Eicosanoid biosynthesis in marine mammals

After 300 million years of evolution, the first land-living mammals reentered the marine environment some 50 million years ago. The driving forces for this dramatic lifestyle change are still a matter of discussion but the struggle for food resources and the opportunity to escape predators probably contributed. Reentering the oceans requires metabolic adaption putting evolutionary pressure on a number of genes. To explore whether eicosanoid signaling has been part of this adaptive response, we first explored whether the genomes of marine mammals involve functional genes encoding for key enzymes of eicosanoid biosynthesis. Cyclooxygenase (COX) and lipoxygenase (ALOX) genes are present in the genome of all marine mammals tested. Interestingly, ALOX12B, which has been implicated in skin development of land-living mammals, is lacking in whales and dolphins and genes encoding for its sister enzyme (ALOXE3) involve premature stop codons and/or frameshifting point mutations, which interrupt the open reading frames. ALOX15 orthologs have been detected in all marine mammals, and the recombinant enzymes exhibit similar catalytic properties as those of land-living species. All marine mammals express arachidonic acid 12-lipoxygenating ALOX15 orthologs, and these data are consistent with the Evolutionary Hypothesis of ALOX15 specificity. These enzymes exhibit membrane oxygenase activity and introduction of big amino acids at the triad positions altered the reaction specificity in favor of arachidonic acid 15-lipoxygenation. Thus, the ALOX15 orthologs of marine mammals follow the Triad concept explaining their catalytic specificity

Functional characterization of a novel arachidonic acid 12S-lipoxygenase in the halotolerant bacterium Myxococcus fulvus exhibiting complex social living patterns

Lipoxygenases are lipid peroxidizing enzymes, which frequently occur in higher plants and mammals. These enzymes are also expressed in lower multicellular organisms but here they are not widely distributed. In bacteria, lipoxygenases rarely occur and evaluation of the currently available bacterial genomes suggested that <0.5% of all sequenced bacterial species carry putative lipoxygenase genes. We recently rescreened the public bacterial genome databases for lipoxygenase‐like sequences and identified two novel lipoxygenase isoforms (MF‐LOX1 and MF‐LOX2) in the halotolerant Myxococcus fulvus. Both enzymes share a low degree of amino acid conservation with well‐characterized eukaryotic lipoxygenase isoforms but they involve the catalytically essential iron cluster. Here, we cloned the MF‐LOX1 cDNA, expressed the corresponding enzyme as N‐terminal hexa‐his‐tag fusion protein, purified the recombinant enzyme to electrophoretic homogeneity, and characterized it with respect to its protein‐chemical and enzymatic properties. We found that M. fulvus expresses a catalytically active intracellular lipoxygenase that converts arachidonic acid and other polyunsaturated fatty acids enantioselectively to the corresponding n‐9 hydroperoxy derivatives. The enzyme prefers C20‐ and C22‐polyenoic fatty acids but does not exhibit significant membrane oxygenase activity. The possible biological relevance of MF‐LOX1 will be discussed in the context of the suggested concepts of other bacterial lipoxygenases

Male guanine-rich RNA sequence binding factor 1 knockout mice (Grsf1−/−) gain less body weight during adolescence and adulthood

The guanine-rich RNA sequence binding factor 1 (GRSF1) is an RNA-binding protein of the heterogenous nuclear ribonucleoprotein H/F (hnRNP H/F) family that binds to guanine-rich RNA sequences forming G-quadruplex structures. In mice and humans there are single copy GRSF1 genes, but multiple transcripts have been reported. GRSF1 has been implicated in a number of physiological processes (e.g. embryogenesis, erythropoiesis, redox homeostasis, RNA metabolism) but also in the pathogenesis of viral infections and hyperproliferative diseases. These postulated biological functions of GRSF1 originate from in vitro studies rather than complex in vivo systems. To assess the in vivo relevance of these findings, we created systemic Grsf1(-/-) knockout mice lacking exons 4 and 5 of the Grsf1 gene and compared the basic functional characteristics of these animals with those of wildtype controls. We found that Grsf1-deficient mice are viable, reproduce normally and have fully functional hematopoietic systems. Up to an age of 15 weeks they develop normally but when male individuals grow older, they gain significantly less body weight than wildtype controls in a gender-specific manner. Profiling Grsf1 mRNA expression in different mouse tissues we observed high concentrations in testis. Comparison of the testicular transcriptomes of Grsf1(-/-) mice and wildtype controls confirmed near complete knock-out of Grsf1 but otherwise subtle differences in transcript regulations. Comparative testicular proteome analyses suggested perturbed mitochondrial respiration in Grsf1(-/-) mice which may be related to compromised expression of complex I proteins. Here we present, for the first time, an in vivo complete Grsf1 knock-out mouse with comprehensive physiological, transcriptomic and proteomic characterization to improve our understanding of the GRSF1 beyond in vitro cell culture models

Atopic Patients Show Increased Interleukin 4 Plasma Levels but the Degree of Elevation Is Not Sufficient to Upregulate Interleukin-4-Sensitive Genes

Background: Atopic diseases constitute a major health challenge for industrialized countries, and elevated levels of interleukin 4 (IL-4) frequently characterize these disorders. Previous in vitro analyses have indicated that IL-4 strongly upregulates the expression of IL-4-sensitive genes in human monocytes. Objective: To explore whether similar expression alterations may contribute to the pathomechanisms of atopic diseases in vivo we carried out a small-scale case-control clinical study (n = 43), in which we quantified the plasma levels of IgE and IL-4 as well as the expression of selected IL4- sensitive genes in blood leukocytes. Methods: 34 allergic patients suffering from allergic rhinitis (n = 11), atopic eczema (n = 11) and allergic asthma (n = 12) as well as 9 healthy control individuals were recruited. IgE and IL-4 plasma levels were determined by ELISA, and the expression of selected IL-4-sensitive gene products in blood leukocytes was quanti-fied by qRT-PCR. In addition, the fatty acid oxygenase activity of isolated monocytes was measured by RP-HPLC analysis of the arachidonic acid oxygenation products (ex vivo activity assays). Results: We found that plasma levels of IgE and IL-4 were significantly elevated in atopic patients but the degree of elevation was not sufficient to upregulate the expression of the selected IL-4-sensitive genes in circulating leukocytes. Moreover, the arachidonic acid oxygenase activity of blood monocytes was not significantly altered in atopic patients. Conclusion: Our data suggest that the IL-4 plasma levels of atopic patients are not high enough to impact the expression of IL-4-sensitive genes

The Reaction Specificity of Mammalian ALOX15 Orthologs is Changed During Late Primate Evolution and These Alterations Might Offer Evolutionary Advantages for Hominidae

Arachidonic acid lipoxygenases (ALOXs) have been implicated in the immune response of mammals. The reaction specificity of these enzymes is decisive for their biological functions and ALOX classification is based on this enzyme property. Comparing the amino acid sequences and the functional properties of selected mammalian ALOX15 orthologs we previously hypothesized that the reaction specificity of these enzymes can be predicted based on their amino acid sequences (Triad Concept) and that mammals, which are ranked in evolution below gibbons, express arachidonic acid 12-lipoxygenating ALOX15 orthologs. In contrast, Hominidae involving the great apes and humans possess 15-lipoxygenating enzymes (Evolutionary Hypothesis). These two hypotheses were based on sequence data of some 60 mammalian ALOX15 orthologs and about half of them were functionally characterized. Here, we compared the ALOX15 sequences of 152 mammals representing all major mammalian subclades expressed 44 novel ALOX15 orthologs and performed extensive mutagenesis studies of their triad determinants. We found that ALOX15 genes are absent in extant Prototheria but that corresponding enzymes frequently occur in Metatheria and Eutheria. More than 90% of them catalyze arachidonic acid 12-lipoxygenation and the Triad Concept is applicable to all of them. Mammals ranked in evolution above gibbons express arachidonic acid 15-lipoxygenating ALOX15 orthologs but enzymes with similar specificity are only present in less than 5% of mammals ranked below gibbons. This data suggests that ALOX15 orthologs have been introduced during Prototheria-Metatheria transition and put the Triad Concept and the Evolutionary Hypothesis on a much broader and more reliable experimental basis

First record of Dothistroma needle blight (Dothistroma septosporum) in the northeast German lowlands

Im April 2015 wurde in einem brandenburgischen Arboretum eine rötliche Bänderung an vorjährigen Nadeln von Pinus jeffreyi Balf. (Jeffrey-Kiefer) und Pinus ponderosa Douglas ex C. Lawson (Gelb-Kiefer) festgestellt. Später konnte diese Symptomatik bei systematischen Kon­trollen auch an Pinus attenuata Lemmon (Höcker-Kiefer) und Pinus thunbergii Parl. (Japanische Schwarz-Kiefer) beobachtet werden. Anhand von mikromorphologischen Untersuchungen und laborativen Analysen bestätigte sich der Verdacht auf eine Infektion durch den Quarantäneschadpilz Dothistroma septosporum (Dorogin) M. More­let (Erreger der Dothistroma-Nadelbräune). Erkrankt waren ausschließlich jüngere Bäume in einem Geländebereich mit anhaltend hoher Luftfeuchtigkeit. Der weltweit vorkommende Krankheitserreger befällt überwiegend Kiefern-Arten (Pinus spp.). Gravierende Schäden verursachte er bislang speziell auf der Südhalbkugel. In letzter Zeit konnte Dothistroma septosporum allerdings auch in einigen europäischen Ländern bemerkenswert oft nachgewiesen werden. Möglicherweise geht die verstärkte Präsenz des Pilzes nördlich des Äquators auf die sich seit einigen Jahrzehnten abzeichnende Klimaveränderung zurück. Da der Krankheitserreger imstande ist, auch an der in Europa heimischen Schwarz-Kiefer (Pinus nigra J.F. Arnold) umfangreiche Schäden hervorzurufen, resultieren aus dem Vorkommen forstwirtschaftliche Risi­ken. Zudem ist unklar, welche Intensität ein Befall von Reinbeständen der Gemeinen Kiefer (Pinus sylvestris L.) erreichen würde.In April 2015, reddish banding at last year's needles of Pinus jeffreyi Balf. (Jeffrey pine) and Pinus ponderosa Douglas ex C. Lawson (Ponderosa Pine) was found in an Arboretum in the federal state of Brandenburg (Germany). These symptoms were observed in systematic controls at Pinus attenuata Lemmon (Knobcone pine) and Pinus thunbergii Parl. (Japanese black pine) later too. Based on micromorphological investigations and laborative tests, the suspected infection by the quarantine fungus Dothi­stroma septosporum (Dorogin) M. Morelet (causative agent of Dothistroma needle blight) was confirmed. Only younger trees in an area with persistently high air humidity were diseased.The worldwide occurring pathogen infects mainly pine species (Pinus spp.). It caused serious damage especially in the southern hemisphere until now. Lately however Dothistroma septosporum was remarkably often detected in several European countries. The increased presence of the fungus north of the Equator is supposed to originate in the recognizable climate change of the last few deca­des. The pathogen is capable of damaging the native to Europe Black pine (Pinus nigra J.F. Arnold) considerably. Therefore forestry risks result. In addition, it is unclear, what intensity an infestation of pure stands of Scots pine (Pinus sylvestris L.) would reach

Virus del síndrome reproductor y respiratorio porcino (PRRSV) en granjas porcinas tecnificadas del Estado de México

Se realizó un estudio transversal por conglomerados en una etapa, de abril a septiembre de 2011, en cerdos de más de 11 semanas de edad, de 11 granjas, para analizar la situación de PRRSV en granjas tecnificadas del Estado de México. Se colectaron muestras de suero (n=220) para ELISA y sangre (n=80) para transcripción en reversa y reacción en cadena de la polimerasa en tiempo real en un solo paso (RTqPCR) para PRRSV, e hisopos nasales (n= 425) para el aislamiento de posibles bacterias patógenas de la enfermedad del complejo respiratorio porcino (CRP). También se aplicó un cuestionario. ELISA mostró una seroprevalencia verdadera de 30.67 %, siendo mayor la probabilidad de ser seropositivos en granjas con antecedentes y calendario de vacunación y de ubicación urbana-semiurbana; los cerdos fueron seroreactivos en 7/11 granjas (63.63 %). RTqPCR mostró viremia en 2/80 (2.5 %) cerdos analizados, de 2/11 granjas (18.18 %); una cerda infectada con PRRSV norteamericano que mostró signos clínicos de la enfermedad y una cerda infectada con PRRSV europeo que no mostró ningún signo clínico evidente de infección. Dos granjas no mostraron cerdos seropositivos o virémicos, no tienen antecedentes ni calendario de vacunación y están ubicadas en una zonasemirural. En todas las granjas se aisló Staphyloccus aureus y Streptococcus suis; en algunas granjas y con menos frecuencia Actinobacillus spp (4/11) y Pasteurella spp (3/11). Es el primer reporte en México de infección por PRRSV europeo

Lipofection improves gene targeting efficiency in E14 TG2a mouse embryonic stem cells

Electroporation has been the method of election for transfection of murine embryonic stem cells for over 15 years; however, it is a time consuming protocol because it requires large amounts of DNA and cells, as well as expensive and delicate equipment. Lipofection is a transfection method that requires lower amounts of cells and DNA than electroporation, and has proven to be effi cient in a large number of cell lines. It has been shown that after lipofection, mouse embryonic stem cells remain pluripotent, capable of forming germ line chimeras and can be transfected with greater effi ciency than with electroporation; however, gene targeting of mouse embryonic stem cells by lipofection has not been reported. The objective of this work was to fi nd out if lipofection can be used as effi ciently as electroporation for regular gene targeting protocols. This context compares gene targeting effi ciency between these techniques in mouse embryonic stem cells E14TG2a, using a gene replacement type vector. No differences were found in gene targeting effi ciency between groups; however, lipofection was three times more effi cient than electroporation in transfection effi ciency, which makes lipofection a less expensive alternative method to produce gene targeting in mouse embryonic stem cells

Prevalence and molecular identification of Chlamydia abortus in commercial dairy goat farms in a hot region in Mexico

The aim of this study was to determine the seroprevalence and presence of Chlamydia abortus in Saanen breed female goats from commercial dairy goat farms under intensive production in the municipality of Guanajuato, Mexico. Sera were collected to determine the prevalence of anti-C. abortus IgG antibodies using recombinant enzymelinked immunosorbent assay (rELISA) and cell culture. Polymerase chain reaction (PCR) was used to prove the presence of the pathogen in swab samples collected from the vagina and rectum of selected animals. Additionally, foetal tissue samples from a sudden abortion were collected. C. abortus prevalence in female goats of commercial milking farms sampled in Guanajuato, Mexico, was 4.87 % (n=246). Seropositive animals were found in six out of nine (66.6 %) dairy goat farms sampled, and prevalence among animals in individual farms ranged between 3.44 and 13.51 %. C. abortus was detected using PCR in spleen tissue from the aborted foetus. PCR-based detection, as well as isolation from vaginal and rectal swabs, was not possible in the present study. Isolation through cell culture was also unsuccessful from aborted foetal tissue samples. In conclusion, the results from rELISA and PCR show that C. abortus is present in dairy goat farms in the state of Guanajuato, Mexico