidation, the mutant is likely to be IBA resistant because it cannot convert IBA to IAA. Adultpxa1 plants grow slowly compared with wild type, with smaller rosettes, fewer leaves, and shorter inflorescence stems, indicating that PXA1 is important throughout development. We identified the molecular defect in pxa1 using a map-based positional approach. PXA1 encodes a predicted peroxisomal ATP-binding cassette transporter that is 42% identical to the human adrenoleukodystrophy (ALD) protein, which is defective in patients with the demyelinating disorder X-linked ALD. Homology to ALD protein and other human and yeast peroxisomal transporters suggests that PXA1 imports coenzyme A esters of fatty acids and IBA into the peroxisome for β-oxidation. The pxa1 mutant makes fewer lateral roots than wild type, both in response to IBA and without exogenous hormones, suggesting that the IAA derived from IBA during seedling development promotes lateral root formation.
Biochemical Evidence for the Activation of Distinct Subsets of Mitogen-Activated Protein Kinases by Voltage and Defense-Related Stimuli
Authors: Link Vinzenz L. , Hofmann Markus G. , Sinha Alok K. , Ehness Rainer , Strnad Miroslav , Roitsch Thomas
Published: November 2008

Activation of mitogen-activated protein (MAP) kinases is a common reaction of plant cells in defense-related signal transduction pathways. To gain insight into the mechanisms that determine specificity in response to a particular stimulus, a biochemical approach has been employed. Photoautotrophic suspension culture cells of tomato (Lycopersicon peruvianum) were used as experimental system to characterize MAP kinase activation by different stress-related stimuli. An elicitor preparation of the tomato-specific pathogen Fusarium oxysporum lycopersiciwas shown to result in the simultaneous induction of four kinase activities that could be separated by ion-exchange chromatography. The simultaneous activation of multiple MAP kinases was further substantiated by distinct pharmacological and immunological properties: a differential sensitivity toward various protein kinase inhibitors and a differential cross-reaction with isoform-specific MAP kinase antibodies. In contrast to the two fungal elicitors chitosan and theF. oxysporum lycopersici preparation, the plant-derived stimuli polygalacturonic acid and salicylic acid were shown to activate distinctly different subsets of MAP kinases. Application of a voltage pulse was introduced as a transient stress-related stimulus that does not persist in the culture. Voltage application activates a distinct set of MAP kinases, resembling those activated by salicylic acid treatment, and generates a refractory state for the salicylic acid response. The inhibitory effect of nifedipine indicates that current application may directly affect voltage-gated calcium channels, thus, providing a tool to study various calcium-dependent pathways.
Altered Levels of Proline Dehydrogenase Cause Hypersensitivity to Proline and Its Analogs in Arabidopsis
Authors: Mani Srikrishnan , Van de Cotte Brigitte , Van Montagu Marc , Verbruggen Nathalie
Published: November 2008

Pro dehydrogenase (PDH) catalyzes the first and rate-limiting step in the Pro catabolic pathway. In Arabidopsis, this enzyme is encoded byAt-PDH. To investigate the role of Pro catabolism in plants, we generated transgenic Arabidopsis plants with altered levels of PDH by sense (PDH-S plants) and antisense (PDH-AS plants) strategies. Free Pro levels were reduced by up to 50% in PDH-S plants under stress and recovery conditions and enhanced by a maximum of 25% in PDH-AS plants, despite large modifications of theAt-PDH transcript and At-PDH protein levels. A similar trend in free Pro levels was observed in the PDH-S and PDH-AS seeds without visible effects on germination or growth. Under stress conditions, PDH transgenic plants showed no signs of change in osmotolerance. However, addition of exogenous Pro increased survival rates of salt-stressed PDH-S plants by 30%. Isotope-labeling studies showed that the conversion of [14C]Pro to Glu was reduced in PDH-AS plants and increased in PDH-S plants, especially under stress conditions. Furthermore, PDH-AS plants were hypersensitive to exogenous Pro, whereas PDH-S plants were sensitive to Pro analogs. These findings demonstrate that altered At-PDH levels lead to weakly modified free Pro accumulation with a limited impact on plant development and growth, suggesting a tight control of Pro homeostasis and/or gene redundancy.
Functional Analysis of the Early Steps of Carotenoid Biosynthesis in Tobacco
Authors: Busch Marco , Seuter Anja , Hain Rüdiger
Published: November 2008

Carotenoids contribute to energy transduction in the light harvesting complexes and serve in protection from excess light fluence. Because of the importance of carotenoids, the genes encoding enzymes of carotenoid biosynthesis in higher plants are potential targets for herbicides. To obtain further insight into tobacco carotenoid biosynthesis and to investigate and prioritize potential herbicide targets in the pathway, the effects of changed phytoene synthase (PSY) and phytoene desaturase (PDS) gene expression were studied in transgenic tobacco (Nicotiana tabacum Petit Havana SR1) plants. Genes for both enzymes were cloned from tobacco, and surprisingly two functional PSY genes were found. Transgenic tobacco plants constitutively expressing these genes in both sense and antisense orientations were examined regarding phenotype, carotenoid content and transcript levels of carotene biosynthesis genes. Overexpression of either psy gene resulted in severe phenotypic effects including dwarfism, altered leaf morphology, and pigmentation. A correlation among phenotype, transcript level, and metabolic profile was demonstrated by comparison of hemizygous and homozygous plants from the same transformation event. Antisense expression of PSY and PDS also caused lethal phenotypes. Transcript levels of other carotene biosynthesis genes remained unaltered in the transgenic mutant. Phytoene accumulated in plants expressing antisense RNA to pds. However, elevated levels of phytoene were detected suggesting an increase in metabolic flux into this pathway.
The Matrix Metalloproteinase GeneGmMMP2Is Activated in Response to Pathogenic Infections in Soybean
Authors: Liu Yongqing , Dammann Christian , Bhattacharyya Madan K.
Published: November 2008

Matrix metalloproteinases (MMPs) play an important role in host defense responses against pathogens in mammals where their activities lead to the production of antimicrobial peptides. We have identified a novel soybean (Glycine max) metalloproteinase gene, GmMMP2, that is transcriptionally up-regulated in infected tissues. The deduced amino acid sequence indicates that this gene belongs to the MMP family. It is a preproprotein containing an N-terminal signal peptide, a cysteine switch, a zinc-binding catalytic motif, and a C-terminal transmembrane domain. The GmMMP2 expressed in and purified from Escherichia coli exhibited an in vitro enzymatic activity in digesting myelin basic protein. All plant metalloproteinases reported so far have no known functions. However, they have been suggested to be involved in extracellular cell matrix degradation during development or senescence. Our investigations demonstrate that the GmMMP2transcript levels were rapidly increased in compatible and incompatible interactions of soybean tissues with the oomycete pathogenPhytophthora sojae or the bacterial pathogenPseudomonas syringae pv. glycinea. In agreement with the GmMMP2 activation, a metalloproteinase activity was gradually increased in suspension-cultured cells following the bacterial infection.GmMMP2 was also activated in response to wounding and dehydration. However, GmMMP2 activation did not correlate with the oxidative burst leading to the hypersensitive response cell death or the tissue senescence progress that involves programmed cell death. Our investigations suggest that GmMMP2 may be involved in a novel defense response of soybean against pathogenic infections.
Functional Regions of Rice Heat Shock Protein, Oshsp16.9, Required for Conferring Thermotolerance inEscherichia coli
Authors: Yeh Ching-Hui , Chen Yih-Ming , Lin Chu-Yung
Published: November 2008

Rice (Oryza sativa) class I low-molecular mass (LMM) heat shock protein (HSP), Oshsp16.9, has been shown to be able to confer thermotolerance in Escherichia coli. To define the regions for this intriguing property, deletion mutants of this hsp have been constructed and overexpressed in E. coliXL1-blue cells after isopropyl β-d-thioglactopyranoside induction. The deletion of amino acid residues 30 through 36 (PATSDND) in the N-terminal domain or 73 through 78 (EEGNVL) in the consensus II domain of Oshsp16.9 led to the loss of chaperone activities and also rendered the E. coli incapable of surviving at 47.5°C. To further investigate the function of these two domains, we determined the light scattering changes of Oshsp16.9 mutant proteins at 320 nm under heat treatment either by themselves or in the presence of a thermosensitive enzyme, citrate synthase. It was observed that regions of amino acid residues 30 through 36 and 73 through 78 were responsible for stability of Oshsp16.9 and its interactions with other unfolded protein substrates, such as citrate synthase. Studies of two-point mutants of Oshsp16.9, GST-N74E73K and GST-N74E74K, indicate that amino acid residues 73 and 74 are an important part of the substrate-binding site of Oshsp16.9. Non-denaturing gel analysis of purified Oshsp16.9 revealed that oligomerization of Oshsp16.9 was necessary but not sufficient for its chaperone activity.
Control of Gibberellin Levels and Gene Expression during De-Etiolation in Pea
Authors: Reid James B. , Botwright Natasha A. , Smith Jennifer J. , O'Neill Damian P. , Kerckhoffs L. Huub J.
Published: November 2008

Gibberellin A1 (GA1) levels drop significantly in wild-type pea (Pisum sativum) plants within 4 h of exposure to red, blue, or far-red light. This response is controlled by phytochrome A (phyA) (and not phyB) and a blue light receptor. GA8 levels are increased in response to 4 h of red light, whereas the levels of GA19, GA20, and GA29 do not vary substantially. Red light appears to control GA1 levels by down-regulating the expression of Mendel's LE (PsGA3ox1) gene that controls the conversion of GA20 to GA1, and by up-regulating PsGA2ox2, which codes for a GA 2-oxidase that converts GA1 to GA8. This occurs within 0.5 to 1 h of exposure to red light. Similar responses occur in blue light. The major GA 20-oxidase gene expressed in shoots, PsGA20ox1, does not show substantial light regulation, but does show up-regulation after 4 h of red light, probably as a result of feedback regulation. Expression of PsGA3ox1 shows a similar feedback response, whereasPsGA2ox2 shows a feed-forward response. These results add to our understanding of how light reduces shoot elongation during de-etiolation.
Temporal and Spatial Expression of a Polygalacturonase during Leaf and Flower Abscission in Oilseed Rape and Arabidopsis
Authors: González-Carranza Zinnia Haydé , Whitelaw Catherine Ann , Swarup Ranjan , Roberts Jeremy Alan
Published: November 2008

During leaf abscission in oilseed rape (Brassica napus), cell wall degradation is brought about by the action of several hydrolytic enzymes. One of these is thought to be polygalacturonase (PG). Degenerate primers were used to isolate a PG cDNA fragment by reverse transcriptase-polymerase chain reaction from RNA extracted from ethylene-promoted leaf abscission zones (AZs), and in turn a full-length clone (CAW471) from an oilseed rape AZ cDNA library. The highest homology of this cDNA (82%) was to an Arabidopsis sequence that was predicted to encode a PG protein. Analysis of expression revealed that CAW471 mRNA accumulated in the AZ of leaves and reached a peak 24 h after ethylene treatment. Ethylene-promoted leaf abscission in oilseed rape was not apparent until 42 h after exposure to the gas, reaching 50% at 48 h and 100% by 56 h. In floral organ abscission, expression of CAW471 correlated with cell separation. Genomic libraries from oilseed rape and Arabidopsis were screened with CAW471 and the respective genomic clones PGAZBRAN and PGAZATisolated. Characterization of these PG genes revealed that they had substantial homology within both the coding regions and in the 5′-upstream sequences. Fusion of a 1,476-bp 5′-upstream sequence ofPGAZAT to β-glucuronidase or green fluorescent protein and transformation of Arabidopsis revealed that this fragment was sufficient to drive expression of these reporter genes in the AZs at the base of the anther filaments, petals, and sepals.
A Calcium-Dependent Protein Kinase Is Systemically Induced upon Wounding in Tomato Plants
Authors: Chico José Manuel , Raı́ces Marcela , Téllez-Iñón Marı́a Teresa , Ulloa Rita Marı́a
Published: November 2008

A full-length cDNA clone (LeCDPK1) from tomato (Lycopersicon esculentum) encoding a calcium-dependent protein kinase (CDPK) was isolated by screening a cDNA library from tomato cell cultures exposed to Cladosporium fulvum elicitor preparations. The predicted amino acid sequence of the cDNA reveals a high degree of similarity with other members of the CDPK family. LeCDPK1 has a putative N-terminal myristoylation sequence and presents a possible palmitoylation site. The in vitro translated protein conserves the biochemical properties of a member of the CDPK family. In addition, CDPK activity was detected in soluble and particulate extracts of tomato leaves. Basal levels of LeCDPK1 mRNA were detected by northern-blot analysis in roots, stems, leaves, and flowers of tomato plants. The expression of LeCDPK1 was rapidly and transiently enhanced in detached tomato leaves treated with pathogen elicitors and H2O2. Moreover, when tomato greenhouse plants were subjected to mechanical wounding, a transient increase of LeCDPK1 steady-state mRNA levels was detected locally at the site of the injury and systemically in distant non-wounded leaves. The increase observed in LeCDPK1 mRNA upon wounding correlates with an increase in the amount and in the activity of a soluble CDPK detected in extracts of tomato leaves, suggesting that this kinase is part of physiological plant defense mechanisms against biotic or abiotic attacks.
Overexpression of a 9-cis-Epoxycarotenoid Dioxygenase Gene in Nicotiana plumbaginifolia Increases Abscisic Acid and Phaseic Acid Levels and Enhances Drought Tolerance
Authors: Qin Xiaoqiong , Zeevaart Jan A.D.
Published: November 2008

The plant hormone abscisic acid (ABA) plays important roles in seed maturation and dormancy and in adaptation to a variety of environmental stresses. An effort to engineer plants with elevated ABA levels and subsequent stress tolerance is focused on the genetic manipulation of the cleavage reaction. It has been shown in bean (Phaseolus vulgaris) that the gene encoding the cleavage enzyme (PvNCED1) is up-regulated by water stress, preceding accumulation of ABA. Transgenic wild tobacco (Nicotiana plumbaginifolia Viv.) plants were produced that overexpress thePvNCED1 gene either constitutively or in an inducible manner. The constitutive expression of PvNCED1 resulted in an increase in ABA and its catabolite, phaseic acid (PA). When thePvNCED1 gene was driven by the dexamethasone (DEX)-inducible promoter, a transient induction ofPvNCED1 message and accumulation of ABA and PA were observed in different lines after application of DEX. Accumulation of ABA started to level off after 6 h, whereas the PA level continued to increase. In the presence of DEX, seeds from homozygous transgenic line TN1 showed a 4-d delay in germination. After spraying with DEX, the detached leaves from line TN1 had a drastic decrease in their water loss relative to control leaves. These plants also showed a marked increase in their tolerance to drought stress. These results indicate that it is possible to manipulate ABA levels in plants by overexpressing the key regulatory gene in ABA biosynthesis and that stress tolerance can be improved by increasing ABA levels.
Manipulation of Strawberry Fruit Softening by Antisense Expression of a Pectate Lyase Gene
Authors: Jiménez-Bermúdez Silvia , Redondo-Nevado José , Muñoz-Blanco Juan , Caballero José L. , López-Aranda José M. , Valpuesta Victoriano , Pliego-Alfaro Fernando , Quesada Miguel A. , Mercado José A.
Published: November 2008

Strawberry (Fragaria ×ananassa, Duch., cv Chandler) is a soft fruit with a short postharvest life, mainly due to a rapid lost of firm texture. To control the strawberry fruit softening, we obtained transgenic plants that incorporate an antisense sequence of a strawberry pectate lyase gene under the control of the 35S promoter. Forty-one independent transgenic lines (Apel lines) were obtained, propagated in the greenhouse for agronomical analysis, and compared with control plants, non-transformed plants, and transgenic lines transformed with the pGUSINT plasmid. Total yield was significantly reduced in 33 of the 41 Apel lines. At the stage of full ripen, no differences in color, size, shape, and weight were observed between Apel and control fruit. However, in most of the Apel lines, ripened fruits were significantly firmer than controls. Six Apel lines were selected for further analysis. In all these lines, the pectate lyase gene expression in ripened fruit was 30% lower than in control, being totally suppressed in three of them. Cell wall material isolated from ripened Apel fruit showed a lower degree of in vitro swelling and a lower amount of ionically bound pectins than control fruit. An analysis of firmness at three different stages of fruit development (green, white, and red) showed that the highest reduction of softening in Apel fruit occurred during the transition from the white to the red stage. The postharvest softening of Apel fruit was also diminished. Our results indicate that pectate lyase gene is an excellent candidate for biotechnological improvement of fruit softening in strawberry.
Selenium Assimilation and Volatilization from Selenocyanate-Treated Indian Mustard and Muskgrass
Authors: de Souza Mark P. , Pickering Ingrid J. , Walla Michael , Terry Norman
Published: November 2008

Selenocyanate (SeCN−) is a major contaminant in the effluents from some oil refineries, power plants, and in mine drainage water. In this study, we determined the potential of Indian mustard (Brassica juncea) and muskgrass (a macroalga,Chara canescens) for SeCN− phytoremediation in upland and wetland situations, respectively. The tolerance of Indian mustard to toxic levels of SeCN− was similar to or higher than other toxic forms of Se. Indian mustard treated with 20 μm SeCN− removed 30% (w/v) of the Se supplied in 5 d, accumulating 554 and 86 μg of Se g−1 dry weight in roots and shoots, respectively. Under similar conditions, muskgrass removed approximately 9% (w/v) of the Se supplied as SeCN− and accumulated 27 μg of Se g−1 dry weight. A biochemical pathway for SeCN− degradation was proposed for Indian mustard. Indian mustard and muskgrass efficiently degraded SeCN− as none of the Se accumulated by either organism remained in this form. Indian mustard accumulated predominantly organic Se, whereas muskgrass contained Se mainly as selenite and organic Se forms. Indian mustard produced volatile Se from SeCN− in the form of less toxic dimethylselenide. Se volatilization by Indian mustard accounted for only 0.7% (w/v) of the SeCN− removed, likely because the biochemical steps in the production of dimethylselenide from organic Se were rate limiting. Indian mustard is promising for the phytoremediation of SeCN−-contaminated soil and water because of its remarkable abilities to phytoextract SeCN−and degrade all the accumulated SeCN− to other Se forms.
Functional Analysis of Tomato Pti4 in Arabidopsis,
Authors: Wu Keqiang , Tian Lining , Hollingworth Jamie , Brown Daniel C.W. , Miki Brian
Published: November 2008

Pti4 is a tomato (Lycopersicon esculentum) transcription factor that belongs to the ERF (ethylene-responsive element binding factor) family of proteins. It interacts with the Pto kinase in tomato, which confers resistance to the Pseudomonas syringae pv tomato pathogen that causes bacterial speck disease. To study the function of Pti4, transgenic Arabidopsis plants were generated that expressed tomatoPti4 driven by the strong constitutive promoters, cauliflower mosaic virus 35S and tCUP. Global gene expression analysis by Affimetric GeneChip indicated that expression of Pti4 in transgenic Arabidopsis plants induced the expression of GCC box-containing PR genes. We also demonstrated that Pti4 enhanced GCC box-mediated transcription of a reporter gene. The data suggests that tomato Pti4 could act as a transcriptional activator to regulate expression of GCC box-containing genes. Furthermore, we show that the expression of tomatoPti4 in transgenic Arabidopsis plants produced a phenotype similar to that seen in plants treated with ethylene, thus providing evidence that the Pti4 gene is involved in the regulation of a subset of ethylene-responsive genes containing the GCC box.
Transgenic Production of Epoxy Fatty Acids by Expression of a Cytochrome P450 Enzyme from Euphorbia lagascaeSeed
Authors: Cahoon Edgar B. , Ripp Kevin G. , Hall Sarah E. , McGonigle Brian
Published: November 2008

Seed oils of a number of Asteraceae and Euphorbiaceae species are enriched in 12-epoxyoctadeca-cis-9-enoic acid (vernolic acid), an unusual 18-carbon Δ12-epoxy fatty acid with potential industrial value. It has been previously demonstrated that the epoxy group of vernolic acid is synthesized by the activity of a Δ12-oleic acid desaturase-like enzyme in seeds of the Asteraceae Crepis palaestina and Vernonia galamensis. In contrast, results from metabolic studies have suggested the involvement of a cytochrome P450 enzyme in vernolic acid synthesis in seeds of the Euphorbiaceae species Euphorbia lagascae. To clarify the biosynthetic origin of vernolic acid in E. lagascae seed, an expressed sequence tag analysis was conducted. Among 1,006 randomly sequenced cDNAs from developingE. lagascae seeds, two identical expressed sequence tags were identified that encode a cytochrome P450 enzyme classified as CYP726A1. Consistent with the seed-specific occurrence of vernolic acid in E. lagascae, mRNA corresponding to theCYP726A1 gene was abundant in developing seeds, but was not detected in leaves. In addition, expression of the E. lagascae CYP726A1 cDNA in Saccharomyces cerevisiae was accompanied by production of vernolic acid in cultures supplied with linoleic acid and an epoxy fatty acid tentatively identified as 12-epoxyoctadeca-9,15-dienoic acid (12-epoxy-18:2Δ9,15) in cultures supplied with α-linolenic acid. Consistent with this, expression of CYP726A1 in transgenic tobacco (Nicotiana tabacum) callus or somatic soybean (Glycine max) embryos resulted in the accumulation of vernolic acid and 12-epoxy-18:2Δ9,15. Overall, these results conclusively demonstrate that Asteraceae species and the Euphorbiaceae E. lagascae have evolved structurally unrelated enzymes to generate the Δ12-epoxy group of vernolic acid.
Stochastic fitting of specular X-ray reflectivity data usingStochFit
Authors: Danauskas Stephen M. , Li Dongxu , Meron Mati , Lin Binhua , Lee Ka Yee C.
Published: November 2008

Specular X-ray reflectivity data provide detailed information on the electron density distribution at an interface. Typical modeling methods involve choosing a generic electron density distribution based on an initial speculation of the electron density profile from the physical parameters of the experimental system. This can lead to a biased set of solutions.StochFitprovides stochastic model-independent and model-dependent methods for analyzing X-ray reflectivities of thin films at an interface.StochFitdivides an electron density profile into many small boxes and stochastically varies the electron density of these boxes to locate the best fit to a measured reflectivity. Additionally, it provides the ability to perform model-dependent fitting with a stochastic search of the parameter space in order to locate the best possible fit. While model-independent profile search algorithms have been described previously, they are difficult to implement because of the heavy computational requirements, and there is a dearth of software available to the general scientific public utilizing these techniques. Several cases that illustrate the usefulness of these techniques are presented, with a demonstration of how they can be used in tandem.
Is your cold-stream working for you or against you? An in-depth look at temperature and sample motion
Authors: Alkire R. W. , Duke N. E. C. , Rotella F. J.
Published: November 2008

It is normally assumed that a commercial gaseous nitrogen cold-stream provides a sample environment near 100 K and that the force of the cold-stream does not induce movement in the sample. As might be expected, the reality is much more complex. Here, an investigation of one cold-stream, starting with the temperature profile, is presented. Using silicon single crystals and flexible mounting loops, an approximate force/vibration profile of the cold-stream is obtained. Results indicate that the center of the temperature profile is offset from the position suggested by the manufacturer-supplied alignment tool and coincides with the area within the cold-stream that has the most consistent force profile. Tests indicate that this region is only about one-third of the width of the cold-stream nozzle opening. To verify that the results were relevant to protein crystallographic data collection, the impact of cold-stream position on the final data quality for lysozyme crystals was analyzed. On the basis of the observations it is recommended that users perform a temperature profile of their cold-streams to ensure proper alignment instead of relying only on the alignment tool for setup. In addition, suggestions are made on what users can look for in data processing to identify problems with loop movement and what users can do to minimize the impact of these problems on their experiments.
The use of blue native PAGE in the evaluation of membrane protein aggregation states for crystallization
Authors: Ma Jichun , Xia Di
Published: November 2008

Crystallization has long been one of the bottlenecks in obtaining structural information at atomic resolution for membrane proteins. This is largely due to difficulties in obtaining high-quality protein samples. One frequently used indicator of protein quality for successful crystallization is the monodispersity of proteins in solution, which is conventionally obtained by size exclusion chromatography (SEC) or by dynamic light scattering (DLS). Although useful in evaluating the quality of soluble proteins, these methods are not always applicable to membrane proteins either because of the interference from detergent micelles or because of the requirement for large sample quantities. Here, the use of blue native polyacrylamide gel electrophoresis (BN–PAGE) to assess aggregation states of membrane protein samples is reported. A strong correlation is demonstrated between the monodispersity measured by BN–PAGE and the propensity for crystallization of a number of soluble and membrane protein complexes. Moreover, it is shown that there is a direct correspondence between the oligomeric states of proteins as measured by BN–PAGE and those obtained from their crystalline forms. When applied to a membrane protein with unknown structure, BN–PAGE was found to be useful and efficient for selecting well behaved proteins from various constructs and in screening detergents. Comparisons of BN–PAGE with DLS and SEC are provided.
Combining precession electron diffraction data with X-ray powder diffraction data to facilitate structure solution
Authors: Xie Dan , Baerlocher Christian , McCusker Lynne B.
Published: November 2008

Information derived from precession electron diffraction (PED) patterns can be used to advantage in combination with high-resolution X-ray powder diffraction data to solve crystal structures that resist solution from X-ray data alone. PED data have been exploited in two different ways for this purpose: (1) to identify weak reflections and (2) to estimate the phases of the reflections in the projection. The former is used to improve the partitioning of the reflection intensities within an overlap group and the latter to provide some starting phases for structure determination. The information was incorporated into a powder charge-flipping algorithm for structure solution. The approaches were first developed using data for the moderately complex zeolite ZSM-5, and then tested on TNU-9, one of the two most complex zeolites known. In both cases, including PED data from just a few projections facilitated structure solution significantly.
Activation of mitogen‐activated protein kinases is involved in the induction of interferon β gene in macrophages infected with Orientia tsutsugamushi
Authors: Koo Jung‐Eun , Yun Ji‐Hyun , Lee Keun‐Hwa , Hyun Jin‐Won , Kang Hee‐Kyoung , Jang Won‐Jong , Park Kyung‐Hee , Koh Young‐Sang
Published: November 2008

We investigated the role of MAPK in IFN‐β gene expression in macrophages after infection with Orientia tsutsugamushi. ERK1/2 became phosphorylated in Orientia‐stimulated macrophages. Selective inhibition of ERK1/2 and p38 MAPK could all significantly reduce Orientia‐stimulated IFN‐β mRNA expression. Orientia inactivation by heat abolished IFN‐β mRNA induction only, whereas cytochalasin D treatment completely blocked both IFN‐β and chemokine expression, suggesting requirement of cellular internalization by viable bacteria for IFN‐β gene induction. In conclusion, our data indicate that MAPK pathways are required to induce maximal IFN‐β gene expression in macrophages during Orientia infection.
Patients’ experience of chronic lymphocytic leukaemia: baseline health‐related quality of life results from the LRF CLL4 trial
Authors: Else Monica , Smith Alastair G. , Cocks Kim , Richards Sue M. , Crofts Shirley , Wade Rachel , Catovsky Daniel ,   on behalf of the UK NCRI CLL Trials Group
Published: November 2008

We examined the effects of active untreated chronic lymphocytic leukaemia (CLL) on health‐related quality of life (HRQoL), measured by the European Organization for Research and Treatment of Cancer quality of life questionnaire (EORTC QLQ‐C30) at randomisation into the Leukaemia Research Fund CLL4 trial. Patients were scored 0–100 within each of 15 domains. A difference between groups of ≥10 points was deemed clinically significant (asterisked * below). 431 valid baseline questionnaires were returned. Compared with population norms, patients had impaired HRQoL in 13/15 domains. The greatest differences were in fatigue*, sleep disturbance*, role functioning and global HRQoL. Fatigue was reported by 81% of patients, compared with the next most common symptoms: sleep disturbance (56%) and dyspnoea (49%). There was no association between spleen, liver or lymph node enlargement, or lymphocytosis and any HRQoL domain. Older age (≥70 years) was associated with poorer physical functioning (P < 0·001) but fewer financial difficulties (P < 0·001*). Impairment of HRQoL at baseline was most apparent in stage A‐progressive patients with B‐symptoms and stage C patients with haemoglobin <120 g/l: compared with all others, these patients had poorer physical, role and social functioning, more fatigue and dyspnoea and poorer global HRQoL (all P ≤ 0·001*). These findings support the recommendation to begin treatment when patients experience symptomatic disease, to improve HRQoL.
Novel archaeal plasmid pAH1 and its interactions with the lipothrixvirus AFV1
Authors: Basta Tamara , Smyth John , Forterre Patrick , Prangishvili David , Peng Xu
Published: November 2008

At present very little is known about interactions between extrachromosomal genetic elements in Archaea. Here we describe an Acidianus strain which carries naturally a novel 28 kb conjugative plasmid‐like element, pAH1, and also serves as a laboratory host for lipothrixvirus AFV1. In an attempt to establish a system for studying plasmid–virus interactions we characterized the genome of pAH1 which closely resembles those of the Sulfolobus conjugative plasmids pARN3 and pARN4. pAH1 integrates site specifically into, and excises from, the host chromosome indicating a dynamic interaction with the latter. Although nucleotide sequence comparisons revealed extensive intergenomic exchange during the evolution of archaeal conjugative plasmids, pAH1 was shown to be stably maintained suggesting that the host system is suitable for studying plasmid–virus interactions. AFV1 infection and propagation leads to a loss of the circular form of pAH1 and this effect correlates positively with the increase in the intracellular quantity of AFV1 DNA. We infer that the virus inhibits plasmid replication since no pAH1 degradation was observed. This mechanism of archaeal viral inhibition of plasmid propagation is not observed in bacteria where relevant bacteriophages either are dependent on a conjugative plasmid for successful infection or are excluded by a resident plasmid.
Facies architecture and depositional processes of the Holocene–Recent accretionary forced regressive Skagen spit system, Denmark
Authors: NIELSEN LARS HENRIK , JOHANNESSEN PETER NIELS
Published: November 2008

Spit systems are seldom recognized in the pre‐Quaternary sedimentary record compared to their common occurrence along present‐day coasts and in Quaternary successions. This lack of recognition may partly be due to the lack of widely accepted depositional models describing the facies characteristics of spit systems and their subaqueous platforms in particular. The Skagen spit system is a large active system that began to form 7150 yr bp and from 5500 bp to Recent times it has prograded 4 m year−1 and accumulated 3·5 × 109 m3 of sand. The spit system provides a unique opportunity for establishing a well‐constrained depositional model because uplift and erosion have made large windows into the preserved facies, while active spit‐forming processes can be examined at the young prograding end of the same system. The depositional model presented here thus builds on excellent outcrops, surface morphology, a well‐defined palaeogeography and detailed C14 age control supplemented with observations from continuous well cores and profiles obtained by ground‐penetrating radar and transient electromagnetic surveys. The factors that have governed the development of the spit system, such as relative sea‐level change, wave and current climate, tidal range, sediment transport and depositional rates are also well‐understood. The sedimentary facies of the spit system are grouped into four principal units consisting from below of thick storm sand beds, dune and bar‐trough deposits, beach deposits and peat beds. These four units form a coarsening and shallowing upward sand‐dominated succession, up to 32 m thick, which overlies offshore silt with a transition zone and is topped by a diastem overlain by young aeolian dune sand. The sedimentary structures and depositional processes are described in detail and integrated into a depositional model, which is compared to other spit systems and linear shoreface models.
Ankyloglossia in Dogs: A Morphological and Immunohistochemical Study
Authors: Karahan S. , ÇInar Kul B.
Published: November 2008

Ankyloglossia is a congenital anomaly of the tongue that is usually characterized by a short and thick lingual frenulum. The genetic mutations such as in TBox genes and other foetal mechanism have still been under investigation as possible causes of ankyloglossia. This study describes morphology of anklyoglossia phenotype found in members of two closely bred Kangal dog families. Morphology of ankyloglossia and immunohistochemical localization of αB‐crystallin, an anti‐apoptotic protein, in the frenulum tissue collected during frenectomy was described. Grossly, the lingual frenulum was observed as it extended up to the tip or near the tip of the tongue. The tip of the tongue was often notched and appeared in ‘W’ shape. No other craniofacial anomalies were associated with ankyloglossia. Histologically, the frenulum tissue was covered by stratified squamous epithelia of variable thickness. Skeletal muscle fibres were often scattered in the vicinity of collagen fibres of the lamina propria. αB‐crystallin was immunolocalized exclusively in skeletal muscle fibres. In conclusion, ankyloglossia in the dog generally occurs as a sole anomaly. The presence of αB‐crystallin immunoreactivity exclusively in skeletal muscle fibres suggests that there may be a connection between occurrences of ankyloglossia in the dog and a delay or interference with apoptosis of the skeletal fibres in the frenulum tissue.
Gross Anatomy of the Female Genital Organs of the Domestic Donkey (Equus asinusLinné, 1758)
Authors: Renner‐Martin T. F. P. , Forstenpointner G. , Weissengruber G. E. , Eberhardt L.
Published: November 2008

Although donkeys play an important role as companion or pack and draught animals, theriogenological studies and anatomical data on the genital organs of the jenny are sparse. To provide anatomical descriptions and morphometric data, the organa genitalia feminina, their arteries and the ligamentum latum uteri of 10 adult but maiden jennies were examined by means of gross anatomical and morphometric techniques. In comparison with anatomical data of horses obtained from literature the genital organs of jennies appear to be more voluminous in relation to the body mass and the position of the ovaries is slightly further cranial than in mares. In asses, the ovaries contain large follicles reaching a diameter of up to 40 mm. The mesosalpinx is much wider than in the horse forming a considerably spacious bursa ovarica. The asinine ligamentum teres uteri reveals a very prominent cranial end, the ‘appendix’. Tortuous mucosal folds occur in the wall of the jenny’s cervical channel. The vascularization of the female genital organs of asses is very similar to that of horses. One of the examined specimens reveals a large mucosal fold dividing the cranial part of the vagina into a left and right compartment.
Prenatal Development of the Human Epicardiac Ganglia
Authors: Saburkina I. , Pauziene N. , Pauza D. H.
Published: November 2008

The aim of this study was to determine the developmental anatomy of intrinsic cardiac ganglia with respect to epicardiac ganglionated nerve plexus in the human fetuses at different gestation stages. Twenty fetal hearts were investigated applying a technique of histochemistry for acetylcholinesterase to visualize the epicardiac neural ganglionated plexus with its subsequent examinations on total (non‐sectioned) hearts. Most epicardiac ganglia embodied multilayered neurons and were oval in shape, but some ganglia involved neurons lying in one layer or had the irregular appearance because of their extensions along inter‐ganglionic nerves. The mean ganglion area of fetuses at gestation stages of 15–40 weeks was 0.03 ± 0.008 mm2. The largest epicardiac ganglia, reaching in area 0.4 mm2, were concentrated on the dorsal surface of both atria. The particular fused or “dual” ganglia were identified at the gestation stages of 23–40 weeks, but they composed only 2.3 ± 0.7% of all found epicardiac ganglia. A direct positive correlation was determined between the fetal age and the ganglion area (mm2) as well as between the fetal age and the number of inter‐ganglionic nerves. The revealed appearance of epicardiac ganglia in the human fetuses at 15–40 weeks of gestation confirms their prenatal development and presumable intrinsic remodelling.
Ovarian Structure in Mice Lines Selected for Weight
Authors: Bernardi S. F. , Brogliatti G. , Oyarzabal M. I.
Published: November 2008

Selection for body weight at 49 day of age (s and h, downward selected lines; s′ and h′, upward selected lines) affected reproductive traits in CF1 mice lines. The objective of this study was to compare ovarian structures in females of these lines, as well as in unselected controls (Line t). The number of ovarian follicles (N), follicle diameter (FD), number of corpora lutea (CL), litter size (LS), and body weight (W), were recorded. There were significant differences among lines for N, FD, CL, LS and W; means values for the lines with the greatest difference for post‐pubertal females were: Ns = 19.3 and Ns′ = 32.7; FDh′ = 161.7 and FDs′ = 178.2; CLh = 10.3 and CLs′ = 21.9; LSs = 6.0 and LSh′ = 11.1; Wh = 18.9 and Ws′ = 32.4. There were also differences between positive lines; Line s′ had a higher proportion of large follicles in pre‐pubertal females, a greater capacity to convert these follicles into CL, but a lower capacity to maintain embryos until term than Line h′. For negative lines, Line h apparently had a reduced incidence of embryonic loss when compared with Line s. In conclusion, selection for body weight modified ovarian structure, as well as reproductive efficiency.
Effect of hCG Treatment on the Oestrous and Ovulation Responses to FSH in Prepubertal Gilts
Authors: Manjarin R , Dominguez JC , Castro MJ , Sprecher DJ , Cassar G , Friendship RM , Kirkwood RN
Published: November 2008

Contents

To ensure sufficient numbers of pregnant females, particularly at hotter times of the year, hormonal induction of gilt oestrus may be necessary. However, the gilt oestrus and ovulation responses to gonadotrophin treatment have often proven unpredictable. The objective of this study was to examine possible reasons for this unpredictability. Prepubertal gilts (approximately 150 days of age, n = 63) were assigned to one of three treatments: injection of 300 IU hCG (n = 15); pre‐treatment with 100 mg FSH in polyvinylpyrrolidinone administered as 2 × 50 mg injections 24 h apart, followed by 600 IU eCG at 24 h after the second FSH injection (n = 23); or FSH pre‐treatment as above followed by 300 IU hCG at 24 h after the second FSH injection (n = 25). To facilitate oestrus detection, gilts were exposed to a mature boar for 15 min daily for 7 days. Blood samples were obtained on the day of eCG or hCG injection and again 10 days later and gilt ovulation responses determined based on elevated progesterone concentrations. The oestrus responses by 7 days were 6.7%, 17.5% and 64.0% for gilts treated with hCG, FSH + eCG and FSH + hCG, respectively (p < 0.001). The oestrous gilt receiving hCG alone and one oestrous FSH + hCG gilt did not ovulate, all other oestrous gilts ovulated. A further two anoestrous FSH + eCG‐treated gilts ovulated. These data suggest that FSH pre‐treatment facilitated the development of ovarian follicles to the point where they became responsive to hCG, but had little effect on the response to eCG.
Acute BRSV Infection in Young AI Bulls: Effect on Sperm Quality
Authors: Alm K , Koskinen E , Vahtiala S , Andersson M
Published: November 2008

Contents

Bovine respiratory syncytial virus (BRSV) infection is an important part of the calf pneumonia complex, occasionally affecting even adult cattle. However, the pathogenicity of BRSV in animals older than 6 months is often neglected. Finland is free of many contagious diseases in farm animals, and this gives a good opportunity to study the effects of specific pathogens on bovine reproduction. This report describes the deteriorating effects of BRSV epizootics on sperm morphology and fertility of young dairy bulls (n = 79) at a bull station. More than half of the young bulls had a clinical respiratory disease caused by BRSV during their quarantine when they were 6 months old. Four of seven subsequent quarantine groups were affected. Six months later, when these seropositive bulls (n = 54) came into semen production, they had poorer sperm morphology, and the proportion of normal spermatozoa was 74.1% in BRSV‐seropositive animals compared with 81.2% in seronegative bulls (n = 25) (p = 0.035). Field fertility was also slightly affected, the 60‐day non‐return rates were 75.2% and 76.8% for BRSV seropositive and seronegative bulls respectively (p = 0.014). Potential reasons for lowered sperm quality are discussed here.
Effect of Entrepreneurial Behaviour on Researchers' Knowledge Production: Evidence from Canadian Universities
Authors: Rherrad Imad
Published: November 2008

The article studies the differences in knowledge production between academic researchers. In this perspective, it attempts at first to answer the following question: what factors explain differences in knowledge production between Canadian researchers in natural sciences and engineering? After a presentation of some of the empirical evidence related to this first question, a distinction between two types of academic institutions, entrepreneurial versus non‐entrepreneurial universities, is introduced. Drawing from this distinction, four empirical models are suggested to test differences in knowledge production between entrepreneurial and non‐entrepreneurial researchers. The results show, first, that funding, time devoted to teaching activities, research team and individual attributes have a similar but differentiated impact on knowledge production of entrepreneurial and non‐entrepreneurial researchers. Second, there are some unbalanced effects of the variables co‐operation, time devoted to research activities, academic fields and university size on the knowledge production of Canadian researchers on natural science and engineering.
Regional differences in fiber characteristics in the rat temporalis muscle
Authors: Tanaka E. , Sano R. , Kawai N. , Korfage J. A. M. , Nakamura S. , Izawa T. , Langenbach G. E. J. , Tanne K.
Published: November 2008

The behavioral differences in muscle use are related to the fiber type composition of the muscles among other variables. The aim of this study was to examine the degree of heterogeneity in the fiber type composition in the rat temporalis muscle. The temporalis muscle was taken from 10‐week‐old Wistar strain male rats (n = 5). Fiber types were classified by immunohistochemical staining according to their myosin heavy chain content. The anterior temporalis revealed an obvious regional difference of the fiber type distribution, whereas the posterior temporalis was homogeneous. The deep anterior temporalis showed a predominant proportion of type IIA fibers and was the only muscle portion displaying slow type fibers (< 10%). The other two muscle portions, the superficial anterior and posterior temporalis, did not differ significantly from each other and contained mainly type IIB fibers. Moreover, the deep anterior temporalis was the only muscle portion showing slow type fibers (< 10%). In the deep portion, type IIX fibers revealed the largest cross‐sectional area (1943.1 ± 613.7 µm2), which was significantly (P < 0.01) larger than those of type IIA and I + IIA fibers. The cross‐sectional area of type IIB fibers was the largest in the remaining two muscle portions and was significantly (P < 0.01) larger than that of type IIX fibers. In conclusion, temporalis muscle in rats showed an obvious heterogeneity of fiber type composition and fiber cross‐sectional area, which suggests multiple functions of this muscle.