Molecular mechanisms of attachment of Rhizobium bacteria to plant roots

Attachment of bacteria to plant cells is one of the earliest steps in many plant-bacterium interactions. This review covers the current knowledge on one of the best-studied examples of bacterium-plant attachment, namely the molecular mechanism by which Rhizobium bacteria adhere to plant roots. Despite differences in several studies with regard to growth conditions of bacteria and plants and to methods used for measuring attachment, an overall consensus can be drawn from the available data. Rhizobial attachment to plant root hairs appears to be a two-step process. A bacterial Ca(2+)-binding protein, designated as rhicadhesin, is involved in direct attachment of bacteria to the surface of the root hair cell. Besides this step, there is another step which results mainly in accumulation and anchoring of the bacteria to the surface of the root hair. This leads to so-called firm attachment. Depending on the growth conditions of the bacteria, the latter step is mediated by plant lectins and/or by bacterial appendages such as cellulose fibrils and fimbriae. The possible role of these adhesions in root nodule formation is discussed.     

Isolation and characterization of sexual sporulation mutants of Aspergillus nidulans.

For the genetic dissection of sexual sporulation in Aspergillus nidulans, we started a collection of ascosporeless mutants. After mutagenization of conidiospores with high doses of UV, we isolated 20 mutants with defects in ascospore formation. We crossed these mutants in two successive rounds with the wild-type strain. Eighteen of the 20 isolated mutants produced progeny with the original mutant phenotype in these crosses, and these mutants were further analyzed. All 18 analyzed mutations were recessive to wild type. We assigned them to 15 complementation groups, based on crosses between mutants. The mutants could be classified as follows according to their cytological phenotype: (1) no croziers, (2) arrest at prekaryogamy, (3) arrest in early meiotic prophase, (4) arrest in late meiotic prophase, (5) arrest in meiotic metaphase I, (6) defective postmeiotic mitosis and/or deliniation of ascospores, and (7) slow progression through the postmeiotic stages of ascospore formation. A large proportion of the mutants, namely 11 of 18, arrested in meiotic prophase or metaphase I. We discuss a possible approach for isolating the wild-type alleles of the genes that carry the sexual sporulation mutations.     

Are Existing Global Scenarios Consistent with Ecological Feedbacks?

Scenarios can help planners and decision makers to think through uncertainties about the future and make decisions that are robust to a variety of possible outcomes. To develop useful scenarios we need to understand the main processes of relevance to the system of interest. Ecological processes, and the feedbacks that they can create between human actions and human well-being, are thought to be important for human societies. Current uncertainties over the long-term resilience of ecosystems and the substitutability of ecosystem goods and services can be translated into three alternative realities: ecosystems may be relatively brittle, relatively resilient, or largely irrelevant. Although these extremes are only rough characterizations of reality, they help us to focus our thinking about the possible outcomes of interactions between humans and the rest of the biosphere. Existing global scenarios can be categorized into a small number of families based on shared themes and assumptions about the future. Considering the internal consistency of four of the main scenario families in relation to the three alternative ecological realities suggests that all existing scenarios make strong, implicit assumptions about the resilience of ecosystems. After a detailed discussion of individual examples, we present a synthesis of the incorporation of ecology in existing scenarios. All current scenarios are inconsistent with at least one possible property of ecosystems and their likely interaction with society. The interrelationships between ecological reality, human views of ecosystems, and social responses to actual and perceived ecological change are complex. For the Millennium Ecosystem Assessment and future scenario exercises, we recommend that essential ecological assumptions should be made explicit to ensure that the details of each scenario are consistent with both the perceived and the actual degree of resilience of ecosystems.     

Continuously improving the practice of cardiology

Guidelines for the management of patients with cardiovascular disease are designed to assist cardiologists and other physicians in their practice. Surveys are conducted to assess whether guidelines are followed in practice. The results of surveys on acute coronary syndromes, coronary revascularisation, secondary prevention, valvular heart disease and heart failure are presented. Comparing surveys conducted between 1995 and 2002, a gradual improvement in use of secondary preventive therapy is observed. Nevertheless, important deviations from established guidelines are noted, with a significant variation among different hospitals in the Netherlands and in other European countries. Measures for further improvement of clinical practice include more rapid treatment of patients with evolving myocardial infarction, more frequent use of clopidogrel and glycoprotein IIb/IIIa receptor blockers in patients with acute coronary syndromes, more frequent use of β-blockers in patients with heart failure and more intense measures to encourage patients to stop smoking. Targets for the proportion of patients who might receive specific therapies are presented.     

Two CYP17 genes in the South African Angora goat (Capra hircus) - the identification of three genotypes that differ in copy number and steroidogenic output

In mammals, cytochrome P450 17alpha-hydroxylase/17-20 lyase (CYP17), which is encoded by a single gene, plays a critical role in the production of mineralocorticoids, glucocorticoids and androgens by the adrenal cortex. Two CYP17 isoforms with unique catalytic properties have been identified in the South African Angora goat (Capra hircus), a subspecies that is susceptible to cold stress because of the inability of the adrenal cortex to produce sufficient levels of cortisol. A real-time-based genotyping assay was used in this study to identify the distribution of the two CYP17 alleles in the South African Angora population. These data revealed that the two CYP17 isoforms were not the product of two alleles of the same gene, but two separate CYP17 genes encoding the two unique CYP17 isoforms. This novel finding was subsequently confirmed by quantitative real-time PCR. Goats were divided into three unique genotypes which differed not only in the genes encoding CYP17, but also in copy number. Furthermore, in vivo assays revealed that the identified genotypes differed in their ability to produce cortisol in response to intravenous insulin injection. This study clearly demonstrates the presence of two CYP17 genes in the South African Angora goat, and further implicates CYP17 as the primary cause of the observed hypocortisolism in this subspecies.     

Flt3 ligand expands dendritic cell numbers in normal and malignant murine prostate

We have developed a murine model that facilitates the structural and functional analysis in vivo of dendritic cell (DC)-mediated phagocytosis of prostate epithelial cells. Recombinant human Flt3 ligand (rhFL) expands the number of dendritic cells in lymphoid and non-lymphoid tissues of mice. We show that rhFL also induced the ingress of dendritic cells into murine prostate, which involutes via epithelial apoptosis after surgical castration. Intact or castrated C57BL/6 and syngeneic transgenic adenocarcinoma of mouse prostate (TRAMP) mice were treated with rhFL or PBS control. Prostate and spleen were then studied by flow cytometry and immunohistochemistry.The number of prostatic CD11c+ and CD11b+ dendritic cells increased significantly in rhFL-treated mice compared with PBS-treated control mice and this effect was greatly augmented by castration of the mice. The immunophenotype of rhFL-mobilized prostatic cells was consistent with that of Langerhans cells (MHC class II+, CD11c+,CD11b+, DEC-205+, CD8 alpha-).MHC class II+ and CD11c+ dendritic cells that were present in the prostate glands of rhFL-treated and castrated C57BL/6 mice were intimately associated with TUNEL+ inclusions, which suggests that Langerhans-type dendritic cells in prostate participated in the clearance of apoptotic cells. Expression of MHC class II, CD54, CD80 and CD86 by prostatic dendritic cells was not up-regulated after castration and freshly isolated rhFL-induced prostate cells were unable to prime allogeneicT cells unless they were activated by culture either on plastic or with recombinant soluble CD40 ligand. Our data suggest that rhFL-mobilized prostatic dendritic cells resemble the functionally immature dendritic cells, which reside in peripheral tissues and contribute to the maintenance of peripheral tolerance.     

Immunization of macaques with formalin-inactivated respiratory syncytial virus (RSV) induces interleukin-13-associated hypersensitivity to subsequent RSV infection

Respiratory syncytial virus (RSV) is a major cause of severe respiratory disease in infants and the elderly. RSV vaccine development has been hampered by results of clinical trials in the 1960s, when formalin-inactivated whole-RSV preparations adjuvated with alum (FI-RSV) were found to predispose infants for enhanced disease following subsequent natural RSV infection. We have reproduced this apparently immunopathological phenomenon in infant cynomolgus macaques and identified immunological and pathological correlates. Vaccination with FI-RSV induced specific virus-neutralizing antibody responses accompanied by strong lymphoproliferative responses. The vaccine-induced RSV-specific T cells predominantly produced the Th2 cytokines interleukin-13 (IL-13) and IL-5. Intratracheal challenge with a macaque-adapted wild-type RSV 3 months after the third vaccination elicited a hypersensitivity response associated with lung eosinophilia. The challenge resulted in a rapid boosting of IL-13-producing T cells in the FI-RSV-vaccinated animals but not in the FI-measles virus-vaccinated control animals. Two out of seven FI-RSV-vaccinated animals died 12 days after RSV challenge with pulmonary hyperinflation. Surprisingly, the lungs of these two animals did not show overt inflammatory lesions. However, upon vaccination the animals had shown the strongest lymphoproliferative responses associated with the most pronounced Th2 phenotype within their group. We hypothesize that an IL-13-associated asthma-like mechanism resulted in airway hyperreactivity in these animals. This nonhuman primate model will be an important tool to assess the safety of nonreplicating candidate RSV vaccines.     

Determination of carbamazepine and carbamazepine 10,11-epoxide in human plasma by tandem liquid chromatography-mass spectrometry with electrospray ionisation

A sensitive method for the determination of carbamazepine and carbamazepine 10,11-epoxide in plasma is described, using high-performance liquid chromatographic separation with tandem mass spectrometry. Samples were purified using liquid-liquid extraction and separated on a Phenomenex Luna C18 5 microm. 150 x 2 mm column with a mobile phase consisting of acetonitrile, methanol and formic acid (0.1%) (10:70:20, v/v). Detection was performed by a Micromass Quattro Ultima mass spectrometer in the MRM mode (LC-MS-MS) using electro spray ionisation (ESI+), monitoring the transition of the protonated molecular ion for carbamazepine at m/z 237.05 and carbamazepine 10,11-epoxide at m/z 253.09 to the predominant ions of m/z 194.09 and 180.04, respectively. The mean recovery was 95% for carbamazepine and 101% for carbamazepine 10,11-epoxide, with a lower limit of quantification of 0.722 ng/ml for carbamazepine and 5.15 ng/ml for carbamazepine 10,11-epoxide, when using 0.5 ml plasma. This high-throughput method was used to quantify 230 samples per day, and is sufficiently sensitive to be employed in pharmacokinetic studies.     

Sensitive and rapid liquid chromatography-tandem mass spectrometry method for the determination of stavudine in human plasma

A sensitive method for the determination of stavudine in plasma was developed, using high-performance liquid chromatographic separation with tandem mass spectrometric detection. The samples were extracted from plasma with Waters, Sep-Pak Vac, 100 mg, tC(18) solid-phase extraction (SPE) columns. Chromatography was performed on a Supelco Discovery C(18), 5 microm, 150 x 2 mm column with a mobile phase consisting of ammonium acetate (0.01 M)-acetonitrile-methanol (800:100:100, v/v/v) at a flow-rate of 0.3 ml/min. Detection was achieved by an Applied Biosystems API 2000 mass spectrometer (LC-MS-MS) set at unit resolution in the multiple reaction monitoring mode (MRM). Atmospheric pressure chemical ionization (APCI) was used for ion production. The mean recovery for stavudine was 94% with a lower limit of quantification set at 4 ng/ml. This assay method makes use of the increased sensitivity and selectivity of mass spectrometric (MS-MS) detection to allow for a more rapid (extraction and chromatography) and selective method for the determination of stavudine in human plasma than has previously been described.