Single Molecule Fluorescence Microscopy on Planar Supported Bilayers

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background in optics and laser physics to a discipline that is now enjoying vivid attention by life-scientists of all venues ¹. This is because single molecule imaging has the unique potential to reveal protein behavior in situ in living cells and uncover cellular organization with unprecedented resolution below the diffraction limit of visible light ². Glass-supported planar lipid bilayers (SLBs) are a powerful tool to bring cells otherwise growing in suspension in close enough proximity to the glass slide so that they can be readily imaged in noise-reduced Total Internal Reflection illumination mode ^3,4. They are very useful to study the protein dynamics in plasma membrane-associated events as diverse as cell-cell contact formation, endocytosis, exocytosis and immune recognition. Simple procedures are presented how to generate highly mobile protein-functionalized SLBs in a reproducible manner, how to determine protein mobility within and how to measure protein densities with the use of single molecule detection. It is shown how to construct a cost-efficient single molecule microscopy system with TIRF illumination capabilities and how to operate it in the experiment.     

Processive cytoskeletal motors studied with single-molecule fluorescence techniques

Processive cytoskeletal motors from the myosin, kinesin, and dynein families walk on actin filaments and microtubules to drive cellular transport and organization in eukaryotic cells. These remarkable molecular machines are able to take hundreds of successive steps at speeds of up to several microns per second, allowing them to effectively move vesicles and organelles throughout the cytoplasm. Here, we focus on single-molecule fluorescence techniques and discuss their wide-ranging applications to the field of cytoskeletal motor research. We cover both traditional fluorescence and sub-diffraction imaging of motors, providing examples of how fluorescence data can be used to measure biophysical parameters of motors such as coordination, stepping mechanism, gating, and processivity. We also outline some remaining challenges in the field and suggest future directions.     

Cauliflower waste incorporation into cane molasses improves ethanol production using <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> MTCC 178

Diluted cane molasses having total sugar and reducing sugar content of 9.60 and 3.80% (w/v) respectively was subjected to ethanol production by Saccharomyces cerevisiae MTCC 178. Incorporation of dried Cauliflower Waste (CW) in molasses at the level of 15 % increased ethanol production by nearly 36 % compared to molasses alone. Addition of 0.2 % yeast extract improved ethanol production by nearly 49 % as compared to molasses alone. When the medium containing diluted molasses and 0.2 % yeast extract was supplemented with 15 % CW, 29 % more ethanol was produced compared to molasses with 0.2 % yeast extract. Cell biomass, ethanol production, final ethanol concentration and fermentation efficiency of 2.65 mg mL⁻¹, 41.2 gL⁻¹, 0.358 gg⁻¹ and 70.11 % respectively were found to be best at 15% CW supplementation level besides reduction in fermentation time but further increase in CW level resulted in decline on account of all the above parameters. This is probably the first report to our knowledge, in which CW was used in enhancing ethanol production significantly using a small quantity of yeast extract.     

新疆沙冬青叶片氨基酸和蛋白质的测试与分离以及抗冻蛋白的鉴定结果分析

提取并纯化了新疆沙冬青(Ammopiptanthus nanus)叶片总蛋白,得到了适合新疆沙冬青叶片总蛋白提取的最佳缓冲体系;用DEAE52-C分离了各蛋白质组分,并用SDS-PAG测定了纯度和分子量(14~90 kD之间);氨基酸组成分析显示了新疆沙冬青属内种遗传的一致性。对抗冻活性分析讨论证实了与蒙古沙冬青同属的新疆沙冬青中也含有抗冻蛋白(AFPs)。     

Endophytic fungi from <Emphasis Type="Italic">Nerium oleander</Emphasis> L (Apocynaceae): main constituents and antioxidant activity

Diverse endophytic fungi exist within plant aerial tissues, with a global estimate of up to a million undescribed species. These endophytes constitute a rich bio-resource for exploration to discover new natural products. Here we investigate fungal endophytes associated with a medicinal plant, Nerium oleander L. (Apocynaceae). A total of 42 endophytic fungal strains were isolated from the host plant. Total antioxidant capacity, xanthine oxidase inhibitory activity, antimicrobial activity, and total phenolic content (TPC) were evaluated for 16 representative fungal cultures grown in improved Czapek’s broth and for the host plant. The total antioxidant capacities and phenolic contents of the fungal cultures ranged from 9.59 to 150.79 μmol trolox/100 mL culture, and from 0.52 to 13.95 mg gallic acid/100 mL culture, respectively. The fungal culture of an endophytic strain Chaetomium sp. showed the strongest antioxidant capacity, contained the highest level of phenolics, and to some extent inhibited xanthine oxidase activity with an IC₅₀ value of 109.8 μg/mL. A significant positive correlation was found between antioxidant capacity and TPC in the tested samples. Most of the endophytic fungal cultures tested have a wide range of antimicrobial activities, which were not very strong, but much better than those of the host plant. The major bioactive constituents of the fungal cultures were investigated using LC-ESI-MS and GC-MS, and preliminary identification detected phenolics (e.g. phenolic acids and their derivatives, flavonoids) and volatile and aliphatic compounds. This study shows that the endophytic fungi isolated from N. oleander can be a potential antioxidant resource.     

The phylogeny of the social Anelosimus spiders (Araneae: Theridiidae) inferred from six molecular loci and morphology

We use fragments of three nuclear genes (Histone 3, 18SrDNA, and 28SrDNA) and three mitochondrial genes (16SrDNA, ND1, and COI) totalling approximately 4.5kb, in addition to morphological data, to estimate the phylogenetic relationships among Anelosimus spiders, well known for their sociality. The analysis includes 67 individuals representing 23 of the 53 currently recognized Anelosimus species and all species groups previously recognized by morphological evidence. We analyse the data using Bayesian, maximum likelihood, and parsimony methods, considering the genes individually as well as combined (mitochondrial, nuclear, and both combined) in addition to a 'total evidence' analysis including morphology. Most of the data partitions are congruent in agreeing on several fundamental aspects of the phylogeny, and the combined molecular data yield a tree broadly similar to an existing morphological hypothesis. We argue that such congruence among data partitions is an important indicator of support that may go undetected by standard robustness estimators. Our results strongly support Anelosimus monophyly, and the monophyly of the recently revised American 'eximius lineage', although slightly altered by excluding A. pacificus. There was consistent support for the scattering of American Anelosimus species in three clades suggesting intercontinental dispersal. Several recently described species are reconstructed as monophyletic, supporting taxonomic decisions based on morphology and behaviour in this taxonomically difficult group. Corroborating previous results from morphology, the molecular data suggest that social species are scattered across the genus and thus that sociality has evolved multiple times, a significant finding for exploring the causes and consequences of social evolution in this group of organisms.     

Multiple Site Directed Mutagenesis Strategy Based on Total RNA and RT-PCR Method

Site-directed PCR-based mutagenesis methods are widely used to generate mutations. All published methods work on DNA clones carrying the target sequence. However, DNA clones are not always available. We have previously published a RT-PCR-based site-directed mutagenesis method starting from total RNA to overcome this problem. In this article, we report an improvement of our previous method to facilitate introduction of multiple mutations into a target sequence. We demonstrate the efficacy and feasibility of this strategy by mutation of the human β-actin gene. BamHI restriction endonuclease cleavage sites were generated within the gene to assist screening. Using three mutagenic primers in a single RT-PCR reaction, seven different clones were produced carrying three single and four multiple mutations. An investigation of the effect of the cycle number and elongation time of the PCR reactions revealed that both have an influence on the ratio of clones carrying single and multiple mutations. An optimized protocol was established for efficient multiple site-directed mutagenesis.     

Association of total tooth loss with smoking, drinking alcohol and nutrition in elderly Japanese: analysis of national database

Objective: Various factors may be associated with edentulism in elderly people. Association of total tooth loss with smoking, alcohol intake and nutrition in non‐institutionalised elderly Japanese was assessed utilising national database. Materials and methods: Records of independent surveys, the Survey of Dental Diseases (SDD) and the National Nutrition Survey (NNS) in 1999 were electronically linked using the household identification number. Results: Among the records of 6903 subjects in the SDD and 12 763 subjects in the NNS, 6805 records were successfully linked. Overall, prevalence of total tooth loss in adults was very similar in males and females at approximately 7.0%, and the smoking rate was 47.6% and 9.9% respectively. Total tooth loss was a rare phenomenon (<2%) in age groups of <60 years. According to the multiple logistic regression analysis involving 2200 subjects aged 60 years or older, significant variables were age, current smokers and vitamin C intake in males, and age and current smokers in females. The variable for current drinkers was significant in females but the odds ratio was <1.0. No significant relationship was detected with respect to former smokers and drinkers, body mass index, vitamin E intake and blood glucose level. Conclusions: Current smoking was associated with total tooth loss, although smoking rate was low in females. Gender difference in the association was suggestive with respect to drinking alcohol and vitamin C intake.