A genetic system for detection of protein nuclear import and export

We have developed a simple genetic assay to detect active nuclear localization (NLS) and export signals (NES) on the basis of their function within yeast cells. The bacterial LexA protein was modified (mLexA) to abolish its intrinsic NLS and fused to the activation domain of the yeast Gal4p (Gal4AD) with or without the SV40 large T-antigen NLS. In the import assay, if a tested protein fused to mLexA-Gal4AD contains a functional NLS, it will enter the cell nucleus and activate the reporter gene expression. In the export assay, if a tested protein fused to mLexA-SV40 NLS-Gal4AD contains a functional NES, it will exit into the cytoplasm, decreasing the reporter gene expression. We tested this system with known NLS and NES and then used it to demonstrate a NES activity of the capsid protein of a plant geminivirus. This approach may help to identify, analyze, and select for proteins containing functional NLS and NES.     

Black carrot anthocyanins exhibit neuroprotective effects against MPP+ induced cell death and cytotoxicity via inhibition of oxidative stress mediated apoptosis

Parkinson’s disease (PD) is a common chronic neurodegenerative disease induced by the death of dopaminergic neurons. Anthocyanins are naturally found antioxidants and well-known for their preventive effects in neurodegenerative disorders. Black carrots (Daucus carota L. ssp. sativus var. atrorubens Alef.) are a rich source of anthocyanins predominantly including acylated cyanidin-based derivatives making them more stable. However, there have been no reports analysing the neuroprotective role of black carrot anthocyanins (BCA) on PD. In order to investigate the potential neuroprotective effect of BCA, human SH-SY5Y cells were treated with MPP+?(1-methyl-4-phenylpyridinium) to induce PD associated cell death and cytotoxicity. Anthocyanins were extracted from black carrots and the composition was determined by HPLC–DAD. SH-SY5Y cells were co-incubated with BCA (2.5, 5, 10, 25, 50, 100 µg/ml) and 0.5 mM MPP+?to determine the neuroprotective effect of BCA against MPP+?induced cell death and cytotoxicity. Results indicate that BCA concentrations did not have any adverse effect on cell viability. BCA revealed its cytoprotective effect, especially at higher concentrations (50, 100 µg/ml) by increasing metabolic activity and decreasing membrane damage. BCA exhibited antioxidant activity via scavenging MPP+?induced reactive oxygen species (ROS) and protecting dopaminergic neurons from ROS mediated apoptosis. These results suggest a neuroprotective effect of BCA due to its high antioxidant and antiapoptotic activity, along with the absence of cytotoxicity. The elevated stability of BCA together with potential neuroprotective effects may shed light to future studies in order to elucidate the mechanism and further neuro-therapeutic potential of BCA which is promising as a neuroprotective agent.

     

Coactosin-Like 1 Antagonizes Cofilin to Promote Lamellipodial Protrusion at the Immune Synapse

Actin depolymerizing factor-homology (ADF-H) family proteins regulate actin filament dynamics at multiple cellular locations. Herein, we have investigated the function of the ADF-H family member coactosin-like 1 (COTL1) in the regulation of actin dynamics at the T cell immune synapse (IS). We initially identified COTL1 in a genetic screen to identify novel regulators of T cell activation, and subsequently found that it associates with F-actin and localizes at the IS in response to TCR+CD28 stimulation. Live cell microscopy showed that depletion of COTL1 protein impaired T cell spreading in response to TCR ligation and abrogated lamellipodial protrusion at the T cell – B cell contact site, producing only a band of F-actin. Significantly, re-expression of wild type COTL1, but not a mutant deficient in F-actin binding could rescue these defects. In addition, COTL1 depletion reduced T cell migration. In vitro studies showed that COTL1 and cofilin compete with each other for binding to F-actin, and COTL1 protects F-actin from cofilin-mediated depolymerization. While depletion of cofilin enhanced F-actin assembly and lamellipodial protrusion at the IS, concurrent depletion of both COTL1 and cofilin restored lamellipodia formation. Taken together, our results suggest that COTL1 regulates lamellipodia dynamics in part by protecting F-actin from cofilin-mediated disassembly.     

Viability of human-derived probiotic lactobacilli in ice cream produced with sucrose and aspartame

A mixture of human-derived probiotic strains of Lactobacillus acidophilus, L. agilis and L. rhamnosus was used as a probiotic culture in ice cream manufacture. Viability and survival of these probiotic cultures were investigated in two different ice cream formulations. Ice cream with sucrose and ice cream with aspartame were prepared and each of these was divided into two subgroups: one with direct addition of the probiotic culture and one with milk fermented by the same probiotic culture. Ice cream samples were stored at −20°C for 6 months and the survival rate of cultures were determined monthly. Probiotic cultures underwent tests for resistance to bile salts, antibiotics, acidic conditions; they were found to be highly resistant to such challenges. Chemical analysis of ice cream samples, such as determination of acidity, pH and solid matter, was also performed. The probiotic cultures remained unchanged in ice cream stored for up to 6 months regardless of the sweeteners used. Using probiotic cultures in ice cream mixes did not alter the characteristics of the product.     

Detection of Adenoviruses and Rotaviruses in Drinking Water Sources Used In Rural Areas of Benin,West Africa

Diseases associated with viruses also found in environmental samples cause major health problems in developing countries. Little is known about the frequency and pattern of viral contamination of drinking water sources in these resource-poor settings. We established a method to analyze 10 liters of water from drinking water sources in a rural area of Benin for the presence of adenoviruses and rotaviruses. Overall, 541 samples from 287 drinking water sources were tested. A total of 12.9% of the sources were positive for adenoviruses and 2.1% of the sources were positive for rotaviruses at least once. Due to the temporary nature of viral contamination in drinking water sources, the probability of virus detection increased with the number of samples taken at one test site over time. No seasonal pattern for viral contaminations was found after samples obtained during the dry and wet seasons were compared. Overall, 3 of 15 surface water samples (20%) and 35 of 247 wells (14.2%) but also 2 of 25 pumps (8%) tested positive for adenoviruses or rotaviruses. The presence of latrines within a radius of 50 m in the vicinity of pumps or wells was identified as being a risk factor for virus detection. In summary, viral contamination was correlated with the presence of latrines in the vicinity of drinking water sources, indicating the importance of appropriate decision support systems in these socioeconomic prospering regions.Although access to safe drinking water is considered a human right, many people suffer from inadequate water supply. Especially in developing countries, improper water quality causes major public health problems affecting mortality rates in highly susceptible people (small children and immunocompromised patients) as well as economic income problems due to disease-related nonproductive time. Only 37.4% of households have access to piped water sources in Benin, West Africa, and in rural areas, even fewer have access (3). Many diseases like diarrhea, gastroenteritis, keratoconjunctivitis, respiratory infections, and hepatitis are associated with viruses, often found in environmental samples like groundwater, surface water, sewage, costal water, shellfish, and tap water (5, 6, 10, 13, 23, 38). Virus concentrations in environmental samples are low both due to the inability to replicate without a host cell and because of continuous degradation and dilution effects. On the other hand and in contrast to most bacterial infections, even small amounts of viruses (as few as 10 PCR-detectable units) are sufficient to establish an infection in the new host (24). Bacterial indicators seem to be inappropriate for analyzing viral contamination, since viruses are more resistant to environmental conditions (2) and spread over a longer distance than bacteria (9). Therefore, viruses are often found without any bacterial indicator for fecal contamination (2, 6). In North America, 15 to 30% of all gastrointestinal diseases were suspected of being related to water (30), whereas worldwide, over 88% of diarrheal diseases are waterborne or water related (18).Routine screening of environmental samples for viral contamination is controversially being discussed at the moment. Furthermore, no standard procedure for the detection of viruses in environmental samples currently exists. In numerous studies, virus concentration from water was achieved by filtration using electropositive filters (1MDS) (12, 15, 25, 26, 33). Further methods using ultrafiltration, glass wool filters (7, 22), or immunomagnetic separation (16, 28) were used to detect small amounts of viruses independently of matrix effects. However, the U.S. Environmental Protection Agency listed adenovirus as one of nine microorganisms on the contamination candidate list for drinking water as a potential indicator virus due to an outstanding resistance to UV disinfection. The 51 presently recognized adenovirus serotypes are responsible for a great variety of human diseases like diarrhea, keratoconjunctivitis, and respiratory infections. However, severe diarrhea, especially in small children and immunocompromised patients, is often caused by rotaviruses. The fatal outcome of infant diarrhea substantially contributes to the high mortality rate of children under the age of 5 years in developing countries (8). In Benin, the probability of dying per 1,000 live births under 5 years of age (under-5 mortality rate) was 152 in 2004, and 17.1% of these deaths were caused by diarrheal diseases (37). To address the frequency and pattern of viral contamination in drinking water sources in rural areas of West Africa, we analyzed water samples during the dry and wet seasons in Benin for contamination with adenoviruses as indicators and rotaviruses as important pathogens in developing countries.     

Changes in patterns of alkaline serine protease and bacilysin formation caused by common effectors of sporulation inBacillus subtilis 168

Bacilysin biosynthesis and alkaline serine protease production inBacillus subtilis 168 were monitored and compared in batch cultures when various effectors of sporulation were added at different stages of growth in a medium containing sucrose and glutamate. Depending on the time of addition, glucose affected sporulation and serine protease formation to the same extent, but had no effect on bacilysin production. Ammonium andl-alanine additions suppressed all three processes. Casamino acids severely interfered with bacilysin formation and sporulation, but not with protease formation. Decoyinine, a well-known inducer of sporulation, induced protease formation as well, but did not affect bacilysin biosynthesis. The extent of the observed effects depended largely on the time of metabolite additions. The results are discussed with reference to a possible coregulation of sporulation and the formation of bacilysin and alkaline serine protease inB. subtilis.     

Effect of reddening of cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis> L.) leaves on the ultrastructure of mesophyll cells

The ultrastructure of cotton leaves, exhibiting reddening as symptom of physiological disorder, was examined by means of transmission electron microscopy. Osmiophilisation of the membrane compartment was established. Massive agglomerations on the tonoplast in the vacuole of cells under the adaxial epidermis were observed, and were referred to as electron-dense osmiophilic substance, most probably of anthocyanin nature. In chloroplast stroma a zone of low electron density enclosing numerous osmiophilic aggregations of unclear chemical character was differentiated. Fragmentation and severe destruction of thylakoids in chloroplasts of reddening cotton leaves was not detected.