REGULATION OF CHAMISE SHOOT GROWTH

Nutritional, hormonal, and environmental control of chamise (Adenostoma fasciculatum H. & A.) shoot growth was investigated. In vitro culture of shoot tips demonstrated that 0.18 M sucrose was required for optimum apical growth. Cytokinin (benzyladenine) promoted shoot growth at otherwise growth-limiting sucrose concentrations and induced uptake of sucrose from the basal medium. Abscisic acid inhibited growth of cultured shoot tips induced by high sucrose concentration or cytokinin. In the field, inhibition of shoot growth was a function of water stress. These studies indicate that the effects of water stress on chamise shoot growth may be mediated by changes in carbohydrate, cytokinin, or growth inhibitor levels at the shoot apex.     

Metabolism of Atrazine in Liquid Cultures and Soil Microcosms by Nocardioides Strains Isolated from a Contaminated Nigerian Agricultural Soil

Atrazine-degrading microorganisms designated EAA-3 and EAA-4, belonging to the genus Nocardioides, were obtained from an agricultural soil in Nigeria. The degradation kinetics of the two strains revealed total disappearance of 25 mg l^?1 of atrazine in less than 72 h of incubation at the rate of 0.42 mg l^?1 h^?1 and 0.35 mg l^?1 h^?1, respectively. Screening for atrazine catabolic genes in these organisms revealed the presence of trzN, atzB, and atzC. Other genes, specifically atzA, atzD, and trzD, were not detected. Potential intermediates of atrazine catabolic route such as hydroxyatrazine, desethylatrazine, and desisopropylatrazine were utilized as sources of carbon and energy, while desisopropyl desethyl-2-hydroxyatrazine and desisopropyl-2-hydroxyatrazine were attacked but in the presence of glucose. A soil microcosm study showed that degradation was faster in microcosms contaminated with 13 mg of atrazine per g^?1 of soil compared with 480 mg g^?1 of soil. In the former, degradation was 10% higher in the inoculated soil than the non-inoculated control (natural attenuation) over the 28-day study period. Corresponding value obtained for the latter was nearly 70% higher. This study has demonstrated that the bacterial strains isolated enhanced atrazine degradation and the catabolic activities of these strains were not affected with increasing soil atrazine concentration.     

Development of a high-volume aerosol collection system for the identification of air-borne micro-organisms

AIMS: A high-volume aerosol collector was developed to efficiently capture airborne bacteria in order to assess levels of diversity in the air. METHODS AND RESULTS: Particulate matter was collected on a device designed to filter 1.4 x 10(6) litres of air in a 24 h period on a 1-microm pore size polyester membrane. Methods were optimized for extraction of genomic DNA from the air filter concentrate. Preparation times of 90 s with 0.5-0. 05 mm diameter zirconia/silica beads yielded the highest concentration genomic DNA that was able to support PCR. A 24-h air sample was taken in Salt Lake City, Utah and the microbial composition was determined by the amplification and sequence analysis of 16S ribosomal DNA fragments. CONCLUSIONS: Sequence analysis revealed a large diversity in the type of microbial species present including clones matching the sequence of Clostridium botulinum. The primary components of the aerosol sample included many different spore-forming bacteria as well as more fragile members of the Proteobacteria division. SIGNIFICANCE AND IMPACT OF STUDY: The high-volume air collection and genomic DNA recovery system allows for the rapid detection of both cultivable as well as culture-resistant organisms in the environment.     

Chromatographic purification and properties of a therapeutic human protein C concentrate

Protein C deficiency (inherited and acquired) has a relatively high incidence rate in the general population worldwide. For many years, protein C deficient patients have been treated with fresh frozen plasma, prothrombin complex concentrates, heparin or oral anticoagulants, which all have clinical drawbacks. We report the production process of a highly purified human protein C concentrate from 1500 l of cryo-poor plasma by a four-step chromatographic procedure. After DEAE-Sephadex adsorption, protein C was separated from clotting factors II, VII and IX by DEAE-Sepharose FF and further purified, using a new strategy, by an on-line chromatographic system combining DMAE-Fractogel and heparin-Sepharose CL-6B. In addition, the product was treated against viral risks by solvent-detergent and nanofiltration on 15-nm membranes. The protein C concentrate was essentially free of other vitamin K-dependent proteins. Proteolytic activity was undetectable. Neither activated protein C, prekallikrein activator, nor activated vitamin K-dependent clotting factors were found resulting in good stability of the protein C activity. In vitro and in vivo animal tests did not reveal any sign of potential thrombogenicity. The final freeze-dried product had a mean protein C concentration of 58 IU/ml and a mean specific activity of 215 IU/mg protein, corresponding to over 12000-fold purification from plasma. Therefore, this concentrate appears to be of potential benefit for the treatment of protein C deficiency.     

End-binding protein 1 (EB1) up-regulation is an early event in colorectal carcinogenesis

End-binding protein (EB1) is a microtubule protein that binds to the tumor suppressor adenomatous polyposis coli (APC). While EB1 is implicated as a potential oncogene, its role in cancer progression is unknown. Therefore, we analyzed EB1/APC expression at the earliest stages of colorectal carcinogenesis and in the uninvolved mucosa (“field effect”) of human and animal tissue. We also performed siRNA-knockdown in colon cancer cell lines. EB1 is up-regulated in early and field carcinogenesis in the colon, and the cellular/nano-architectural effect of EB1 knockdown depended on the genetic context. Thus, dysregulation of EB1 is an important early event in colon carcinogenesis.     

Synthesis and characterization of new porphyrazine-Gd(III) conjugates as multimodal MR contrast agents

Magnetic resonance imaging (MRI) has long been used clinically and experimentally as a diagnostic tool to obtain three-dimensional, high-resolution images of deep tissues. These images are enhanced by the administration of contrast agents such as paramagnetic Gd(III) complexes. Herein, we describe the preparation of a series of multimodal imaging agents in which paramagnetic Gd(III) complexes are conjugated to a fluorescent tetrapyrrole, namely, a porphyrazine (pz). Zinc metalated pzs conjugated to one, four, or eight paramagnetic Gd(III) complexes are reported. Among these conjugates, Zn-Pz-8Gd(III) exhibits an ionic relaxivity four times that of the monomeric Gd(III) agent, presumably because of increased molecular weight and a molecular relaxivity that is approximately thirty times larger, while retaining the intense electronic absorption and emission of the unmodified pz. Unlike current clinical MR agents, Zn-Pz-1Gd(III) is taken up by cells. This probe demonstrates intracellular fluorescence by confocal microscopy and provides significant contrast enhancement in MR images, as well as marked phototoxicity in assays of cellular viability. These results suggest that pz agents possess a new potential for use in cancer imaging by both MRI and near-infrared (NIR) fluorescence, while acting as a platform for photodynamic therapy.     

Induced root-secreted phenolic compounds as a belowground plant defense

Rhizosphere is the complex place of numerous interactions between plant roots, microbes and soil fauna. Whereas plant interactions with aboveground organisms are largely described, unravelling plant belowground interactions remains challenging. Plant root chemical communication can lead to positive interactions with nodulating bacteria, mycorriza or biocontrol agents or to negative interactions with pathogens or root herbivores. A recent study¹ suggested that root exudates contribute to plant pathogen resistance via secretion of antimicrobial compounds. These findings point to the importance of plant root exudates as belowground signalling molecules, particularly in defense responses. In our report,² we showed that under Fusarium attack the barley root system launched secretion of phenolic compounds with antimicrobial activity. The secretion of de novo biosynthesized t-cinnamic acid induced within 2 days illustrates the dynamic of plant defense mechanisms at the root level. We discuss the costs and benefits of induced defense responses in the rhizosphere. We suggest that plant defense through root exudation may be cultivar dependent and higher in wild or less domesticated varieties.Key words: root exudates, plant defense, t-cinnamic acid, fusarium, induced defensePlants grow and live in very complex and changing ecosystems. Because plants lack the mobility to escape from attack by pathogens or herbivores, they have developed constitutive and in addition inducible defenses that are triggered by spatiotemporally dynamic signaling mechanisms. These defenses counteract the aggressor directly via toxins or defense plant structures or indirectly by recruitment of antagonists of aggressors. Whereas induced defenses are well described in aboveground interactions, evidence of the occurrence of such mechanisms in belowground interactions remains limited. The biosynthesis of a defensive molecule could be both constitutive and inducible with a low level of a preformed pool (Fig. 1). In addition, upon encounter of an attacking organism, those levels could be induced to rise locally to a high level of active compound that is able to disarm the pathogen.^2,3 Only a few examples show that root exudates play a role in induced plant defense. Hairy roots of Ocimum basilicum secrete rosmarinic acid only when challenged by the pathogenic fungus Pythium ultimum.⁴ Wurst et al.⁵ reported on the induction of irridoid glycosides in root exudates of Plantago lanceolata in presence of nematodes. In vivo labelling experiments² with ¹³CO₂ showed the induction of phenolic compounds secreted by barley roots after Fusarium graminearum infection and the de novo biosynthesis of root secreted t-cinnamic acid within 2 days. These results show that the pool of induced t-cinnamic acid originated from both pre-formed and newly formed carbon pools (Fig. 1), highlighting a case of belowground induced defense inside and outside the root system.Open in a separate windowFigure 1Suggested mechanisms for the induction of root defense exudates in barley in response to Fusarium attack. Upon pathogen attack by Fusarium, the initial preformed pool of phenolic compounds is increased by the addition of inducible, de novo biosynthesized t-cinnamic acid. Both, the preformed pool and the de novo biosynthesized pool fuel the exudation of defense compounds from infected roots.The concept of fitness costs is frequently presented to explain the coexistence of both constitutive and induced defense.⁶ In the case of induced defense, resources are invested in defenses only when the plant is under attack. In the absence of an infection, plants can optimize allocation of their resources to reproduction and growth to compete with neighbours.⁷ Constitutive defenses are thought to be more beneficial when the probability of attack is high, whereas adjustable, induced defenses are more valuable to fight against an unpredictable pathogen. Non disturbed soil is a heterogeneous matrix where biodiversity is very high and patchy^8,9 and organism motility is rather restricted.¹⁰ As a consequence of the patchiness, belowground environment is expected to be favourable to selection for induced responses.¹¹ The absence of defense root exudates between two infections may form an unpredictable environment for soil pathogens and reduce the chance for adaptation of root attackers. Plants may also use escape strategies to reduce the effect of belowground pathogens. Henkes et al. (unpublished) showed that Fusarium-infected barley plants reduced carbon allocation towards infected roots within a day and increased allocation carbon to uninfected roots. These results illustrate how reallocation of carbon toward non infected root parts represents a way to limit the negative impact of root infection.We have demonstrated the potential of barley plants to defend themselves against soil pathogen by root exudation.² Even the barley cultivar ‘Barke’ used in our study, a modern cultivated variety, was able to launch defense machinery via exudation of antimicrobial compounds when infected by F. graminearum. We suggest that plant defense through root exudation might be cultivar dependent and perhaps higher in wild or less domesticated varieties. Taddei et al.¹² reported that constitutivelyproduced root exudates from a resistant Gladiolus cultivar inhibit spore germination of Fusarium oxysporum whereas root exudates from a susceptible cultivar do not affect F. oxysporum germination. Root exudates from the resistant cultivar contained higher amounts of aromaticphenolic compounds compared to the susceptible cultivar and these compounds may be responsible for the inhibition of spore germination. Metabolic profiling of wheat cultivars, ‘Roblin’ and ‘Sumai3’, respectively, susceptible and resistant to Fusarium Head Blight, showed that t-cinnamic acid was a discriminating factor responsible for resistance/defense function.¹³ Therefore it is likely that wild barley varieties hold higher defense capacities compare to cultivated varieties selected for high yield. In the future, plant breeders in organic and low-input farming could use root-system defense ability as new trait in varietal variation.     

Optimisation of a quantitative polymerase chain reaction-based strategy for the detection and quantification of human herpesvirus 6 DNA in patients undergoing allogeneic haematopoietic stem cell transplantation

Human herpesvirus 6 (HHV-6) may cause severe complications after haematopoietic stem cell transplantation (HSCT). Monitoring this virus and providing precise, rapid and early diagnosis of related clinical diseases, constitute essential measures to improve outcomes. A prospective survey on the incidence and clinical features of HHV-6 infections after HSCT has not yet been conducted in Brazilian patients and the impact of this infection on HSCT outcome remains unclear. A rapid test based on real-time quantitative polymerase chain reaction (qPCR) has been optimised to screen and quantify clinical samples for HHV-6. The detection step was based on reaction with TaqMan^® hydrolysis probes. A set of previously described primers and probes have been tested to evaluate efficiency, sensitivity and reproducibility. The target efficiency range was 91.4% with linearity ranging from 10-10⁶ copies/reaction and a limit of detection of five copies/reaction or 250 copies/mL of plasma. The qPCR assay developed in the present study was simple, rapid and sensitive, allowing the detection of a wide range of HHV-6 loads. In conclusion, this test may be useful as a practical tool to help elucidate the clinical relevance of HHV-6 infection and reactivation in different scenarios and to determine the need for surveillance.     

Association of 25-Hydroxyvitamin D status and genetic variation in the vitamin D metabolic pathway with FEV1 in the Framingham Heart Study

Background

Vitamin D is associated with lung function in cross-sectional studies, and vitamin D inadequacy is hypothesized to play a role in the pathogenesis of chronic obstructive pulmonary disease. Further data are needed to clarify the relation between vitamin D status, genetic variation in vitamin D metabolic genes, and cross-sectional and longitudinal changes in lung function in healthy adults.

Methods

We estimated the association between serum 25-hydroxyvitamin D [25(OH)D] and cross-sectional forced expiratory volume in the first second (FEV₁) in Framingham Heart Study (FHS) Offspring and Third Generation participants and the association between serum 25(OH)D and longitudinal change in FEV₁ in Third Generation participants using linear mixed-effects models. Using a gene-based approach, we investigated the association between 241 SNPs in 6 select vitamin D metabolic genes in relation to longitudinal change in FEV₁ in Offspring participants and pursued replication of these findings in a meta-analyzed set of 4 independent cohorts.

Results

We found a positive cross-sectional association between 25(OH)D and FEV₁ in FHS Offspring and Third Generation participants (P = 0.004). There was little or no association between 25(OH)D and longitudinal change in FEV₁ in Third Generation participants (P = 0.97). In Offspring participants, the CYP2R1 gene, hypothesized to influence usual serum 25(OH)D status, was associated with longitudinal change in FEV₁ (gene-based P < 0.05). The most significantly associated SNP from CYP2R1 had a consistent direction of association with FEV₁ in the meta-analyzed set of replication cohorts, but the association did not reach statistical significance thresholds (P = 0.09).

Conclusions

Serum 25(OH)D status was associated with cross-sectional FEV₁, but not longitudinal change in FEV₁. The inconsistent associations may be driven by differences in the groups studied. CYP2R1 demonstrated a gene-based association with longitudinal change in FEV₁ and is a promising candidate gene for further studies.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-015-0238-y) contains supplementary material, which is available to authorized users.     

WATER RELATIONS OF TWO CALIFORNIA CHAPARRAL SHRUBS

Water use patterns of two California chaparral shrub species, chamise (Adenostoma fasciculatum H. and A.) and Stanford manzanita (Arctostaphylos stanfordiana Parry), were compared during summer drought. Observations of diurnal and seasonal courses of shoot water potential, leaf conductance and transpiration revealed that chamise was more conservative in water use than manzanita. Evidence obtained cast doubt on a hypothesis previously proposed to explain an anomalous pattern of shoot water potential in chamise.