bluuebunny/crossref_metadata_2025 huggingface dataset:
The role of the MAPK pathway alterations in GM-CSF modulated human neutrophil apoptosis with aging
Authors: Larbi Anis , Douziech Nadine , Fortin Carl , Linteau Annie , Dupuis Gilles , Fulop Tamas
Published: March 2005

Background

Neutrophils represent the first line of defence against aggressions. The programmed death of neutrophils is delayed by pro-inflammatory stimuli to ensure a proper resolution of the inflammation in time and place. The pro-inflammatory stimuli include granulocyte-macrophage colony-stimulating factor (GM-CSF). Recently, we have demonstrated that although neutrophils have an identical spontaneous apoptosis in elderly subjects compared to that in young subjects, the GM-CSF-induced delayed apoptosis is markedly diminished. The present study investigates whether an alteration of the GM-CSF stimulation of MAPKs play a role in the diminished rescue from apoptosis of PMN of elderly subjects.

Methods

Neutrophils were separated from healthy young and elderly donors satisfying the SENIEUR protocol. Neutrophils were stimulated with GM-CSF and inhibitors of the MAPKinase pathway. Apoptosis commitment, phosphorylation of signaling molecules, caspase-3 activities as well as expression of pro- and anti-apoptotic molecules were performed in this study. Data were analyzed using Student's two-tailedt-test for independent means. Significance was set for p ≤ 0.05 unless stated otherwise.

Results

In this paper we present evidence that an alteration in the p42/p44 MAPK activation occurs in PMN of elderly subjects under GM-CSF stimulation and this plays a role in the decreased delay of apoptosis of PMN in elderly. We also show that p38 MAPK does not play a role in GM-CSF delayed apoptosis in PMN of any age-groups, while it participates to the spontaneous apoptosis. Our results also show that the alteration of the p42/p44 MAPK activation contributes to the inability of GM-CSF to decrease the caspase-3 activation in PMN of elderly subjects. Moreover, GM-CSF converts the pro-apoptotic phenotype to an anti-apoptotic phenotype by modulating the bcl-2 family members Bax and Bcl-xL in PMN of young subjects, while this does not occur in PMN of elderly. However, this modulation seems MAPK independent.

Conclusion

Our results show that the alteration of p42/p44 MAPK activation contributes to the GM-CSF induced decreased PMN rescue from apoptosis in elderly subjects. The modulation of MAPK activation in PMN of elderly subjects might help to restore the functionality of PMN with aging.
Wearable kinesthetic system for capturing and classifying upper limb gesture in post-stroke rehabilitation
Authors: Tognetti Alessandro , Lorussi Federico , Bartalesi Raphael , Quaglini Silvana , Tesconi Mario , Zupone Giuseppe , De Rossi Danilo
Published: March 2005

Background

Monitoring body kinematics has fundamental relevance in several biological and technical disciplines. In particular the possibility to exactly know the posture may furnish a main aid in rehabilitation topics. In the present work an innovative and unobtrusive garment able to detect the posture and the movement of the upper limb has been introduced, with particular care to its application in post stroke rehabilitation field by describing the integration of the prototype in a healthcare service.

Methods

This paper deals with the design, the development and implementation of a sensing garment, from the characterization of innovative comfortable and diffuse sensors we used to the methodologies employed to gather information on the posture and movement which derive from the entire garments. Several new algorithms devoted to the signal acquisition, the treatment and posture and gesture reconstruction are introduced and tested.

Results

Data obtained by means of the sensing garment are analyzed and compared with the ones recorded using a traditional movement tracking system.

Conclusion

The main results treated in this work are summarized and remarked. The system was compared with a commercial movement tracking system (a set of electrogoniometers) and it performed the same accuracy in detecting upper limb postures and movements.
Novel G-protein-coupled receptor-like proteins in the plant pathogenic fungus Magnaporthe grisea
Authors: Kulkarni Resham D , Thon Michael R , Pan Huaqin , Dean Ralph A
Published: March 2005

Background

The G-protein-coupled receptors (GPCRs) are one of the largest protein families in human and other animal genomes, but no more than 10 GPCRs have been characterized in fungi. Do fungi contain only this handful or are there more receptors to be discovered? We asked this question using the recently sequenced genome of the fungal plant pathogen Magnaporthe grisea.

Results

Proteins with significant similarity to fungus-specific and other eukaryotic GPCRs were identified in M. grisea. These included homologs of known fungal GPCRs, the cAMP receptors from Dictyostelium, and a steroid receptor mPR. We also identified a novel class of receptors typified by PTH11, a cell-surface integral membrane protein required for pathogenicity. PTH11 has seven transmembrane regions and an amino-terminal extracellular cysteine-rich EGF-like domain (CFEM domain), a characteristic also seen in human GPCRs. Sixty-one PTH11-related proteins were identified in M. grisea that shared a common domain with homologs in Neurospora crassa and other fungi belonging to this subphylum of the Ascomycota (the Pezizomycotina). None was detected in other fungal groups (Basidiomycota or other Ascomycota subphyla, including yeasts) or any other eukaryote. The subclass of PTH11 containing the CFEM domain is highly represented in M. grisea.

Conclusion

In M. grisea we identified homologs of known GPCRs and a novel class of GPCR-like receptors specific to filamentous ascomycetes. A member of this new class, PTH11, is required for pathogenesis, thus suggesting roles in pathogenicity for other members. The identified classes constitute the largest number of GPCR-like proteins reported in fungi to date.
Making very similar embryos with divergent genomes: conservation of regulatory mechanisms ofOtxbetween the ascidiansHalocynthia roretziandCiona intestinalis
Authors: Oda-Ishii Izumi , Bertrand Vincent , Matsuo Isao , Lemaire Patrick , Saiga Hidetoshi
Published: March 2005

Ascidian embryos develop with a fixed cell lineage into simple tadpoles. Their lineage is almost perfectly conserved, even between the evolutionarily distant species Halocynthia roretzi and Ciona intestinalis,which show no detectable sequence conservation in the non-coding regions of studied orthologous genes. To address how a common developmental program can be maintained without detectable cis-regulatory sequence conservation, we compared in both species the regulation of Otx, a gene with a shared complex expression pattern. We found that in Halocynthia, the regulatory logic is based on the use of very simple cell line-specific regulatory modules, the activities of which are conserved,in most cases, in the Ciona embryo. The activity of each of these enhancer modules relies on the conservation of a few repeated crucial binding sites for transcriptional activators, without obvious constraints on their precise number, order or orientation, or on the surrounding sequences. We propose that a combination of simplicity and degeneracy allows the conservation of the regulatory logic, despite drastic sequence divergence. The regulation of Otx in the anterior endoderm by Lhx and Fox factors may even be conserved with vertebrates.
Clonal analysis of the relationships between mechanosensory cells and the neurons that innervate them in the chicken ear
Authors: Satoh Takunori , Fekete Donna M.
Published: March 2005

In vertebrates, hair-cell-bearing mechanosensory organs and the neurons that innervate them share a common placodal origin. In the inner ear, the peripheral neurons for both auditory and vestibular systems emigrate from the otic placode as neuroblasts, and divide, differentiate and innervate only one of six to eight distinct sensory organs. How these neurons find their correct target is unknown, although one suggestion is that they synapse with clonally related cells. To test this idea for both the middle and inner ears of chicken embryos, lineage analysis was initiated at the time of neuroblast delamination by labeling progenitors with replication-defective retroviruses. The vast majority (89%) of clones were restricted to a single anatomical subdivision of the sensory periphery or its associated ganglia, indicating limited clonal dispersion. Among the remaining clones, we found evidence of a shared neurosensory lineage in the middle ear. Likewise, in the inner ear, neurons could be related to cells of the otic epithelium, although the latter cells were not widely distributed. Rather, they were restricted to a region in or near the utricular macula. None of the other seven sensory organs was related to the ganglion neurons, suggesting that a common lineage between neurons and their targets is not a general mechanism of establishing synaptic connections in the inner ear. This conclusion is further strengthened by finding a shared lineage between the vestibular and acoustic ganglia, revealing the presence of a common progenitor for the two functional classes of neurons.
α-Synuclein Targets the Plasma Membrane via the Secretory Pathway and Induces Toxicity in Yeast
Authors: Dixon Cheryl , Mathias Neal , Zweig Richard M , Davis Donnie A , Gross David S
Published: March 2005

A pathological feature of Parkinson's disease is the presence of Lewy bodies within selectively vulnerable neurons. These are ubiquitinated cytoplasmic inclusions containing α-synuclein, an abundant protein normally associated with presynaptic terminals. Point mutations in the α-synuclein gene (A30P and A53T), as well as triplication of the wild-type (WT) locus, have been linked to autosomal dominant Parkinson's. How these alterations might contribute to disease progression is unclear. Using the genetically tractable yeast Saccharomyces cerevisiae as a model system, we find that both the WT and the A53T isoforms of α-synuclein initially localize to the plasma membrane, to which they are delivered via the classical secretory pathway. In contrast, the A30P mutant protein disperses within the cytoplasm and does not associate with the plasma membrane, and its intracellular distribution is unaffected by mutations in the secretory pathway. When their expression is elevated, WT and A53T, but not A30P, are toxic to cells. At moderate levels of expression, WT and A53T induce the cellular stress (heat-shock) response and are toxic to cells bearing mutations in the 20S proteasome. Our results reveal a link between plasma membrane targeting of α-synuclein and its toxicity in yeast and suggest a role for the quality control (QC) system in the cell's effort to deal with this natively unfolded protein.
Synthetic Lethality of Retinoblastoma Mutant Cells in the Drosophila Eye by Mutation of a Novel Peptidyl Prolyl Isomerase Gene
Authors: Edgar Kyle A , Belvin Marcia , Parks Annette L , Whittaker Kellie , Mahoney Matt B , Nicoll Monique , Park Christopher C , Winter Christopher G , Chen Feng , Lickteig Kim , Ahmad Ferhad , Esengil Hanife , Lorenzi Matthew V , Norton Amanda , Rupnow Brent A , Shayesteh Laleh , Tabios Mariano , Young Lynn M , Carroll Pamela M , Kopczynski Casey , Plowman Gregory D , Friedman Lori S , Francis-Lang Helen L
Published: March 2005

Mutations that inactivate the retinoblastoma (Rb) pathway are common in human tumors. Such mutations promote tumor growth by deregulating the G1 cell cycle checkpoint. However, uncontrolled cell cycle progression can also produce new liabilities for cell survival. To uncover such liabilities in Rb mutant cells, we performed a clonal screen in the Drosophila eye to identify second-site mutations that eliminate Rbf− cells, but allow Rbf+ cells to survive. Here we report the identification of a mutation in a novel highly conserved peptidyl prolyl isomerase (PPIase) that selectively eliminates Rbf− cells from the Drosophila eye.
A Genetic Screen in Drosophila for Identifying Novel Components of the Hedgehog Signaling Pathway
Authors: Collins Russell T , Cohen Stephen M
Published: March 2005

The Hedgehog signaling pathway plays an essential role in the pattern formation and development of metazoan animals. Misregulation of Hedgehog signaling has also been associated with the formation of multiple types of cancer. For these reasons, the Hedgehog pathway has attracted considerable interest. Many proteins required in the Hedgehog pathway have been identified, and while much has been learned about their function in signal transduction, it is clear that this complement of proteins does not comprise the full set necessary for Hedgehog signal transduction. Because significant gaps remain in our knowledge of the molecules required for Hedgehog signaling, we performed an enhancer/suppressor screen in Drosophila melanogaster to identify novel components of the pathway. In addition to the isolation of new alleles of the known pathway components patched and smoothened, this screen identified 14 novel complementation groups and a larger number of loci represented by single alleles. These groups include mutations in the genes encoding the translation factors eRF1 and eIF1A and the kinesin-like protein Pavarotti. It also identified mutations in a gene whose product is necessary for the movement of Hedgehog protein through tissues.
Maternal Transmission Ratio Distortion at the Mouse Om Locus Results From Meiotic Drive at the Second Meiotic Division
Authors: Wu Guangming , Hao Lanping , Han Zhiming , Gao Shaorong , Latham Keith E , de Villena Fernando Pardo-Manuel , Sapienza Carmen
Published: March 2005

We have observed maternal transmission ratio distortion (TRD) in favor of DDK alleles at the Ovum mutant (Om) locus on mouse chromosome 11 among the offspring of (C57BL/6 × DDK) F1 females and C57BL/6 males. Although significant lethality occurs in this backcross (∼50%), differences in the level of TRD found in recombinant vs. nonrecombinant chromosomes among offspring argue that TRD is due to nonrandom segregation of chromatids at the second meiotic division, i.e., true meiotic drive. We tested this hypothesis directly, by determining the centromere and Om genotypes of individual chromatids in zygote stage embryos. We found similar levels of TRD in favor of DDK alleles at Om in the female pronucleus and TRD in favor of C57BL/6 alleles at Om in the second polar body. In those embryos for which complete dyads have been reconstructed, TRD was present only in those inheriting heteromorphic dyads. These results demonstrate that meiotic drive occurs at MII and that preferential death of one genotypic class of embryo does not play a large role in the TRD.
Preliminary 1H NMR Study on Archaeological Waterlogged Wood
Authors: Maccotta Antonella , Fantazzini Paola , Garavaglia Carla , Donato Ines D. , Perzia Patrizia , Brai Maria , Morreale Filippa
Published: March 2005

Magnetic Resonance Relaxation (MRR) and Magnetic Resonance Imaging (MRI) are powerful tools to obtain detailed information on the pore space structure that one is unlikely to obtain in other ways. These techniques are particularly suitable for Cultural Heritage materials, because they use water 1H nuclei as a probe. Interaction with water is one of the main causes of deterioration of materials. Porous structure in wood, for example, favours the penetration of water, which can carry polluting substances and promote mould growth. A particular case is waterlogged wood from underwater discoveries and moist sites; in fact, these finds are very fragile because of chemical, physical and biological decay from the long contact with the water. When wood artefacts are brought to the surface and directly dried in air, there is the collapse of the cellular structures, and wood loses its original form and dimensions and cannot be used for study and museum exhibits. In this work we have undertaken the study of some wood finds coming from Ercolano's harbour by MRR and MRI under different conditions, and we have obtained a characterization of pore space in wood and images of the spatial distribution of the confined water in the wood.
Further Applications of a New Biosensor Method for Dating Cellulosic Finds
Authors: Campanella L. , Chicco F. , Favero G. , Gatta T. , Tomassetti M.
Published: March 2005

Using a cellulosic material dating method recently proposed by three of the authors of the present article further applications to real samples are discussed. In the first instance, to wood samples, that is, to a type of sample for which the method was specifically developed but with the samples differing widely in age, and then to textile or paper samples. Of course in the latter two cases the results obtained are still quite preliminary, above all because of the difficulty of procuring certainly dated samples of this type.
Environmental Accounting for the Lagoon of Venice and the Case of Fishing
Authors: Bastianoni Simone , Marchettini Nadia , Niccolucci Valentina , Pulselli Federico M.
Published: March 2005

This paper presents the application of the eMergy evaluation, introduced by H.T. Odum as an efficient methodology of environmental accounting. The results of this analysis are a part of a wider sustainability assessment for the Province of Venice realized in 2003. Within a territorial sustainability analysis, it is necessary to implement an environmental accounting method, which is able: to precisely consider the boundaries of the investigated system (spatial scale); to arbitrarily separate the life of the investigated system into periods (temporal scale); to account for both economic and natural support to a given territorial system in order to implement a sustainable management of resources; to assess all the items supporting the system on the basis of a physical unit that is objective (while the traditional economic evaluation method is based on individual preferences, hence it is subjective). The Province of Venice is a very complex system due, on one hand, to the heavy and consolidated presence of industrial activity since the beginning of the 20th century and, on the other hand, to the principal natural ecosystem which characterizes it: the lagoon, the largest wetland in the Mediterranean Sea. The role of the lagoon, as a provider of resources and services is represented by the analysis of two aspects: the lagoon as an ecosystem on the basis of the evaluation of all flows that cyclically support its biotic activity and the lagoon as a productive system on the basis of the contribution of both natural and “economic” inputs that support human activity. In particular, the case of clam fishing will be presented to put on evidence the importance of natural inputs that are systematically neglected by the traditional accounting methodologies.
Penetration of Solar Radiation into the Waters of Messina Strait (Italy)
Authors: Dattilo Arduino Massimo , Decembrini Franco , Bracchini Luca , Focardi Silvia , Mazzuoli Stefania , Rossi Claudio
Published: March 2005

The optical properties of the waters of five different stations, three located in the Messina Strait and two near the Strait (open sea), were analysed. Direct spectral measurements of the downward solar irradiance (290 — 800 nm) at different depths (0.5 m, 7 m, 10 m, 13 m, 20 m) were made using a cosine sensor connected to a spectroradiometer. Water samples were collected in the surface layer and their absorption spectra were analysed. The natural fluorescence profiles, along the water column, were determined using a fluorometer (SBE 911plus — Sea Teach). The spectral attenuation coefficient (Kλ), the variation of Kλ in different wavelength ranges (ΔKΔλ), the wavelength corresponding to minimum value of Kλ, the spectral depths of penetration of both 1 % and 10 % of the sub‐surface irradiance values (Pλ), the depths of 1 % of penetration of UVB, UVA and PAR, the depth ranges of the maxim concentration of Chl a and superficial CDOM were measured at each station. The maximum solar UVB penetration was about 65 % of the photic zone and the maximum UVA penetration was nearly 100 % (data of the Ionic sea station). Thus, a large part of the photic zone was exposed to UV radiation sufficient to cause a possible reduction in the photosynthetic activity of phytoplankton. The spectral penetration of solar radiation, especially UVB radiation, was significantly different in the three stations of the Strait with respect to the two stations studied in the open sea. This shows that variations in the spectral attenuation along the water column can be used as an indicator of properties of the water body.
Optimisation of a Photochemical Sensor for Total Organic Carbon Measurement
Authors: Visco Giovanni , Bellanti Francesco , Campanella Luigi , Mazzella Tommaso , Nobili Valentina
Published: March 2005

A new tool for the measurement of TOC based on the heterogeneous photocatalysis activated by two different light sources has been planned. The instrument is constituted by a cell where the degradation occurs thanks to both the UV irradiation and the presence of a powered catalyst and by a measurement cell lodging CO2 gas permeable membrane electrode (detector), thermometer, inlet and outlet ways for the samples. One of the sources has a maximum emission at 254nm, while the other simulates the solar spectrum. A suspension (1 g/L) of titanium dioxide (Degussa P25) has been used to catalyse the process. A magnetic stirrer and a circulating pump avoid the deposition of the catalyst on the bottom of the reactor. The online measurement of produced CO2 was performed on 11 molecules (commercial names: Parachlorophenol, D‐Glucose, Acid Blue 29, Atraton, Aspirin, Dimethoate, Aldicarb sulfone, Hydroquinone, (R)‐Propranolol, Citric acid, Atrazine) to test the system efficiency. The experimental CO2 value was compared with the theoretical TOC value calculated by means of the structure's formula.
Optimization of Supercritical Fluid Extraction by Carbon Dioxide with Organic Modifiers of Polycyclic Aromatic Hydrocarbons from Urban Particulate Matter
Authors: Librando Vito , Tomaselli Gaetano , Tringali Giuseppe
Published: March 2005

The main advantages of using supercritical fluids for the extractions of organic pollutants from environmental matrix is that they are inexpensive, contaminant free, and less costly to dispose safely than organic solvents. In this work, a series of extraction experiments were carried out using CO2 as supercritical fluid on a certified sample of “Urban dust” (NIST S.R.M. 1649a) to optimize the analytical parameters with the aim of investigating the extraction limit of organic pollutant by using an almost “organic solvent‐free” technique. The certified sample contains small concentrations of several organic pollutants, as PAH and PCB. The initial tests of extraction were carried out with only CO2 in supercritical phase, by maintaining the temperature at 50 °C and 80 °C and by making the pressure vary between 230 bar and 600 bar. The effect of three organic modifiers (methanol, nhexane and toluene), added at 5 % in volume, has been considered. The yield of recovery has been estimated for anthracene, fluoranthene, chrysene, benzo[a]pyrene, indeno[1, 2, 3‐cd]pyrene by GC‐MS according to the increasing molecular weight.
The Raman Spectrum of Hematite: Possible Indicator for a Compositional or Firing Distinction among Terra Sigillata Wares
Authors: Zoppi Angela , Lofrumento Cristiana , Castellucci Emilio Mario , Migliorini Maria Grazia
Published: March 2005

The Raman spectrum of hematite (α‐Fe2O3) is here discussed as a distinctive feature for Terra Sigillata sample wares. The broadening of the Raman lines and the appearance and increasing of the disorder band at 660 cm‐1, while moving from the external slip to the inner body, are proposed as valuation parameters for the study of the mineralogical composition and firing process of pottery productions.
Heavy Metal Contamination in Compost. A Possible Solution
Authors: Zennaro Mariachiara , Cristofori Fabrizio , Formigoni Daniele , Frignani Franco , Pavoni Bruno
Published: March 2005

With the objective of improving qualitative characteristics of compost, an analytical survey was carried out in a composting plant in Lombardy (Italy) in all process of production, with particular reference to heavy metals (HM) Zn and Pb. The investigation was principally aimed to study the contents and the accumulation of HM during composting process and to identify a technological solution for reducing HM content in the final product. A merceological analysis of Municipal Solid Waste (MSW) input to the composting plant, a chemical analysis of the organic fraction of MSW after mechanical separation, and a comparison with values reported by some authors, showed that Zn and Pb are significant contaminants, even though concentrations have recently decreased in comparison to previous years. On the basis of Zn and Pb content in raw material input to the plant, an estimate of the theoretical value of Zn and Pb in produced compost was made. The comparison of theoretical values with the real ones, experimentally determined, confirmed that at the end of composting process the concentration is 2.6 times the initial value for Zn and 1.6 times the initial value for Pb, as suggested by some authors. Finally, the analytical investigation of Zn and Pb contents in the compost refining line, carried out by means of sieving tests, showed that by eliminating a fraction of compost < 1 mm, both Zn and Pb, which is the more critical one, can be largely removed, without a substantial yield loss (only 10 % of the final product is eliminated).
Display of lipase on the cell surface of Escherichia coli using OprF as an anchor and its application to enantioselective resolution in organic solvent
Authors: Lee Seung Hwan , Choi Jong‐il , Han Mee‐Jung , Choi Jong Hyun , Lee Sang Yup
Published: March 2005

We have developed a new cell surface display system using a major outer membrane protein of Pseudomonas aeruginosa OprF as an anchoring motif. Pseudomonas fluorescens SIK W1 lipase gene was fused to the truncated oprF gene by C‐terminal deletion fusion strategy. The truncated OprF‐lipase fusion protein was successfully displayed on the surface of Escherichia coli. Localization of the truncated OprF‐lipase fusion protein was confirmed by western blot analysis, immunofluorescence microscopy, and whole‐cell lipase activity. To examine the enzymatic characteristics of the cell surface displayed lipase, the whole‐cell enzyme activity and stability were determined under various conditions. Cell surface displayed lipase showed the highest activity at 37°C and pH 8.0. It retained over 80% of initial activity after incubation for a week in both aqueous solution and organic solvent. When the E. coli cells displaying lipases were used for enantioselective resolution of racemic 1‐phenylethanol in hexane, (R)‐phenyl ethyl acetate was successfully obtained with the enantiomeric excess of greater than 96% in 36 h of reaction. These results suggest that E. coli cells displaying lipases using OprF as an anchoring motif can be employed for various biotechnological applications both in aqueous and nonaqueous phases. © 2005 Wiley Periodicals, Inc.
Large‐scale production of pharmaceuticals by marine sponges: Sea, cell, or synthesis?
Authors: Sipkema Detmer , Osinga Ronald , Schatton Wolfgang , Mendola Dominick , Tramper Johannes , Wijffels René H.
Published: March 2005

Marine sponges are known to produce an overwhelming array of secondary metabolites with pharmaceutical potential. The technical and economical potential of using marine sponges for large‐scale production of these compounds was assessed for two cases: the anticancer molecule halichondrin B from a Lissodendoryx sp., and avarol from Dysidea avara for its antipsoriasis activity. An economic and technical analysis was done for three potential production methods: mariculture, ex situ culture (in tanks), and cell culture. We concluded that avarol produced by mariculture or ex situ culture could become a viable alternative to currently used pharmaceuticals for the treatment of psoriasis. Production of halichondrin B from sponge biomass was found to not be a feasible process, mainly due to the extremely low concentration of the compound in the sponge. Technical feasibility was also analyzed for five alternatives: chemical synthesis, wild harvest, primmorph culture, genetic modification and semi‐synthesis. It was concluded that the latter two approaches could prove to be valuable methods for the production of pharmaceuticals, based on chemical structures of secondary metabolites present in trace amounts in marine sponges. © 2005 Wiley Periodicals, Inc.
Solubility and proteolysis of the Zb‐MalE and Zb‐MalE31 proteins during overproduction in Escherichia coli
Authors: Sandén Anna Maria , Boström Maria , Markland Katrin , Larsson Gen
Published: March 2005

From the hypothesis that the rate of expression of a nascent polypeptide controls the accumulation of soluble full‐length protein, accumulation of the model fusion proteins Zb‐MalE and Zb‐MalE31, were studied. MalE and MalE31 are two isoforms of the maltose binding protein, differing only in two consecutive amino acids. Parameters controlling the expression rate were the transcription rate, which was controlled by IPTG induction of the lacUV5 promoter and the substrate addition levels during fed‐batch cultivation.

Results show that the two product proteins appear in both soluble and insoluble fractions during cultivation and are both subjected to proteolysis. However, the accumulation of the soluble form of Zb‐MalE31 protein is radically lower, at all conditions, due to the small difference in primary structure.

It was shown that both proteolysis and inclusion body formation could be influenced by the selected parameters although a change in feed rate had a considerably higher effect. A high concentration of inducer and a “high” feed rate result in a low accumulation of soluble product, due to a high proteolysis. The concentration of inducer leading to different levels of transcription is, however, an efficient tool to influence inclusion body formation. At low IPTG concentrations (≤5 μM), this formation is almost abolished while at a comparatively high concentration (≥300 μM) 50% of the total product accumulated was in the form of inclusion bodies. © 2005 Wiley Periodicals, Inc.
How can domestic households become part of the solution to England's recycling problems?
Authors: Smallbone Teresa
Published: March 2005

A waste disposal problem of looming proportions, coupled with a lack of sufficient public engagement in the preferred alternative to disposal, which is recycling, continues to perplex English policy‐makers. Based on both a literature review across a wide range of disciplines and a national survey of consumer attitudes towards their own participation in recycling, this paper finds that past efforts at increasing recycling have been based on an implied model of consumer recycling behaviour that is not supported by what happens in practice. By disentangling thinking about recycling behaviour from academic thinking about green consumerism, the paper considers the waste and recycling problem from a different angle. It suggests that research on the personal values of people who recycle could be utilized in marketing communications that show these values being fulfilled by recycling. Focusing greater marketing attention on people who already claim to recycle, and helping them through better communication and improved practical help, could achieve much higher levels of reclaimed materials. Copyright © 2005 John Wiley & Sons, Ltd and ERP Environment.
The carboxyl terminus of the chemokine receptor CCR3 contains distinct domains which regulate chemotactic signaling and receptor down‐regulation in a ligand‐dependent manner
Authors: Sabroe Ian , Jorritsma Annelies , Stubbs Victoria E. L. , Xanthou Georgina , Jopling Louise A. , Ponath Paul D. , Williams Timothy J. , Murphy Philip M. , Pease James E.
Published: March 2005

The chemokine receptor CCR3 regulates the chemotaxis of leukocytes implicated in allergic disease, such as eosinophils. Incubation of eosinophils with CCL11, CCL13 or CCL5 resulted in a rapid decrease of cell‐surface CCR3 which was replicated using CCR3 transfectants. Progressive truncation of the CCR3 C terminus by 15 amino acids produced three constructs, Δ340, Δ325 and Δ310. Δ340 and Δ325 were able to bind CCL11 with affinities similar to wild‐type CCR3. Δ340 transfectants exhibited enhanced migration and reduced receptor down‐regulation in response to CCL11 and CCL13. Δ325 transfectants displayed chemotactic responses to CCL11 and CCL13 similar to wild‐type CCR3, and had impaired down‐regulation when stimulated with CCL13 but not CCL11. In contrast, neither the Δ325 nor Δ340 truncation affected chemotaxis or receptor down‐regulation induced by CCL5. Δ310 transfectants bound CCL11 poorly and were biologically inactive. Inhibitors of p38 mitogen‐activated protein kinase and PI3‐kinase antagonized eosinophil shape change responses and chemotaxis of transfectants to CCL11 and CCL13. In contrast, shape change but not chemotaxis was sensitive to inhibition of the extracellular signal‐regulated kinase kinase pathway suggesting differential regulation of the two responses. Thus, the CCR3 C terminus contains distinct domains responsible for the regulation of receptor desensitization and for coupling to chemotactic responses.
Protection against tetanus toxin using a plant‐based vaccine
Authors: Tregoning John S. , Clare Simon , Bowe Frances , Edwards Lorna , Fairweather Neil , Qazi Omar , Nixon Peter J. , Maliga Pal , Dougan Gordon , Hussell Tracy
Published: March 2005

Plant‐expressed vaccines may provide a unique opportunity for generating anti‐pathogen immunity, especially in countries where cold storage is lacking. In the following study, we show that soluble protein from tobacco leaves expressing fragment C of tetanus toxin protected mice against a lethal tetanus toxin challenge. More importantly, we show that a single intranasal (i.n.) vaccination was as efficient as oral delivery, inducing high levels of activated CD4+ T cells and anti‐toxin antibody. Unlike the oral route, i.n. delivery did not require the presence of adjuvant (cholera toxin). Indeed, addition of cholera toxin induced bystander immune responses to plant proteins as well. This is the first study documenting protective immunity by a single i.n. dose of plant vaccine. Plant‐based vaccines are promising because they are more heat stable, are easy to produce, cheap and do not require needles.
Scant activation of CD8 T cells by antigen loaded on heat shock protein
Authors: Winau Florian , Sponaas Anne‐Marit , Weber Stephan , Schwierzeck Vera , Winter Ralf , Hurwitz Robert , Kaufmann Stefan H. E.
Published: March 2005

Heat shock proteins (HSP) not only function as chaperones for denatured proteins but also for antigenic peptides, thus inducing protective T cell responses. Here we show that vaccination with peptide‐loaded HSP70 causes initial interferon‐γ production by murine CD8 T cells but no T cell expansion. These CD8 T cells lacked cytotoxic activity in vitro and in vivo, which was not due to apoptosis. Restimulation with peptide‐pulsed dendritic cells both bypassed the proliferative block and suspended the non‐protective state of CD8 T lymphocytes in an infection model with the bacterial pathogen, Listeria monocytogenes. Cotransfer of antigen‐specific CD4 T cells circumvented the proliferative arrest of CD8 T cells. Our data suggest that HSP vaccines induce CD8 T cell unresponsiveness unless proficient help is provided. Assuming that this model reflects the antigenically experienced human condition where immunological space is restricted and any T cell response possibly leads to suppression of heterologous reactions, our findings bear implications for rational vaccination protocols including those for immunocompromised patients.
Airway inflammation: chemokine‐induced neutrophilia and the class I phosphoinositide 3‐kinases
Authors: Thomas Matthew J. , Smith Alexandra , Head Denise H. , Milne Laura , Nicholls Andrew , Pearce Wayne , Vanhaesebroeck Bart , Wymann Matthias P. , Hirsch Emilio , Trifilieff Alexandre , Walker Christoph , Finan Peter , Westwick John
Published: March 2005

Class I phosphoinositide 3‐kinases (PI3K) are known to play a significant role in neutrophil chemotaxis. However, the relative contributions of different PI3K isoforms, and how these impact on lung inflammation, have not been addressed. In vitro studies using wild‐type and PI3Kγ knockout neutrophils demonstrated the major role of the γ isoform in chemotactic but not chemokinetic events. This was confirmed by a model of direct chemokine instillation into the airways in vivo. Within all studies, a low yet significant degree of neutrophil movement in the absence of PI3Kγ could be observed. No role for the δ isoform was demonstrated both in vitro and in vivo using PI3Kδ kinase‐dead knock‐in mice. Moreover, further studies using the broad‐spectrum PI3K inhibitors wortmannin or LY294002 showed no other class I PI3K isoforms to be involved in these chemotactic processes. Here, we identify a contributory PI3K‐independent mechanism of neutrophil movement, yet demonstrate PI3Kγ as the pivotal mediator through which the majority of neutrophils migrate into the lung in response to chemokines. These data resolve the complexities of chemokine‐induced neutrophilia and PI3K signaling and define the γ isoform as a promising target for new therapeutics to treat airway inflammatory diseases.
Integrin activation by bacterial fimbriae through a pathway involving CD14, Toll‐like receptor 2, and phosphatidylinositol‐3‐kinase
Authors: Harokopakis Evlambia , Hajishengallis George
Published: March 2005

CD11b–CD18 and other integrins play important roles in immunity and inflammation and require prior activation through inside‐out signaling to efficiently bind their ligands. We present evidence for a novel TLR2‐dependent signaling pathway that leads to CD11b–CD18 activation in human monocytes or neutrophils upon recognition of Porphyromonas gingivalis fimbriae through CD14. The activated binding‐state of CD11b–CD18, which involves induction of conformational changes, was monitored through detection of an activation‐specific epitope of CD11b. The ability of fimbriae to induce this activation epitope was significantly inhibited by a mAb to TLR2, but not to TLR4 or unrelated surface molecules. Moreover, the ability of fimbriae to activate CD11b–CD18 was significantly inhibited by pharmacological inhibitors of phosphatidylinositol‐3‐kinase but not of PKC or of p38 mitogen‐activated protein kinase. The signaling pathway activated by fimbriae is distinct from that which is activated by N‐formyl‐Met–Leu–Phe, a prototypical integrin activator, since the former was insensitive to pertussis toxin. This novel function of TLR2 as a signaling receptor for pathogen‐induced activation of CD11b–CD18 may play a significant role in infection‐driven chronic inflammatory conditions, such as periodontal disease or atherosclerosis, where P. gingivalis has been implicated.
The granzyme B inhibitor proteinase inhibitor 9 (PI9) is expressed by human mast cells
Authors: Bladergroen Bellinda A. , Strik Merel C. M. , Wolbink Angela M. , Wouters Dorine , Broekhuizen Roel , Kummer J. Alain , Hack C. Erik
Published: March 2005

The activity of granzyme B, a main effector molecule of cytotoxic T lymphocytes (CTL) and natural killer cells, is regulated by the human intracellular serpin proteinase inhibitor 9 (PI9). This inhibitor is particularly expressed by CTL and dendritic cells, in which it serves to protect these cells against endogenous and locally released granzyme B. Moreover, PI9 expression by neoplastic cells may constitute one of the mechanisms for tumors to escape immune surveillance. Here we show that PI9 is also expressed by human mast cells. In immunohistochemical studies using a PI9‐specific monoclonal antibody, strong cytoplasmic staining for PI9 was found in normal mast cells in various tissues throughout the body. In addition, in 80% of all cases of cutaneous and systemic mastocytosis tested the majority of the mast cells expressed PI9. As an in vitro model for PI9 expression by mast cells, we studied expression by the human mast cell line HMC‐1. Stimulation of HMC‐1 with PMA and the calcium ionophore A23187 resulted in a marked increase of PI9 expression. Thus, PI9 is expressed by activated mast cells. We suggest that this expression serves to protect these cells against apoptosis induced by granzyme B released during initiation of the local inflammatory response.
A new approach for sequencing human IRS1 phosphotyrosine‐containing peptides using CapLC‐Q‐TOFmicro
Authors: Lu Xiao‐Ming , Lu Mary Y. , Fischman Alan J. , Tompkins Ronald G.
Published: March 2005

Reversible phosphorylation of proteins functions as a biological switching network for activation and inhibition of downstream biological processes. Since phosphorylations of these sites are often transient processes, and hence sub‐stoichiometric, systematic characterization of phosphorylation sites is a formidable challenge. In this work, a new approach was developed to pinpoint phosphotyrosine sites on tyrosine‐containing peptides. This required (1) the development of a new and highly sensitive nano‐electrospray assembly and (2) validation of the concept that the specificity and detection limit for trace levels of phosphotyrosine immonium ion in peptide mixtures from protein digests can be increased by varying the collision energy. With our method, an automatic tandem mass spectrometric analysis of peptides eluted from a C18 capillary liquid chromatographic column is triggered by a positive confirmation of phosphotyrosine immonium ion in a time‐of‐flight mass spectrometric survey. The approach was tested by analyzing the phosphorylation of human IRS‐1 peptides that interact with the Src‐homology 2 domain and mixtures of these peptides with tryptic digests of bovine serum albumin and horse heart myoglobin. Copyright © 2005 John Wiley & Sons, Ltd.
Analysis of FeII‐mediated decomposition of a linoleic acid‐derived lipid hydroperoxide by liquid chromatography/mass spectrometry
Authors: Lee Seon Hwa , Oe Tomoyuki , Arora Jasbir S. , Blair Ian A.
Published: March 2005

Intracellular FeII, which is up‐regulated during oxidative stress and during iron overload, induces the formation of a hydroxyl radical by Fenton chemistry. The hydroxyl radical can convert the prototypic ω‐6 polyunsaturated fatty acid, linoleic acid, to 13‐hydroperoxy‐9,11‐(Z,E)‐octadecadienoic acid (13‐HPODE). Cyclooxygenases can also convert linoleic acid to 13(S)‐HPODE during oxidative stress. Subsequent FeII‐mediated decomposition to protein‐ and DNA‐reactive bifunctional electrophiles was examined by normal‐phase liquid chromatography (LC)/atmospheric pressure chemical ionization (APCI)/mass spectrometry. The potential individual bifunctional electrophiles trans‐4,5‐epoxy‐2(E)‐decenal (EDE), cis‐EDE, 4‐oxo‐2(E)‐nonenal (ONE) and 4‐hydroxy‐2(E)‐nonenal (HNE) exhibited protonated molecular ions at m/z 169, 169, 155 and 157, respectively. The MH+ ion at m/z 173 for 4‐hydroperoxy‐2(E)‐nonenal (HPNE) was very weak with an ion corresponding to the loss of OH at m/z 156 as the major ion in the APCI mass spectrum. The bifunctional electrophiles were all separated under normal‐phase LC conditions. Interestingly, ions corresponding to ONE and HNE were detected at the same retention time as HPNE, suggesting that it decomposed in the source of the mass spectrometer to ONE and HNE. All five bifunctional electrophiles were formed when 13‐HPODE was treated with 50 µM FeII. At this concentration of FeII, the addition of vitamin C resulted in increased bifunctional electrophile formation. At higher concentrations of FeII (500 µM to 2 mM), no HPNE was detected and there was no additive effect of vitamin C. Additional experiments with synthetic HPNE revealed that it was quantitatively converted to a mixture of ONE and HNE by FeII. The HNE is thought to arise from a one‐electron reduction of an alkoxy radical derived from HPNE. In contrast, ONE can arise through an α‐cleavage of the HPNE‐derived alkoxy radical or by direct dehydration of HPNE. Copyright © 2005 John Wiley & Sons, Ltd.
A new high‐performance liquid chromatographic/electrospray ionization tandem mass spectrometric method for the simultaneous determination of cyclophosphamide, methotrexate and 5‐fluorouracil as markers of surface contamination for occupational exposure monitoring
Authors: Sabatini Laura , Barbieri Anna , Tosi Marco , Violante Francesco Saverio
Published: March 2005

A new high‐performance liquid chromatographic/electrospray ionization tandem mass spectrometric (HPLC/ESI‐MS/MS) method was developed for the simultaneous quantification of 5‐fluorouracil (5FU), methotrexate (MTX) and cyclophosphamide (CP) in environmental samples. These compounds, commonly used in the treatment of cancer, are recognized as genotoxic. In order to estimate the occupational exposure of hospital personnel handling these drugs, wipe samples were taken from the working surfaces and directly analyzed (with trophosphamide as internal standard) using a reversed‐phase capillary column and MS/MS detection. This is the first HPLC/MS/MS method for the simultaneous determination of 5FU, MTX and CP. The present method offers high sensitivity, with detection limits of 1.1 µg l−1 for MTX and CP and 33.3 µg l−1 for 5FU, avoiding any sample preconcentration procedure. Rapidity, specificity, high accuracy (mean values between 92.4 and 99.9%) and precision (mean RSD values between 3.4 and 12.1%) make the method suitable for the routine determination of these three antineoplastic drugs. Copyright © 2005 John Wiley & Sons, Ltd.
Direct exposure electron ionization mass spectrometry and gas chromatography/mass spectrometry techniques to study organic coatings on archaeological amphorae
Authors: Colombini Maria Perla , Modugno Francesca , Ribechini Erika
Published: March 2005

Two different analytical approaches, direct exposure electron ionization mass spectrometry (DE‐MS) and gas chromatography/mass spectrometry (GC/MS), were compared in a study of archaeological resinous materials. DE‐MS was found to be an efficient fingerprinting tool for the fast screening of organic archaeological samples and for providing information on the major components. GC/MS appeared to be more efficient in unravelling the sample composition at a molecular level, despite the long analysis time and the need for a wet chemical pretreatment. Both procedures were applied to characterize the organic material present as coatings in Roman and Egyptian amphorae. DE‐MS successfully identified abietanic compounds, hence a diterpenic resinous material could be identified and its degree of oxidation assessed. GC/MS enabled us to identify dehydroabietic acid, 7‐oxodehydroabietic acid, 15‐hydroxy‐7‐oxodehydroabietic acid, 15‐hydroxydehydroabietic acid, retene, tetrahydroretene, norabietatriene, norabietatetraene and methyl dehydroabietate. These oxidized and aromatized abietanes provided evidence that the amphorae examined were waterproofed with a pitch produced from resinous wood of plants from thePinaceaefamily. The chemometric evaluation of the GC/MS data highlighted significant chemical differences between the pitches found in the two archaeological sites, basically related to differences in the production techniques of the materials and in their degradation pathways. Copyright © 2005 John Wiley & Sons, Ltd.