Functional and immunological characterization of a natural polymorphic variant of a heat-labile toxin (LT-I) produced by enterotoxigenic Escherichia coli (ETEC)

Heat-labile toxins (LT) encompass at least 16 natural polymorphic toxin variants expressed by wild-type enterotoxigenic Escherichia coli (ETEC) strains isolated from human beings, but only one specific form, produced by the reference ETEC H10407 strain (LT1), has been intensively studied either as a virulence-associated factor or as a mucosal/transcutaneous adjuvant. In the present study, we carried out a biological/immunological characterization of a natural LT variant (LT2) with four polymorphic sites at the A subunit (S190L, G196D, K213E, and S224T) and one at the B subunit (T75A). The results indicated that purified LT2, in comparison with LT1, displayed similar in vitro toxic activities (adenosine 3`,5`-cyclic monophosphate accumulation) on mammalian cells and in vivo immunogenicity following delivery via the oral route. Nonetheless, the LT2 variant showed increased adjuvant action to ovalbumin when delivered to mice via the transcutaneous route while antibodies raised in mice immunized with LT2 displayed enhanced affinity and neutralization activity to LT1 and LT2. Taken together, the results indicate that the two most frequent LT polymorphic forms expressed by wild ETEC strains share similar biological features, but differ with regard to their immunological properties.

The three-dimensional structure of bothropasin, the main hemorrhagic factor from Bothrops jararaca venom: Insights for a new classification of snake venom metalloprotease subgroups

Bothropasin is a 48 kDa hemorrhagic PIII snake venom metalloprotease (SVMP) isolated from Bothrops jararaca, containing disintegrin/cysteine-rich adhesive domains. Here we present the crystal structure of bothropasin complexed with the inhibitor POL647. The catalytic domain consists of a scaffold of two subdomains organized similarly to those described for other SVMPs, including the zinc and calcium-binding sites. The free cysteine residue Cys(189) is located within a hydrophobic core and it is not available for disulfide bonding or other interactions. There is no identifiable secondary structure for the disintegrin domain, but instead it is composed mostly of loops stabilized by seven disulfide bonds and by two calcium ions. The ECD region is in a loop and is structurally related to the RGD region of RGD disintegrins, which are derived from I`ll SVMPs. The ECD motif is stabilized by the Cys(117)_Cys(310) disulfide bond (between the disintegrin and cysteine-rich domains) and by one calcium ion. The side chain of Glu(276) of the ECD motif is exposed to solvent and free to make interactions. In bothropasin, the HVR (hyper-variable region) described for other Pill SVMPs in the cysteine-rich domain, presents a well-conserved sequence with respect to several other Pill members from different species. We propose that this subset be referred to as PIII-HCR (highly conserved region) SVMPs. The differences in the disintegrin-like, cysteine-rich or disintegrin-like cysteine-rich domains may be involved in selecting target binding, which in turn could generate substrate diversity or specificity for the catalytic domain. (C) 2008 Elsevier Ltd. All rights reserved.

Functional Analysis of saxophone, the Drosophila Gene Encoding the BMP Type I Receptor Ortholog of Human ALK1/ACVRL1 and ACVR1/ALK2

In metazoans, bone morphogenetic proteins (BMPS) direct a myriad of developmental and adult homeostatic evens through their heterotetrameric type I and type II receptor complexes. We examined 3 existing and 12 newly generated mutations in the Drosophila type I receptor gene, saxophone (sax), the ortholog of the human Activin Receptor-Like. Kinasel and -2 (ALK1/ACVR1 and ALK2/ACVR1) genes. Our genetic analyses identified two distinct classes of sax alleles. The first class consists of homozygous viable gain-of-function (GOF) alleles that exhibit (1) synthetic lethality in combination with mutations in BMP pathway components, and (2) significant maternal effect lethality that can be rescued by an increased dosage of the BMP encoding gene, dpp(+). In contrast, the second class consists of alleles that are recessive lethal and do not exhibit lethality in combination with mutations in other BMP pathway components. The alleles in this second class are clearly loss-of-function (LOF) with both complete and partial loss-of-function mutations represented. We find that one allele in the second class of recessive lethals exhibits dominant-negative behavior, albeit distinct from the GOF activity of the first class of viable alleles. On the basis of the fact that the first class of viable alleles can be reverted to lethality and on our ability to independently generate recessive lethal sat mutations, our analysis demonstrates that sax is an essential gene. Consistent with this conclusion, we find that a normal sax transcript is produced by sax(P), a viable allele previously reported to be mill, and that this allele can be reverted to lethality. Interestingly, we determine that two mutations in the first: class of sax alleles show the same amino acid substitutions as mutations in the human receptors ALK1/ACVR1-1 and ACVR1/ALK2, responsible for cases of hereditary hemorrhagic telangiectasia type 2 (HHT2) and fibrodysplasia ossificans progressiva (FOP), respectively. Finally, the data presented here identify different functional requirements for the Sax receptor, support the proposal that Sax participates in a heteromeric receptor complex, and provide a mechanistic framework for future investigations into disease states that arise from defects in BMP/TGF-beta signaling.

Role of sigma(54) in the regulation of genes involved in type I and type IV pili biogenesis in Xylella fastidiosa

The phytopathogen Xylella fastidiosa produces long type IV pili and short type I pili involved in motility and adhesion. In this work, we have investigated the role of sigma factor sigma(54) (RpoN) in the regulation of fimbrial biogenesis in X. fastidiosa. An rpoN null mutant was constructed from the non-pathogenic citrus strain J1a12, and microarray analyses of global gene expression comparing the wild type and rpoN mutant strains showed few genes exhibiting differential expression. In particular, gene pilA1 (XF2542), which encodes the structural pilin protein of type IV pili, showed decreased expression in the rpoN mutant, whereas two-fold higher expression of an operon encoding proteins of type I pili was detected, as confirmed by quantitative RT-PCR (qRT-PCR) analysis. The transcriptional start site of pilA1 was determined by primer extension, downstream of a sigma(54)-dependent promoter. Microarray and qRT-PCR data demonstrated that expression of only one of the five pilA paralogues, pilA1, was significantly reduced in the rpoN mutant. The rpoN mutant made more biofilm than the wild type strain and presented a cell-cell aggregative phenotype. These results indicate that sigma(54) differentially regulates genes involved in type IV and type I fimbrial biogenesis, and is involved in biofilm formation in X. fastidiosa.

Ixolaris, a tissue factor inhibitor, blocks primary tumor growth and angiogenesis in a glioblastoma model

Background: The expression levels of the clotting initiator protein Tissue Factor (TF) correlate with vessel density and the histological malignancy grade of glioma patients. Increased procoagulant tonus in high grade tumors (glioblastomas) also indicates a potential role for TF in progression of this disease, and suggests that anticoagulants could be used as adjuvants for its treatment. Objectives: We hypothesized that blocking of TF activity with the tick anticoagulant Ixolaris might interfere with glioblastoma progression. Methods and results: TF was identified in U87-MG cells by flow-cytometric and functional assays (extrinsic tenase). In addition, flow-cytometric analysis demonstrated the exposure of phosphatidylserine in the surface of U87-MG cells, which supported the assembly of intrinsic tenase (FIXa/FVIIIa/FX) and prothrombinase (FVa/FXa/prothrombin) complexes, accounting for the production of FXa and thrombin, respectively. Ixolaris effectively blocked the in vitro TF-dependent procoagulant activity of the U87-MG human glioblastoma cell line and attenuated multimolecular coagulation complexes assembly. Notably, Ixolaris inhibited the in vivo tumorigenic potential of U87-MG cells in nude mice, without observable bleeding. This inhibitory effect of Ixolaris on tumor growth was associated with downregulation of VEGF and reduced tumor vascularization. Conclusion: Our results suggest that Ixolaris might be a promising agent for anti-tumor therapy in humans.

Expression of human papillomavirus type 16 E7 oncoprotein alters keratinocytes expression profile in response to tumor necrosis factor-alpha

Acute expression of E7 oncogene from human papillomavirus (HPV) 16 or HPV18 is sufficient to overcome tumor necrosis factor (TNF)-alpha cytostatic effect on primary human keratinocytes. In the present study, we investigated the molecular basis of E7-induced TNF resistance through a comparative analysis of the effect of this cytokine on the proliferation and global gene expression of normal and E7-expressing keratinocytes. Using E7 functional mutants, we show that E7-induced TNF resistance correlates with its ability to mediate pRb degradation and cell transformation. On the other hand, this effect does not depend on E7 sequences required to override DNA damage-induced cell cycle arrest or extend keratinocyte life span. Furthermore, we identified a group of 66 genes whose expression pattern differs between normal and E7-expressing cells upon cytokine treatment. These genes are mainly involved in cell cycle regulation suggesting that their altered expression may contribute to sustained cell proliferation even in the presence of a cytostatic stimulus. Differential expression of TCN1 (transcobalamin I), IFI44 (Interferon-induced protein 44), HMGB2 (high-mobility group box 2) and FUS [Fusion (involved in t(12; 16) in malignant liposarcoma)] among other genes were further confirmed by western-blot and/or real-time polymerase chain reaction. Moreover, FUS upregulation was detected in HPV-positive cervical high-grade squamous intraepithelial lesions when compared with normal cervical tissue. Further evaluation of the role of such genes in TNF resistance and HPVassociated disease development is warranted.

Functional Expression of Chemoreceptors with the Help of a Guanine Nucleotide Exchange Factor

Odorant receptors and other chemoreceptors are usually poorly expressed in the plasma membrane of heterologous cells. A key point of regulation in G protein-mediated signaling is the interconversion between the active GTP-bound and inactive GDP-bound states of the G alpha subunit, which regulatory proteins, such as guanine nucleotide exchange factors (GEFs), can control. GEFs stimulate formation of the GTP-bound state of G alpha and therefore are considered to work as positive regulators of G protein-coupled receptor signaling. Ric-8B, a GEF that is specifically expressed in olfactory sensory neurons, promotes functional expression of odorant receptors in HEK293T cells because it amplifies the initially low receptor signaling through G alpha olf. This same strategy could be used to functionally express other types of chemoreceptors.

Sample stacking in CZE using dynamic thermal junctions I. Analytes with low dpK(a)/dT crossing a single thermally induced pH junction in a BGE with high dpH/dT

The possibility to compress analyte bands at the beginning of CE runs has many advantages. Analytes at low concentration can be analyzed with high signal-to-noise ratios by using the so-called sample stacking methods. Moreover, sample injections with very narrow initial band widths (small initial standard deviations) are sometimes useful, especially if high resolutions among the bands are required in the shortest run time. In the present work, a method of sample stacking is proposed and demonstrated. It is based on BGEs with high thermal sensitive pHs (high dpH/dT) and analytes with low dpK(a)/dT. High thermal sensitivity means that the working pK(a) of the BGE has a high dpK(a)/dT in modulus. For instance, Tris and Ethanolamine have dpH/dT = -0.028/degrees C and -0.029/degrees C, respectively, whereas carboxylic acids have low dpK(a)/dT values, i.e. in the -0.002/degrees C to+0.002/degrees C range. The action of cooling and heating sections along the capillary during the runs affects also the local viscosity, conductivity, and electric field strength. The effect of these variables on electrophoretic velocity and band compression is theoretically calculated using a simple model. Finally, this stacking method was demonstrated for amino acids derivatized with naphthalene-2,3-dicarboxaldehyde and fluorescamine using a temperature difference of 70 degrees C between two neighbor sections and Tris as separation buffer. In this case, the BGE has a high pH thermal coefficient whereas the carboxylic groups of the analytes have low pK(a) thermal coefficients. The application of these dynamic thermal gradients increased peak height by a factor of two (and decreased the standard deviations of peaks by a factor of two) of aspartic acid and glutamic acid derivatized with naphthalene-2,3-dicarboxaldehyde and serine derivatized with fluorescamine. The effect of thermal compression of bands was not observed when runs were accomplished using phosphate buffer at pH 7 (negative control). Phosphate has a low dpH/dT in this pH range, similar to the dK(a)/dT of analytes. It is shown that vertical bar dK(a)/dT-dpH/dT vertical bar >> 0 is one determinant factor to have significant stacking produced by dynamic thermal junctions.

Arbetsorganisation i träbränsleeldande fjärrvärmeverk

The objective of this study has been to describe and analyse existing forms of organisation in heating plants using wood fuels, regarding work tasks, organisational structure, skill demands, crew recruitment, working hours and wage conditions. Sixteen plants ranging from 10 to 120 MW have been studied by means of interviews, work place observations and written material. The job of the operator of heating plants is fairly qualified, independent and varied. The most negative factor is shift work. Some of the bigger plants (enterprises) have a relatively hierarchic, segmented and perhaps also an oversized organisation. However, modern concepts of organisation, such as customer orientation, ”flat organisation”, integration of production and maintenance etc, are gaining ground. Blue collar and white collar tasks are increasingly being integrated. Some of the medium sized enterprises have reached very far and may serve as models for bigger enterprises.

Medarbetarundersökningens roll i chefernas arbetsmiljöarbete : En kvalitativ studie hos Trafikverket

En god arbetsmiljö är en lagstadgad rättighet för arbetstagaren enligt Arbetsmiljölagen. Arbetet med att säkerställa en god arbetsmiljö kallas arbetsmiljöarbete och är en nyckelfaktor för att skapa hälsosamma och attraktiva arbetsplatser. Medarbetarundersökningar är en undersökningsmetod i det systematiska arbetsmiljöarbetet men kan också användas i syfte att ta reda på medarbetarnas uppfattningar om sin arbetssituation med målet att skapa verksamhetsförbättringar. I denna studie har syftet varit att undersöka vilken roll som medarbetarundersökningen spelar i chefernas arbetsmiljöarbete. Studien undersökte hur cheferna arbetar med arbetsmiljöarbete och hur arbetet med resultatet från en medarbetarundersökning genomförts. Studien genomfördes på en funktion med tre organisationsnivåer och bygger på 12 kvalitativa intervjuer med funktions-, avdelnings- och enhetschefer. Studien visar att medarbetarundersökningen spelar en viktig roll i chefernas arbetsmiljöarbete på alla organisationsnivåer både som diskussionsunderlag i ledningsgrupper och i dialogen med medarbetarna samt som bevis på att genomförda arbetsmiljöinsatser fungerar. Enhetscheferna använder både medarbetarundersökningen och andra undersökningsmetoder i sitt arbetsmiljöarbete.

Elbesparing med pelletkaminer och solvärme i direktelvärmda småhus

The aim of this study was to investigate how electricallyheated houses can be converted to using wood pellet and solarheating. There are a large number of wood pellet stoves on themarket. Many stoves have a water jacket, which gives anopportunity to distribute the heat to domestic hot water and aradiator heating system. Three typical Swedish houses with electric resistanceheating have been studied. Fourteen different system conceptsusing wood pellet stoves and solar heating systems have beenevaluated. The systems and the houses have been simulated indetail using TRNSYS. The houses have been divided in up to 10different zones and heat transfer by air circulation throughdoorways and open doors have been simulated. The pellet stoveswere simulated using a recently developed TRNSYS component,which models the start- and stop phases, emissions and thedynamic behaviour of the stoves. The model also calculates theCO-emissions. Simulations were made with one stove without awater jacket and two stoves with different fractions of thegenerated heat distributed in the water circuit. Simulations show that the electricity savings using a pelletstove are greatly affected by the house plan, the systemchoice, if the internal doors are open or closed and thedesired level of comfort. Installing a stove with awater-jacket connected to a radiator system and a hot waterstorage has the advantage that heat can be transferred todomestic hot water and be distributed to other rooms. Suchsystems lead to greater electricity savings, especially inhouses having a traditional layout. It was found that not allrooms needed radiators and that it was more effective in mostcases t use a stove with a higher fraction of the heatdistributed by the water circuit. The economic investigation shows that installing a woodpellet stove without a water jacket gives the lowest totalenergy- and capital costs in the house with an open plan (fortoday's energy prices and the simulated comfort criteria). Inthe houses with a traditional layout a pellet stove givesslightly higher costs than the reference house having onlyelectrical resistance heating due to the fact that less heatingcan be replaced. The concepts including stoves with a waterjacket all give higher costs than the reference system, but theconcept closest to be economical is a system with a bufferstore, a stove with a high fraction of the heat distributed bythe water circuit, a new water radiator heating system and asolar collector. Losses from stoves can be divided into: flue gas lossesincluding leakage air flow when the stove is not in operation;losses during start and stop phases; and losses due to a highair factor. An increased efficiency of the stoves is importantboth from a private economical point of view, but also from theperspective that there can be a lack of bio fuel in the nearfuture also in Sweden. From this point of view it is alsoimportant to utilize as much solar heat as possible. Theutilization of solar heat is low in the simulated systems,depending on the lack of space for a large buffer store. The simulations have shown that the annual efficiency ismuch lower that the nominal efficiency at full power. Thesimulations have also shown that changing the control principlefor the stove can improve efficiency and reduce theCO-emissions. Today's most common control principle for stovesis the on/off control, which results in many starts and stopsand thereby high CO-emissions. A more advanced control varyingthe heating rate from maximum to minimum to keep a constantroom temperature reduces the number of starts and stops andthereby the emissions. Also the efficiency can be higher withsuch a control, and the room temperature will be kept at a moreconstant temperature providing a higher comfort.

Biolinguistics or Physicolinguistics? Is The Third Factor Helpful Or Harmful In Explaining Language?

Noam Chomsky (2005) proposed that a ‘third factor’, consisting of general principles and natural laws, may explain core properties of language in a principled manner, minimizing the need for either genetic endowment or experience. But the focus on third-factor patterns in much recent bio-linguistic work is misguided for several reasons: First, ‘the’ third factor is a vague and disparate collection of unrelated components, useless as an analytical tool. Second, the vagueness of the third factor, together with the desire for principled explanations, too often leads to sweeping claims, such as syntax “coming for free, directly from physics”, that are unwarranted without a case-by-case causal analysis. Third, attention is diverted away from a proper causal analysis of language as a biological feature. The point with biolinguistics is to acknowledge the language faculty as a biological feature. The best way forward towards an understanding of language is to take the biology connection seriously, instead of dabbling with physics.

Tid som välfärdsfaktor : förändrade motiv i svensk arbetstidspolicy

Time as a welfare factor This article presents the results of an archive study of working time development in Sweden. It investigates how structural policy change is moving from a social discourse closely related to Swedish welfare reforms, towards an economic discourse motivated by financial arguments. By doing so, the political measures to solve working time related problems in today’s flexible working life appear to be contradictory. On one side we find time-poor people on the labour market mainly supported by tax-reductions and private time saving solutions. On the other side we find time-rich people mainly supported by activation programs and/or welfare benefits. This is a system and a policy strategy that obviously disregards the other side of the coin.

Hårdare tag i arbetslivet? : Åtgärder mot arbetsrelaterat våld i facklig press 1978–2004

Preventing violence at work: A study of descriptions of safety measures in Swedish trade union journals 1978–2004 The purpose of this study is to examine if perceptions of interventions aimed at violence in the workplace have changed since the 1970s. In the beginning of the study period, structural factors are seen as the dominating explanation for workplace violence. The crime perspective rises in the 1990’s and methods of intervention becomes the control- and justice functions of larger society. The result shows search for accountability to be a salient factor for understanding the development towards an increasing use of penal sanctions.

Kundernas behov i butiksmiljöer : Skillnaderna mellan handlares och kunders uppfattning av kundernas behov i klädbutiker

Konkurrensen inom detaljhandeln blir allt hårdare, både från andra butiker men även från e-handeln, vilket sätter press på fysiska butiker att ha nöjda kunder som fortsätter handla i butiken. Ett tillvägagångssätt för att möta den hårdnande konkurrensen är att ha en butiksmiljö s.k. servicescape som är utformad efter kundernas behov, vilket dock förutsätter att handlarna vet vilka behov sina kunder har. Har handlarna en felaktig uppfattning om kundernas behov föreligger det ett gap som minskar chansen att kunderna blir nöjda och därmed butikens konkurrenskraft. En avgränsning gjordes till klädbutiker, då klädbutiker är bland de butikstyper som är mest konkurrensutsatt. Syftet med uppsatsen var att kartlägga hur kundernas behov överensstämmer eller skiljer sig från handlarnas uppfattning om deras behov. För att uppfylla syftet tillämpades en kvalitativ metod men med en statistisk bearbetning dvs. en kvantitativ bearbetning då resultatet redovisades med hjälp av frekvenser och värden på en skala. Datainsamlingsverktyget som användes var enkäter och utformades efter de tio faktorerna som sammanställdes i den teoretiska referensramen. Enkäterna delades ut till både kunder och handlare i Borlänges stadskärna och Faluns stadskärna. Resultatet av uppsatsen visade att det för faktorerna ljus (4,2), trängsel (4.5) (4.6), Ljud (4.7), toaletter (4.9), (4.10) finns en skillnad ett s.k. gap mellan handlarnas uppfattning om kundernas behov och vad kundernas behov egentligen är. Det framkom även att handlarna ansåg att kunderna har större behov av att faktorerna rent & städat (4.3), (4.4) in & utgångar (4.8) är tillfredsställande än vad kunderna egentligen har. Det framkom precis som behandlades i den teoretiska referensramen att kundernas behov tenderar att variera utifrån deras demografiska faktorer ålder och kön. Om handlarna har en låg, medel eller hög prisnivå påverkade även det hur viktigt det är för kunderna att faktorerna är tillfredsställande. Slutsatsen som författarna identifierade var att det föreligger ett gap mellan handlarnas uppfattning om kundernas behov och vad kunderna anser att deras behov är gällande flera av faktorerna. Detta är dock något som kunderna säger vilket inte behöver stämma överrens med hur de verkligen tycker. Det är dock svårt att urskilja något mönster gällande gapen utan det är nästan enbart tendenser man kan urskilja, vilket gör att varje faktor bör analyseras individuellt.