Molecular basis and regulation of insect pathogenicity in plant-beneficial pseudomonads

Les bactéries du genre Pseudomonas ont la capacité étonnante de s'adapter à différents habitats et d'y survivre, ce qui leur a permis de conquérir un large éventail de niches écologiques et d'interagir avec différents organismes hôte. Les espèces du groupe Pseudomonas fluorescens peuvent être facilement isolées de la rhizosphère et sont communément connues comme des Pseudomonas bénéfiques pour les plantes. Elles sont capables d'induire la résistance systémique des plantes, d'induire leur croissance et de contrer des phytopathogènes du sol. Un sous-groupe de ces Pseudomonas a de plus développé la capacité d'infecter et de tuer certaines espèces d'insectes. Approfondir les connaissances sur l'interaction de ces bactéries avec les insectes pourraient conduire au développement de nouveaux biopesticides pour la protection des cultures. Le but de cette thèse est donc de mieux comprendre la base moléculaire, l'évolution et la régulation de la pathogénicité des Pseudomonas plante-bénéfiques envers les insectes. Plus spécifiquement, ce travail a été orienté sur l'étude de la production de la toxine insecticide appelée Fit et sur l'indentification d'autres facteurs de virulence participant à la toxicité de la bactérie envers les insectes. Dans la première partie de ce travail, la régulation de la production de la toxine Fit a été évaluée par microscopie à épifluorescence en utilisant des souches rapportrices de Pseudomonas protegens CHA0 qui expriment la toxine insecticide fusionnée à une protéine fluorescente rouge, au site natif du gène de la toxine. Celle-ci a été détectée uniquement dans l'hémolymphe des insectes et pas sur les racines des plantes, ni dans les milieux de laboratoire standards, indiquant une production dépendante de l'hôte. L'activation de la production de la toxine est contrôlée par trois protéines régulatrices dont l'histidine kinase FitF, essentielle pour un contrôle précis de l'expression et possédant un domaine "senseur" similaire à celui de la kinase DctB qui régule l'absorption de carbone chez les Protéobactéries. Il est donc probable que, durant l'évolution de FitF, un réarrangement de ce domaine "senseur" largement répandu ait contribué à une production hôte-spécifique de la toxine. Les résultats de cette étude suggèrent aussi que l'expression de la toxine Fit est plutôt réprimée en présence de composés dérivés des plantes qu'induite par la perception d'un signal d'insecte spécifique. Dans la deuxième partie de ce travail, des souches mutantes ciblant des facteurs de virulence importants identifiés dans des pathogènes connus ont été générées, dans le but d'identifier ceux avec une virulence envers les insectes atténuée. Les résultats ont suggéré que l'antigène O du lipopolysaccharide (LPS) et le système régulateur à deux composantes PhoP/PhoQ contribuent significativement à la virulence de P. protegens CHA0. La base génétique de la biosynthèse de l'antigène O dans les Pseudomonas plante-bénéfiques et avec une activité insecticide a été élucidée et a révélé des différences considérables entre les lignées suite à des pertes de gènes ou des acquisitions de gènes par transfert horizontal durant l'évolution de certaines souches. Les chaînes latérales du LPS ont été montrées comme vitales pour une infection des insectes réussie par la souche CHA0, après ingestion ou injection. Les Pseudomonas plante-bénéfiques, avec une activité insecticide sont naturellement résistants à la polymyxine B, un peptide antimicrobien modèle. La protection contre ce composé antimicrobien particulier dépend de la présence de l'antigène O et de la modification du lipide A, une partie du LPS, avec du 4-aminoarabinose. Comme les peptides antimicrobiens cationiques jouent un rôle important dans le système immunitaire des insectes, l'antigène O pourrait être important chez les Pseudomonas insecticides pour surmonter les mécanismes de défense de l'hôte. Le système PhoP/PhoQ, connu pour contrôler les modifications du lipide A chez plusieurs bactéries pathogènes, a été identifié chez Pseudomonas chlororaphis PCL1391 et P. protegens CHA0. Pour l'instant, il n'y a pas d'évidence que des modifications du lipide A contribuent à la pathogénicité de cette bactérie envers les insectes. Cependant, le senseur-kinase PhoQ est requis pour une virulence optimale de la souche CHA0, ce qui suggère qu'il régule aussi l'expression des facteurs de virulence de cette bactérie. Les découvertes de cette thèse démontrent que certains Pseudomonas associés aux plantes sont de véritables pathogènes d'insectes et donnent quelques indices sur l'évolution de ces microbes pour survivre dans l'insecte-hôte et éventuellement le tuer. Les résultats suggèrent également qu'une recherche plus approfondie est nécessaire pour comprendre comment ces bactéries sont capables de contourner ou surmonter la réponse immunitaire de l'hôte et de briser les barrières physiques pour envahir l'insecte lors d'une infection orale. Pour cela, les futures études ne devraient pas uniquement se concentrer sur le côté bactérien de l'interaction hôte-microbe, mais aussi étudier l'infection du point de vue de l'hôte. Les connaissances gagnées sur la pathogénicité envers les insectes des Pseudomonas plante-bénéfiques donnent un espoir pour une future application en agriculture, pour protéger les plantes, non seulement contre les maladies, mais aussi contre les insectes ravageurs. -- Pseudomonas bacteria have the astonishing ability to survive within and adapt to different habitats, which has allowed them to conquer a wide range of ecological niches and to interact with different host organisms. Species of the Pseudomonas fluorescens group can readily be isolated from plant roots and are commonly known as plant-beneficial pseudomonads. They are capable of promoting plant growth, inducing systemic resistance in the plant host and antagonizing soil-borne phytopathogens. A defined subgroup of these pseudomonads evolved in addition the ability to infect and kill certain insect species. Profound knowledge about the interaction of these particular bacteria with insects could lead to the development of novel biopesticides for crop protection. This thesis thus aimed at a better understanding of the molecular basis, evolution and regulation of insect pathogenicity in plant-beneficial pseudomonads. More specifically, it was outlined to investigate the production of an insecticidal toxin termed Fit and to identify additional factors contributing to the entomopathogenicity of the bacteria. In the first part of this work, the regulation of Fit toxin production was probed by epifluorescence microscopy using reporter strains of Pseudomonas protegens CHAO that express a fusion between the insecticidal toxin and a red fluorescent protein in place of the native toxin gene. The bacterium was found to express its insecticidal toxin only in insect hemolymph but not on plant roots or in common laboratory media. The host-dependent activation of Fit toxin production is controlled by three local regulatory proteins. The histidine kinase of this regulatory system, FitF, is essential for the tight control of toxin expression and shares a sensing domain with DctB, a sensor kinase regulating carbon uptake in Proteobacteria. It is therefore likely that shuffling of a ubiquitous sensor domain during the evolution of FitF contributed to host- specific production of the Fit toxin. Findings of this study additionally suggest that host-specific expression of the Fit toxin is mainly achieved by repression in the presence of plant-derived compounds rather than by induction upon perceiving an insect-specific signal molecule. In the second part of this thesis, mutant strains were generated that lack factors previously shown to be important for virulence in prominent pathogens. A screening for attenuation in insect virulence suggested that lipopolysaccharide (LPS) O-antigen and the PhoP-PhoQ two-component regulatory system significantly contribute to virulence of P. protegens CHAO. The genetic basis of O-antigen biosynthesis in plant-beneficial pseudomonads displaying insect pathogenicity was elucidated and revealed extensive differences between lineages due to reduction and horizontal acquisition of gene clusters during the evolution of several strains. Specific 0 side chains of LPS were found to be vital for strain CHAO to successfully infect insects by ingestion or upon injection. Insecticidal pseudomonads with plant-beneficial properties were observed to be naturally resistant to polymyxin B, a model antimicrobial peptide. Protection against this particular antimicrobial compound was dependent on the presence of O-antigen and modification of the lipid A portion of LPS with 4-aminoarabinose. Since cationic antimicrobial peptides play a major role in the immune system of insects, O-antigenic polysaccharides could be important for insecticidal pseudomonads to overcome host defense mechanisms. The PhoP-PhoQ system, which is well-known to control lipid A modifications in several pathogenic bacteria, was identified in Pseudomonas chlororaphis PCL1391 and P. protegens CHAO. No evidence was found so far that lipid A modifications contribute to insect pathogenicity in this bacterium. However, the sensor kinase PhoQ was required for full virulence of strain CHAO suggesting that it additionally regulates the expression of virulence factors in this bacterium. The findings of this thesis demonstrate that certain plant-associated pseudomonads are true insect pathogens and give some insights into how these microbes evolved to survive within and eventually kill the insect host. Results however also point out that more in-depth research is needed to know how exactly these fascinating bacteria manage to bypass or overcome host immune responses and to breach physical barriers to invade insects upon oral infection. To achieve this, future studies should not only focus on the bacterial side of the microbe-host interactions but also investigate the infection from a host-oriented view. The knowledge gained about the entomopathogenicity of plant-beneficial pseudomonads gives hope for their future application in agriculture to protect plants not only against plant diseases but also against insect pests.

Mutations in the heat-shock protein A9 (HSPA9) gene cause the EVEN-PLUS syndrome of congenital malformations and skeletal dysplasia.

We and others have reported mutations in LONP1, a gene coding for a mitochondrial chaperone and protease, as the cause of the human CODAS (cerebral, ocular, dental, auricular and skeletal) syndrome (MIM 600373). Here, we delineate a similar but distinct condition that shares the epiphyseal, vertebral and ocular changes of CODAS but also included severe microtia, nasal hypoplasia, and other malformations, and for which we propose the name of EVEN-PLUS syndrome for epiphyseal, vertebral, ear, nose, plus associated findings. In three individuals from two families, no mutation in LONP1 was found; instead, we found biallelic mutations in HSPA9, the gene that codes for mHSP70/mortalin, another highly conserved mitochondrial chaperone protein essential in mitochondrial protein import, folding, and degradation. The functional relationship between LONP1 and HSPA9 in mitochondrial protein chaperoning and the overlapping phenotypes of CODAS and EVEN-PLUS delineate a family of "mitochondrial chaperonopathies" and point to an unexplored role of mitochondrial chaperones in human embryonic morphogenesis.

Bartonella apis sp. nov., a honey bee gut symbiont of the class Alphaproteobacteria.

Here, we report the culture and characterization of an alphaproteobacterium of the order Rhizobiales, isolated from the gut of the honey bee Apis mellifera. Strain PEB0122T shares >95 % 16S rRNA gene sequence similarity with species of the genus Bartonella, a group of mammalian pathogens transmitted by bloodsucking arthropods. Phylogenetic analyses showed that PEB0122T and related strains from the honey bee gut form a sister clade of the genus Bartonella. Optimal growth of strain PEB0122T was obtained on solid media supplemented with defibrinated sheep blood under microaerophilic conditions at 35-37 °C, which is consistent with the cultural characteristics of other species of the genus Bartonella. Reduced growth of strain PEB0122T also occurred under aerobic conditions. The rod-shaped cells of strain PEB0122T had a mean length of 1.2-1.8 μm and revealed hairy surface structures. Strain PEB0122T was positive for catalase, cytochrome c oxidase, urease and nitrate reductase. The fatty acid composition was comparable to those of other species of the genus Bartonella, with palmitic acid (C16 : 0) and isomers of 18- and 19-carbon chains being the most abundant. The genomic DNA G+C content of PEB0122T was determined to be about 45.5 mol%. The high 16S rRNA gene sequence similarity with species of Bartonella and its close phylogenetic position suggest that strain PEB0122T represents a novel species within the genus Bartonella, for which we propose the name Bartonella apis sp. nov. The type strain is PEB0122T ( = NCIMB 14961T = DSM 29779T).

Treatment of cancer with oral drugs: a position statement by the Spanish Society of Medical Oncology

Cancer treatment involves the participation of multiple medical specialties and, as our knowledge of the disease increases, this fact becomes even more apparent. The degree of multidisciplinarity is determined by several factors, which include the severity and type of disease, the increasing diversity in the available pharmacological and non-pharmacological therapies, and the range of specialists involved in cancer therapy, such as medical oncologists, radiotherapists, gynecologists, gastroenterologists, urologists, surgeons, and pneumologists, among others. Across Europe, the situation of cancer care can be variable due to the diversity of health systems, differences in drug reimbursement, and the degree of establishment of Medical Oncology as a medical specialty in the European Union states.

The forced adoption of a fast-track appraisal process for a breast cancer treatment in UK

Trastuzumab (Herceptin ®, Roche) is approved in UK for the treatment of the metastatic breast cancer since 2001. As of 2005, concomitantly with the publication of 3 studies that showed it produces a 50% reduction of the recurrence rates of breast cancer, trastuzumab started to be prescribed in the earlt adjuvant treatrnent of this disease. Und June 2006, trastuzumab did not have both: 1) regulatory approval and 2) NICE [National Institute for Health and Clinical Excellence] recommendation for the use in early stages of breast cancer. During the period until June 2006, the trastuzumab use in those patients was not reimbursed and because the cost of trastuzumab is equal with the yearly UK average income, most of patients could not self fund their treatrnent. Before the publication of the final NICE guidance, the new data of trastuzumab in early breast cancer raised enormous patient and professional interest and expectations. A great volume of public and professional pressure was generated to transcend a system by which Primary Care Trusts can reimburse a treatment only after a formal guidance was issued. This paper draw on a case study depicting and analyzing the process by which regulatory approval and NICE recommendations were achieved in a record time and how trastuzumab became a standard treatment on early adjuvant breast cancer. According to the data we gathered in this work we were witnessing one of the fastest processes of adoption of a health care technology since the creation of NICE, in 1999. This study addresses the following research question: How and why does the adoption pattern of trastuzumab differ from the rational decision-making model of the reimbursement process in UK? [Author, p. 4]

A feasability study of the analysis of the economic outcomes of specialized nutrition in obesity and weight management in Switzerland

The increasing prevalence of obesity and its associated complications requires specialized care to improve outcomes and control health care costs. Obesity is associated with numerous serious and costly medical problems requiring specialized care in managing health. The economic burden of obesity includes increased inpatient and outpatient medical expenditures as well as employer-related issues of absenteeism and associate costs. The objectives of this study are: - To describe the health consequences and the economic burden of obesity, - To review the existing treatment - To argue in favor of a specialized nutritional intervention that has shown to improve health and reduce obesity related health care costs. Therefore, expose the possibility of introducing the specialized nutrition in Switzerland and the feasibility of this project considering the medical trends and reimbursement system in Switzerland The benefits and outcomes for the patients will be the significant weight loss which reduces the severity and risk factors for complications and the improved health and quality of life. Weight loss will be a combination of a diet, exercise and behavioral interventions which are the basic recommendations for obesity treatment in addition to the specialized nutritional support. By nutritional support, we mean products that are intended to provide nutritional support in the dietary management of people with specific diseases and conditions when adequate intake of regular foods is compromised. These products are called, Food for special medical purposes FSMP. They are not intended to treat, cure, prevent, mitigate or have a direct impact on disease in a manner similar to drugs or other medical treatments and should be used under medical supervision. They also provide a low cost alternative to surgery. From a health care system perspective, the specialized nutrition will drive its advantage by reducing the utilization of medical services for obesity associated complications like medication, physician's consultations and surgical interventions arriving to a cost effective care for the hospitals, the health care organizations and the third party payers which are the health insurances. [Author, p. 4]

Maritime transportation in the Gulf of Finland in 2007 and in 2015

The Gulf of Finland is said to be one of the densest operated sea areas in the world. It is a shallow and economically vulnerable sea area with dense passenger and cargo traffic of which petroleum transports have a share of over 50 %. The winter conditions add to the risks of maritime traffic in the Gulf of Finland. It is widely believed that the growth of maritime transportation will continue also in the future. The Gulf of Finland is surrounded by three very different national economies with, different maritime transportation structures. Finland is a country of high GDP/per capita with a diversified economic structure. The number of ports is large and the maritime transportation consists of many types of cargoes: raw materials, industrial products, consumer goods, coal and petroleum products, and the Russian transit traffic of e.g. new cars and consumer goods. Russia is a large country with huge growth potential; in recent years, the expansion of petroleum exports has lead to a strong economic growth, which is also apparent in the growth of maritime transports. Russia has been expanding its port activities in the Gulf of Finland and it is officially aiming to transport its own imports and exports through the Russian ports in the future; now they are being transported to great extend through the Finnish, Estonian and other Baltic ports. Russia has five ports in the Gulf of Finland. Estonia has also experienced fast economic growth, but the growth has been slowing down already during the past couples of years. The size of its economy is small compared to Russia, which means the transported tonnes cannot be very massive. However, relatively large amounts of the Russian petroleum exports have been transported through the Estonian ports. The future of the Russian transit traffic in Estonia looks nevertheless uncertain and it remains to be seen how it will develop and if Estonia is able to find replacing cargoes if the Russian transit traffic will come to an end in the Estonian ports. Estonia’s own import and export consists of forestry products, metals or other raw materials and consumer goods. Estonia has many ports on the shores of the Gulf of Finland, but the port of Tallinn dominates the cargo volumes. In 2007, 263 M tonnes of cargoes were transported in the maritime traffic in the Gulf of Finland, of which the share of petroleum products was 56 %. 23 % of the cargoes were loaded or unloaded in the Finnish ports, 60 % in the Russian ports and 17 % in the Estonian ports. The largest ports were Primorsk (74.2 M tonnes) St. Petersburg (59.5 M tonnes), Tallinn (35.9 M tonnes), Sköldvik (19.8 M tonnes), Vysotsk (16.5 M tonnes) and Helsinki (13.4 M) tonnes. Approximately 53 600 ship calls were made in the ports of the Gulf of Finland. The densest traffic was found in the ports of St. Petersburg (14 651 ship calls), Helsinki (11 727 ship calls) and Tallinn (10 614 ship calls) in 2007. The transportation scenarios are usually based on the assumption that the amount of transports follows the development of the economy, although also other factors influence the development of transportation, e.g. government policy, environmental aspects, and social and behavioural trends. The relationship between the development of transportation and the economy is usually analyzed in terms of the development of GDP and trade. When the GDP grows to a certain level, especially the international transports increase because countries of high GDP produce, consume and thus transport more. An effective transportation system is also a precondition for the economic development. In this study, the following factors were taken into consideration when formulating the future scenarios: maritime transportation in the Gulf of Finland 2007, economic development, development of key industries, development of infrastructure and environmental aspects in relation to maritime transportation. The basic starting points for the three alternative scenarios were: • the slow growth scenario: economic recession • the average growth scenario: economy will recover quickly from current instability • the strong growth scenario: the most optimistic views on development will realize According to the slow growth scenario, the total tonnes for the maritime transportation in the Gulf of Finland would be 322.4 M tonnes in 2015, which would mean a growth of 23 % compared to 2007. In the average growth scenario, the total tonnes were estimated to be 431.6 M tonnes – a growth of 64 %, and in the strong growth scenario 507.2 M tonnes – a growth of 93%. These tonnes were further divided into petroleum products and other cargoes by country, into export, import and domestic traffic by country, and between the ports. For petroleum products, the share of crude oil and oil products was estimated and the number of tanker calls in 2015 was calculated for each scenario. However, the future development of maritime transportation in the GoF is dependent on so many societal and economic variables that it is not realistic to predict one exact point estimate value for the cargo tonnes for a certain scenario. Plenty of uncertainty is related both to the degree in which the scenario will come true as well as to the cause-effect relations between the different variables. For these reasons, probability distributions for each scenario were formulated by an expert group. As a result, a range for the total tonnes of each scenario was formulated and they are as follows: the slow growth scenario: 280.8 – 363 M tonnes (expectation value 322.4 M tonnes)

Investigating the impact of small versus large firms on economic performance of countries and industries

Following earlier work by Audretsch et al. (2002), we assume that an optimal size-class structure exists, in terms of achieving maximal economic growth rates. Such an optimal structure is likely to exist as economies need a balance between the core competences of large firms (such as exploitation of economies of scale) and those of smaller firms (such as flexibility and exploration of new ideas). Accordingly, changes in size-class structure (i.e., changes in the relative shares in economic activity accounted for by micro, small, medium-sized and large firms) may affect macro-economic growth. Using a unique data base of the EU-27 countries for the period 2002-2008 for five broad sectors of economic activity and four size-classes, we find empirical support which suggests that, on average for these countries over this period, the share of micro and large firms may have been ‘above optimum’ (particularly in lower income EU countries) whereas the share of medium-sized firms may have been ‘below optimum’ (particularly in higher income EU countries). This evidence suggests that the transition from a ‘managed’ to an ‘entrepreneurial’ economy (Audretsch and Thurik, 2001) has not been completed yet in all countries of the EU-27. Keywords: small firms, large firms, size-classes, macro-economic performance

Knowledge Sharing between Generations in an Organisation - Retention of the Old or Building the New?

The study explores knowledge transfer between retiring employees and their successors in expert work. My aim is to ascertain whether there is knowledge development or building new knowledge related to this organisational knowledge transfer between generations; in other words, is the transfer of knowledge from experienced, retiring employees to their successors merely retention of the existing organisational knowledge by distributing it from one individual to another or does this transfer lead to building new and meaningful organisational knowledge. I call knowledge transfer between generations and the possibly related knowledge building in this study knowledge sharing between generations. The study examines the organisation and knowledge management from a knowledge-based and constructionist view. From this standpoint, I see knowledge transfer as an interactive process, and the exploration is based on how the people involved in this process understand and experience the phenomenon studied. The research method is organisational ethnography. I conducted the analysis of data using thematic analysis and the articulation method, which has not been used before in organisational knowledge studies. The primary empirical data consists of theme interviews with twelve employees involved in knowledge transfer in the organisation being studied and five follow-up theme interviews. Six of the interviewees are expert duty employees due to retire shortly, and six are their successors. All those participating in the follow-up interviews are successors of those soon to retire from their expert responsibilities. The organisation in the study is a medium-sized Finnish firm, which designs and manufactures electrical equipment and systems for the global market. The results of the study show that expert work-related knowledge transfer between generations can mean knowledge building which produces new, meaningful knowledge for the organisation. This knowledge is distributed in the organisation to all those that find it useful in increasing the efficiency and competitiveness of the whole organisation. The transfer and building of knowledge together create an act of knowledge sharing between generations where the building of knowledge presupposes transfer. Knowledge sharing proceeds between the expert and the novice through eight phases. During the phases of knowledge transfer the expert guides the novice to absorb the knowledge to be transferred. With the expert’s help the novice gradually comes to understand the knowledge and in the end he or she is capable of using it in his or her work. During the phases of knowledge building the expert helps the novice to further develop the knowledge being transferred so that it becomes new, useful knowledge for the organisation. After that the novice takes the built knowledge to use in his or her work. Based on the results of the study, knowledge sharing between generations takes place in interaction and ends when knowledge is taken to use. The results I obtained in the interviews by the articulation method show that knowledge sharing between generations is shaped by the novices’ conceptions of their own work goals, knowledge needs and duties. These are not only based on the official definition of the work, but also how the novices find their work or how they prioritise the given objectives and responsibilities. The study shows that the novices see their work primarily as maintenance or development. Those primarily involved in maintenance duties do not necessarily need knowledge defined as transferred between generations. Therefore, they do not necessarily transfer knowledge with their assigned experts, even though this can happen in favourable circumstances. They do not build knowledge because their view of their work goals and duties does not require the building of new knowledge. Those primarily involved in development duties, however, do need knowledge available from their assigned experts. Therefore, regardless of circumstances they transfer knowledge with their assigned experts and also build knowledge because their work goals and duties create a basis for building new knowledge. The literature on knowledge transfer between generations has focused on describing either the knowledge being transferred or the means by which it is transferred. Based on the results of this study, however, knowledge sharing between generations, that is, transfer and building is determined by how the novice considers his or her own knowledge needs and work practices. This is why studies on knowledge sharing between generations and its implementation should be based not only on the knowledge content and how it is shared, but also on the context of the work in which the novice interprets and shares knowledge. The existing literature has not considered the possibility that knowledge transfer between generations may mean building knowledge. The results of this study, however, show that this is possible. In knowledge building, the expert’s existing organisational knowledge is combined with the new knowledge that the novice brings to the organisation. In their interaction this combination of the expert’s “old” and the novice’s “new” knowledge becomes new, meaningful organisational knowledge. Previous studies show that knowledge development between the members of an organisation is the prerequisite for organisational renewal which in turn is essential for improved competitiveness. Against this background, knowledge building enables organisational renewal and thus enhances competitiveness. Hence, when knowledge transfer between generations is followed by knowledge building, the organisation kills two birds with one stone. In knowledge transfer the organisation retains the existing knowledge and thus maintains its competitiveness. In knowledge building the organisation developsnew knowledge and thus improves its competitiveness.

Prokaryotic expression of a truncated form of bovine herpesvirus 1 glycoprotein E (gE) and its use in an ELISA for gE antibodies

This article describes the expression of a truncated form of bovine herpesvirus 1 (BoHV-1) glycoprotein E (gE) for use as immunodiagnostic reagent. A 651 nucleotide fragment corresponding to the amino-terminal third (217 amino acids) of BoHV-1 gE - that shares a high identity with the homologous BoHV-5 counterpart - was cloned as a 6×His-tag fusion protein in an Escherichia coli expression vector. A soluble protein of approximately 25 kDa purified from lysates of transformed E. coli was recognized in Western blot (WB) by anti-6xHis-tag and anti-BoHV-1 gE monoclonal antibodies. In addition, the recombinant protein was specifically recognized in WB by antibodies present in the sera of cattle seropositive to BoHV-1 and BoHV-5. An indirect ELISA using the expressed protein as coating antigen performed comparably to a commercial anti-gE ELISA and was able to differentiate serologically calves vaccinated with a gE-deleted BoHV-5 strain from calves infected with BoHV-1. Thus, the truncated gE may be useful for serological tests designed to differentiate BoHV-1/BoHV-5 infected animals from those vaccinated with gE-negative marker vaccines.

New species of Aulonemia and Chusquea (Poaceae: Bambusoideae: Bambuseae) from southeastern Brazil

Aulonemia fimbriatifolia and Chusquea longispiculata, two woody bamboo species from the Atlantic forest of southeastern Brazil, are described as new and key characters are illustrated. Aulonemia fimbriatifolia is compared and contrasted with four other similar species, but it is considered unique within the genus and possibly among Neotropical woody bamboos due to its basally fimbriate foliage leaf blades. Chusquea longispiculata shares extremely reduced glumes I and II and reflexed lower inflorescence branches with a number of other Brazilian species of the genus, but is distinguished based on its spikelets that reach nearly 2 cm in length, as well as several vegetative features.

Floristic diversity of two crystalline rocky outcrops in the Brazilian northeast semi-arid region

Floristic composition and structure of vegetation were studied in two rocky outcrop areas in the semi-arid region of northeastern Brazil. From April 2007 to September 2008, 18 monthly field trips were carried out. Vascular plants were randomly collected throughout the outcrop areas. For structural analysis, 30 plots of 1 × 1 m were set in the vegetation islands. The checklist presented combines 211 species (69 families and 168 genera), although only 56 species were collected in the plots. Fabaceae (18 spp.; 8.5%), Asteraceae (17 spp.; 8%), Orchidaceae (13 spp.; 6.1%), Euphorbiaceae (13 spp.; 6.1%), Bromeliaceae (10 spp.; 4.7%), and Poaceae (eight spp.; 3.8%) are the richest families. Overall, 1,792 shrub and herbaceous specimens were counted in the plots. The Shannon-Wiener (H) diversity index values were 2.572 and 2.547 nats individual-1. The species that presented the highest absolute abundance values (number of plants) had low frequencies in the plots and vice-versa. The biological spectrum had a high proportion of phanerophytes and therophytes, followed by cryptophytes, chamaephytes, and hemicryptophytes. The studied flora shares floristic components similar to other rocky outcrop areas of the semi-arid region in northeastern Brazil, including in relation to dominant groups in the vegetation structure.

Floristic composition of a conservation area in the Federal District of Brazil

Cerradão vegetation shares many species with savanna and forest areas and is one of the most vulnerable phytophysiognomies in the Cerrado (Brazilian savanna) biome. The floristic composition of the Cerradão Biological Reserve was examined between September/2007 and November/2008. A total of 282 species distributed among 194 genera and 75 families were encountered, demonstrating proportions of 0.91 herbaceous species and 0.54 shrub species for each tree species. Fabaceae, Asteraceae, Rubiaceae, Poaceae, Myrtaceae, Malpighiaceae, and Melastomataceae were the most species-rich families. Fully 72.3% of the species of this dystrophic cerradão were shared by cerrado and forest vegetations, while 60.43% were shared by other cerradão sites, although the largest proportion of species (91%) were shared with cerrado sensu stricto. No species was found to be exclusive to this cerradão site, but approximately 95% of all species were native to the Cerrado biome.

Molluscicidal activity of Punica granatum bark and Canna indica root

The molluscicidal activity of Punica granatum Linn. (Punicaceae) and Canna indica Linn. (Cannaceae) against the snail Lymnaea acuminata was studied. The molluscicidal activity of P. granatum bark and C. indica root was found to be both time and dose dependent. The toxicity of P. granatum bark was more pronounced than that of C. indica. The 24 h LC50 of the column-purified root of C. indica was 6.54 mg/l whereas that of the column-purified bark of P. granatum was 4.39 mg/l. The ethanol extract of P. granatum (24 h LC50: 22.42 mg/l) was more effective than the ethanol extract of C. indica (24 h LC50: 55.65 mg/l) in killing the test animals. P. granatum and C. indica may be used as potent molluscicides since the concentrations used to kill the snails were not toxic for the fish Colisa fasciatus, which shares the same habitat with the snail L. acuminata.

Identification of the divergent calmodulin binding motif in yeast Ssb1/Hsp75 protein and in other HSP70 family members

Yeast soluble proteins were fractionated by calmodulin-agarose affinity chromatography and the Ca2+/calmodulin-binding proteins were analyzed by SDS-PAGE. One prominent protein of 66 kDa was excised from the gel, digested with trypsin and the masses of the resultant fragments were determined by MALDI/MS. Twenty-one of 38 monoisotopic peptide masses obtained after tryptic digestion were matched to the heat shock protein Ssb1/Hsp75, covering 37% of its sequence. Computational analysis of the primary structure of Ssb1/Hsp75 identified a unique potential amphipathic alpha-helix in its N-terminal ATPase domain with features of target regions for Ca2+/calmodulin binding. This region, which shares 89% similarity to the experimentally determined calmodulin-binding domain from mouse, Hsc70, is conserved in near half of the 113 members of the HSP70 family investigated, from yeast to plant and animals. Based on the sequence of this region, phylogenetic analysis grouped the HSP70s in three distinct branches. Two of them comprise the non-calmodulin binding Hsp70s BIP/GR78, a subfamily of eukaryotic HSP70 localized in the endoplasmic reticulum, and DnaK, a subfamily of prokaryotic HSP70. A third heterogeneous group is formed by eukaryotic cytosolic HSP70s containing the new calmodulin-binding motif and other cytosolic HSP70s whose sequences do not conform to those conserved motif, indicating that not all eukaryotic cytosolic Hsp70s are target for calmodulin regulation. Furthermore, the calmodulin-binding domain found in eukaryotic HSP70s is also the target for binding of Bag-1 - an enhancer of ADP/ATP exchange activity of Hsp70s. A model in which calmodulin displaces Bag-1 and modulates Ssb1/Hsp75 chaperone activity is discussed.