Identification of Botryticidal Proteins with Similarity to NBS–LRR Proteins in Rosemary Pepper (<Emphasis Type="Italic">Lippia sidoides</Emphasis> Cham.) Flowers

Heavy agricultural losses are closely related to attacks by insect-pests and phytopathogens such as bacteria and fungi. Among them, the fungus Botrytis cinerea can cause gray mold in more than 200 different species of plants, and is considered a challenging problem for agribusiness. Fungicides are commonly used to control this pathogen because they are fast-working and easy to apply. However, the continuous use of fungicides may promote the selection of resistant fungi and can also cause profound contamination in ecosystems. Aiming to find alternative strategies to solve these problems, several studies have focused on searching for plant proteins and peptides with antifungal activities (AFPs). With this in mind, this report shows the isolation and characterization of two novels antifungal proteins from flowers of rosemary pepper (Lippia sidoides Cham.) with 10 and 15 kDa. Isolation was performed by using an Octyl-Sepharose hydrophobic column. In vitro bioassays indicated that isolated proteins were able to inhibit B. cinerea development, but were not effective against all bacteria tested. Moreover, N-termini sequences indicate that both proteins showed sequence homology with NBS–LRR R proteins with a lower molecular mass, suggesting possible protein fragmentation. Data reported here could help in the development of biotechnological products for crop protection against phytopathogenic fungi in the near future.     

Assessment of the genetic risks of a metallic alloy used in medical implants

The use of artificial implants provides a palliative or permanent solution for individuals who have lost some bodily function through disease, an accident or natural wear. This functional loss can be compensated for by the use of medical devices produced from special biomaterials. Titanium alloy (Ti-6Al-4V) is a well-established primary metallic biomaterial for orthopedic implants, but the toxicity of the chemical components of this alloy has become an issue of concern. In this work, we used the MTT assay and micronucleus assay to examine the cytotoxicity and genotoxicity, respectively, of an extract obtained from this alloy. The MTT assay indicated that the mitochondrial activity and cell viability of CHO-K1 cells were unaffected by exposure to the extract. However, the micronucleus assay revealed DNA damage and an increase in micronucleus frequency at all of the concentrations tested. These results show that ions released from Ti-6Al-4V alloy can cause DNA and nuclear damage and reinforce the importance of assessing the safety of metallic medical devices constructed from biomaterials.     

Sunlight-Exposed Biofilm Microbial Communities Are Naturally Resistant to Chernobyl Ionizing-Radiation Levels

Background

The Chernobyl accident represents a long-term experiment on the effects of exposure to ionizing radiation at the ecosystem level. Though studies of these effects on plants and animals are abundant, the study of how Chernobyl radiation levels affect prokaryotic and eukaryotic microbial communities is practically non-existent, except for a few reports on human pathogens or soil microorganisms. Environments enduring extreme desiccation and UV radiation, such as sunlight exposed biofilms could in principle select for organisms highly resistant to ionizing radiation as well.

Methodology/Principal Findings

To test this hypothesis, we explored the diversity of microorganisms belonging to the three domains of life by cultivation-independent approaches in biofilms developing on concrete walls or pillars in the Chernobyl area exposed to different levels of radiation, and we compared them with a similar biofilm from a non-irradiated site in Northern Ireland. Actinobacteria, Alphaproteobacteria, Bacteroidetes, Acidobacteria and Deinococcales were the most consistently detected bacterial groups, whereas green algae (Chlorophyta) and ascomycete fungi (Ascomycota) dominated within the eukaryotes. Close relatives to the most radio-resistant organisms known, including Rubrobacter species, Deinococcales and melanized ascomycete fungi were always detected. The diversity of bacteria and eukaryotes found in the most highly irradiated samples was comparable to that of less irradiated Chernobyl sites and Northern Ireland. However, the study of mutation frequencies in non-coding ITS regions versus SSU rRNA genes in members of a same actinobacterial operational taxonomic unit (OTU) present in Chernobyl samples and Northern Ireland showed a positive correlation between increased radiation and mutation rates.

Conclusions/Significance

Our results show that biofilm microbial communities in the most irradiated samples are comparable to non-irradiated samples in terms of general diversity patterns, despite increased mutation levels at the single-OTU level. Therefore, biofilm communities growing in sunlight exposed substrates are capable of coping with increased mutation rates and appear pre-adapted to levels of ionizing radiation in Chernobyl due to their natural adaptation to periodical desiccation and ambient UV radiation.     

Failure of CD4 T-cells to respond to liver-derived antigen and to provide help to CD8 T-cells

CD4 T-cell help is required for the induction of efficient CD8 T-cells responses and the generation of memory cells. Lack of CD4 T-cell help may contribute to an exhausted CD8 phenotype and viral persistence. Little is known about priming of CD4 T-cells by liver-derived antigen. We used TF-OVA mice expressing ovalbumin in hepatocytes to investigate CD4 T-cell priming by liver-derived antigen and the impact of CD4 T-cell help on CD8 T-cell function. Naïve and effector CD4 T-cells specific for ovalbumin were transferred into TF-OVA mice alone or together with naïve ovalbumin-specific CD8 T-cells. T-cell activation and function were analyzed. CD4 T-cells ignored antigen presented by liver antigen-presenting cells (APCs) in vitro and in vivo but were primed in the liver-draining lymph node and the spleen. No priming occurred in the absence of bone-marrow derived APCs capable of presenting ovalbumin in vivo. CD4 T-cells primed in TF-OVA mice displayed defective Th1-effector function and caused no liver damage. CD4 T-cells were not required for the induction of hepatitis by CD8 T-cells. Th1-effector but not naïve CD4 T-cells augmented the severity of liver injury caused by CD8 T-cells. Our data demonstrate that CD4 T-cells fail to respond to liver-derived antigen presented by liver APCs and develop defective effector function after priming in lymph nodes and spleen. The lack of CD4 T-cell help may be responsible for insufficient CD8 T-cell function against hepatic antigens.     

Adelphocorisella Miyamoto and Yasunaga newly recorded from Australia, with the description of a new species (Heteroptera: Miridae: Mirinae)

Adelphocorisella australis sp. n. is described from north Queensland. This, the first representative of the genus known from Australia, is compared with the two previously described species, both from Japan.     

Morphological and oxidative alterations on Sertoli cells cytoskeleton due to retinol-induced reactive oxygen species

Retinol (vitamin A) is involved in several cellular processes, like cell division, differentiation, transformation and apoptosis. Although it has been shown that retinol is a limitant factor for all these processes, the precise mechanisms by which retinol acts are still unknown. In the present study we hypothesised that alterations in the cytoskeleton of Sertoli cells induced by retinol supplementation could indicate an adaptive maintenance of its functions, since it plays an important role in the transformation process that we observed. Previous results demonstrated that Sertoli cells treated with retinol showed an oxidative imbalance, that leads the cell to two phenotypes: apoptosis or transformation. Our group has identified characteristics of Sertoli cells transformed by retinol which results in normal cell functions modification. In the present study the actin filament fluorescence assay and the deformation coefficient showed a modification in the morphology induced by retinol. We also observed an oxidative alteration in isolated cytoskeleton proteins and did not show alterations when these proteins are analyzed by electrophoreses. Our results showed an increase in mitochondria superoxide production and a decrease in nitric oxide levels. All results were partially or completely reverted by co-treatment of the antioxidant Trolox®. These findings suggest that the cytoskeleton components suffer individual alterations in different levels and that these alterations generate a global phenotype modification and that these processes are probably ROS dependent. We believe that the results from this study indicate an adaptation of the cytoskeleton to oxidative imbalance since there was not a loss of its function. (Mol Cell Biochem 271: 189–196, 2005)     

Role of pressor mechanisms from the NTS and CVLM in control of arterial pressure

In the present study, we investigated the effects of inhibition of the caudal ventrolateral medulla (CVLM) with the GABA(A) agonist muscimol combined with the blockade of glutamatergic mechanism in the nucleus of the solitary tract (NTS) with kynurenic acid (kyn) on mean arterial pressure (MAP), heart rate (HR), and regional vascular resistances. In male Holtzman rats anesthetized intravenously with urethane/chloralose, bilateral injections of muscimol (120 pmol) into the CVLM or bilateral injections of kyn (2.7 nmol) into the NTS alone increased MAP to 186 +/- 11 and to 142 +/- 6 mmHg, respectively, vs. control: 105 +/- 4 mmHg; HR to 407 +/- 15 and to 412 +/- 18 beats per minute (bpm), respectively, vs. control: 352 +/- 12 bpm; and renal, mesenteric and hindquarter vascular resistances. However, in rats with the CVLM bilaterally blocked by muscimol, additional injections of kyn into the NTS reduced MAP to 88 +/- 5 mmHg and mesenteric and hindquarter vascular resistances below control baseline levels. Moreover, in rats with the glutamatergic mechanisms of the NTS blocked by bilateral injections of kyn, additional injections of muscimol into the CVLM also reduced MAP to 92 +/- 2 mmHg and mesenteric and hindquarter vascular resistances below control baseline levels. Simultaneous blockade of NTS and CVLM did not modify the increase in HR but also abolished the increase in renal vascular resistance produced by each treatment alone. The results suggest that important pressor mechanisms arise from the NTS and CVLM to control vascular resistance and arterial pressure under the conditions of the present study.