A rapid mini-prep method for isolation of ectomycorrhizal DNA from Tuber species

A rapid procedure has been developed to isolate DNA from the ectomycorrhizae of Tuber spp. for use in PCR experiments. The method described is fast and sensitive and can overcome the amplification problems that can arise in the presence of inhibitors. For this reason it can be used to type ectomycorrhizae even starting from a single root tip and make mycorrhizae identification much more rapid.     

Angiopoietin decoy secreted at tumor site impairs tumor growth and metastases by inducing local inflammation and altering neoangiogenesis

The extracellular domain of the receptor tyrosine kinase Tie2/TEK (exTEK) has been used as an angiopoietin decoy to study the role of angiopoietins in the tumor–host interactions, using a syngeneic model of experimental metastases and subcutaneous tumor. Soluble exTEK secreted by transfected tumor cells inhibited HUVECs from forming tubes in Matrigel. ExTEK-transfected C26 colon carcinoma and TS/A mammary tumor cells displayed reduced growth rate when injected subcutaneously, and reduced ability to form experimental metastases when injected intravenously. Immunohistochemical analysis of tumors and metastases showed increased leukocytes infiltration and signs of inflammation in exTEK-secreting compared to parental tumor, as well as impairment in neo-vessel growth and organization. However, while neoangiogenesis eventually rescued in the subcutis, it failed to organize in the experimental metastases of exTEK-secreting tumor, contributing to the hampering of metastatic growth and to increased mice survival. The reactive infiltrate of C26TEK contained a different percentage of leukocytes and was responsible for the tumor inhibition. In fact, leukopenia induced by -irradiation of recipient mice or injection into interferon gamma (IFN-) gene knockout (GKO) mice resulted in reduced mouse survival and an increased number of lung metastases. On the other hand, interleukin (IL)-12 treatment prolonged the survival of mice bearing subcutaneous C26TEK but not of those bearing lung metastases, suggesting that IL-12 could exert further antiangiogenic effects at the site where the tumor can restore neoangiogenesis. These results show in vivo that reduced angiopoietin availability at the tumor site induces a local inflammatory response and impairment of neoangiogenesis which act synergistically to limit tumor growth and metastasis.Abbreviations AEC amino-ethylcarbazole - ELISA enzyme-linked immunosorbent assay - HRP horseradish peroxidase - HUVEC human umbilical vascular endothelial cell - i.v. intravenous - s.c. subcutaneous - TBS Tris-HCl buffered solution     

Changes in public order after the opening of a medically supervised safer injecting facility for illicit injection drug users

Background

North America''s first medically supervised safer injecting facility for illicit injection drug users was opened in Vancouver on Sept. 22, 2003. Although similar facilities exist in a number of European cities and in Sydney, Australia, no standardized evaluations of their impact have been presented in the scientific literature.

Methods

Using a standardized prospective data collection protocol, we measured injection-related public order problems during the 6 weeks before and the 12 weeks after the opening of the safer injecting facility in Vancouver. We measured changes in the number of drug users injecting in public, publicly discarded syringes and injection-related litter. We used Poisson log-linear regression models to evaluate changes in these public order indicators while considering potential confounding variables such as police presence and rainfall.

Results

In stratified linear regression models, the 12-week period after the facility''s opening was independently associated with reductions in the number of drug users injecting in public (p < 0.001), publicly discarded syringes (p < 0.001) and injection-related litter (p < 0.001). The predicted mean daily number of drug users injecting in public was 4.3 (95% confidence interval [CI] 3.5–5.4) during the period before the facility''s opening and 2.4 (95% CI 1.9–3.0) after the opening; the corresponding predicted mean daily numbers of publicly discarded syringes were 11.5 (95% CI 10.0–13.2) and 5.4 (95% CI 4.7–6.2). Externally compiled statistics from the city of Vancouver on the number of syringes discarded in outdoor safe disposal boxes were consistent with our findings.

Interpretation

The opening of the safer injecting facility was independently associated with improvements in several measures of public order, including reduced public injection drug use and public syringe disposal.Many cities are experiencing epidemics of bloodborne diseases as a result of illicit injection drug use,^1,2,3 and drug overdoses have become a leading cause of death in many urban areas.^4,5,6 Public drug use also plagues many inner city neighbourhoods, and the unsafe disposal of syringes in these settings is a major community concern.^{7,8,9,10,11,12,13}In over 2 dozen European cities and, more recently, in Sydney, Australia, medically supervised safer injecting facilities, where injection drug users (IDUs) can inject previously obtained illicit drugs under the supervision of medical staff, have been established in an effort to reduce the community and public health impacts of illicit drug use.¹⁴ Inside these facilities IDUs are typically provided with sterile injecting equipment, emergency care in the event of overdose, as well as primary care services and referral to addiction treatment.^13,15 Although anecdotal reports have suggested that such sites may improve public order,¹² reduce the number of deaths from overdose¹⁶ and improve access to care,¹⁷ no standardized evaluations of their impact are available in the scientific literature.¹⁸On Sept. 22, 2003, health officials in Vancouver opened a government-sanctioned safer injecting facility as pilot project. The facility, the first in North America, is centrally located in Vancouver''s Downtown Eastside, which is the most impoverished urban neighbourhood in Canada and home to well-documented overdose and HIV epidemics among the estimated 5000 IDUs who reside there.^19,20 Federal approval for the 3-year project was granted on the condition that the health and social impacts of the facility be rigorously evaluated. Although evaluation of the facility''s impact on certain outcomes (e.g., HIV incidence) is ongoing and will take several years, it is now possible to examine the impacts of the site on public order. Therefore, we conducted this study to test the hypothesis that changes in improperly discarded syringes and public drug use would be observed after the opening of the safer injecting facility.     

Diterpenes from the leaves of Croton zambesicus

Two new trachylobane- and one isopimarane-type diterpenoids: ent-18-hydroxy-trachyloban-3-one; ent-trachyloban-3-one; isopimara-7,15-dien-3beta-ol, were isolated from the leaves of Croton zambesicus, together with trans-phytol, beta-sitosterol, alpha-amyrin and stigmasterol. The structures were determined by extensive NMR techniques and X-ray analysis. The cytotoxicity of these compounds has been evaluated on cancer and non-cancer cell-lines.     

Tuber borchii fruit body: 2-dimensional profile and protein identification

The formation of the fruit body represents the final phase of the ectomycorrhizal fungus T. borchii life cycle. Very little is known concerning the molecular and biochemical processes involved in the fructification phase. 2-DE maps of unripe and ripe ascocarps revealed different protein expression levels and the comparison of the electropherograms led to the identification of specific proteins for each developmental phase. Associating micropreparative 2-DE to microchemical approaches, such as N-terminal sequencing and 2-D gel-electrophoresis mass-spectrometry, proteins playing pivotal roles in truffle physiology were identified.     

In vitro exposure of human lymphocytes to 900 MHz CW and GSM modulated radiofrequency: studies of proliferation, apoptosis and mitochondrial membrane potential

The aim of this study was to investigate the nonthermal effects of radiofrequency (RF) fields on human immune cells exposed to a Global System for Mobile Communication (GSM) signal generated by a commercial cellular phone and by a sinusoidal non-modulated signal. To assess whether mobile phone RF-field exposure affects human immune cell functions, peripheral blood mononuclear cells (PBMCs) from healthy donors were exposed in vitro to a 900 MHz GSM or continuous-wave (CW) RF field 1 h/day for 3 days in a transverse electromagnetic mode (TEM) cell system (70-76 mW/kg average specific absorption rate, SAR). The cells were cultured for 48 or 72 h, and the following end points were studied: (1) mitogen-induced proliferation; (2) cell cycle progression; (3) spontaneous and 2-deoxy-D-ribose (dRib)-induced apoptosis; (4) mitochondrial membrane potential modifications during spontaneous and dRib-induced-apoptosis. Data obtained from cells exposed to a GSM-modulated RF field showed a slight decrease in cell proliferation when PBMCs were stimulated with the lowest mitogen concentration and a slight increase in the number of cells with altered distribution of phosphatidylserine across the membrane. On the other hand, cell cycle phases, mitochondrial membrane potential and susceptibility to apoptosis were found to be unaffected by the RF field. When cells were exposed to a CW RF field, no significant modifications were observed in comparison with sham-exposed cells for all the end points investigated.     

The copper(II) binding properties of the cyclic peptide c(HGHK)

The new cyclic tetrapeptide c(HGHK) was synthesised in the solid phase and its complexes with copper(II) were studied in aqueous solution at various pH values by means of potentiometric and spectroscopic methods (UV, EPR, CD). Six mononuclear coordination species were clearly identified within the pH range 3-11. Spectroscopic data strongly suggest sequential formation of N, 2N, 3N and 4N equatorial donor sets around the copper(II) centre from the lowest to the highest pH, involving both imidazole nitrogens and amide nitrogens. A detailed comparison with the copper(II) binding properties of HGHG and Ac-HGHG ligands is also reported.     

The deletion of the succinate dehydrogenase gene KlSDH1 in Kluyveromyces lactis does not lead to respiratory deficiency

We have isolated a Kluyveromyces lactis mutant unable to grow on all respiratory carbon sources with the exception of lactate. Functional complementation of this mutant led to the isolation of KlSDH1, the gene encoding the flavoprotein subunit of the succinate dehydrogenase (SDH) complex, which is essential for the aerobic utilization of carbon sources. Despite the high sequence conservation of the SDH genes in Saccharomyces cerevisiae and K. lactis, they do not have the same relevance in the metabolism of the two yeasts. In fact, unlike SDH1, KlSDH1 was highly expressed under both fermentative and nonfermentative conditions. In addition to this, but in contrast with S. cerevisiae, K. lactis strains lacking KlSDH1 were still able to grow in the presence of lactate. In these mutants, oxygen consumption was one-eighth that of the wild type in the presence of lactate and was normal with glucose and ethanol, indicating that the respiratory chain was fully functional. Northern analysis suggested that alternative pathway(s), which involves pyruvate decarboxylase and the glyoxylate cycle, could overcome the absence of SDH and allow (i) lactate utilization and (ii) the accumulation of succinate instead of ethanol during growth on glucose.