Eukaryotic phylotypes in aquatic moss pillars inhabiting a freshwater lake in East Antarctica, based on 18S rRNA gene analysis

Aquatic mosses of Leptobryum species form unique tower-like pillars of vegetation termed “moss pillars” in Antarctic lakes. Moss pillars have distinct redox-affected sections: oxidative exteriors and reductive interiors. We have proposed that a “pillar” is a community and habitat of functionally interdependent organisms and may represent a mini-biosphere. Batteries of 16S rRNA genotypes, or phylotypes, of eubacteria and cyanobacteria, but no archaea, have been identified in moss pillars. However, detailed identification or phylogenetic analyses of the moss and their associated eukaryotic microbiota have not been performed. This study analyzed near-full-length 18S rRNA gene sequences obtained from two whole moss pillars. In total, 28 PCR clone libraries from two whole moss pillars were constructed, and 96 clones from each library (total 2,688 clones) were randomly selected and sequenced. Molecular phylogenetic analysis revealed that the phylotype belonging to Bryophyta, considered to be derived from moss, was closely related (99.9?%) to the 18S rRNA gene sequence from Leptobryum pyriforme. Unexpectedly, phylotypes belonging to a novel clade of fungi dominated (approximately 27–75?%) the moss pillar libraries. This suggests that fungi may contribute to carbon cycling in the moss pillar as parasites or decomposers. In addition, phylotypes related to ciliates and tardigrades were subdominant in the exterior, while the phylotype of the ameba-like, single-celled eukaryote, Cercomonas (Cercozoa), was detected only in the interior. These features were shared by both moss pillars. The 18S rRNA gene-based profiles demonstrated that redox-related factors may control distribution of some eukaryotic microbes in a whole moss pillar.     

Nanobiotechnology: quantum dots in bioimaging

Many biological systems, including protein complexes, are natural nanostructures. To better understand these structures and to monitor them in real time, it is becoming increasingly important to develop nanometer-scale signaling markers. Single-molecule methods will play a major role in elucidating the role of all proteins and their mutual interactions in a given organism. Fluorescent semiconductor nanocrystals, known as quantum dots, have several advantages of optical and chemical features over the traditional fluorescent labels. These features make them desirable for long-term stability and simultaneous detection of multiple signals. Here, we review current approaches to developing a biological application for quantum dots.     

Analysis of metabolic and physiological responses to gnd knockout in Escherichia coli by using C-13 tracer experiment and enzyme activity measurement

The physiological and metabolic responses to gnd knockout in Escherichia coli K-12 was quantitatively investigated by using the (13)C tracer experiment (GC-MS/NMR) together with the enzyme activity analysis. It was shown that the general response to the gene knockout was the local flux rerouting via Entner-Doudoroff pathway and the direction reversing via non-oxidative pentose phosphate pathway (PPP). The mutant was found to direct higher flux to phosphoglucose isomerase reaction as compared to the wild-type, but the respiratory metabolism was comparable in both strains. The anaplerotic pathway catalyzed by malic enzyme was identified in the mutant, which was accompanied with an up-regulation of phosphoenolpyruvate carboxylase and down-regulation of phosphoenolpyruvate carboxykinase. The presented results provide first evidence that compensatory mechanism existed in PPP and anaplerotic pathway in response to the gnd deletion.     

Progression of flagellar stages during artificially delayed motility initiation in sea urchin sperm

Transition from immotile to motile flagella may involve a series of states, in which some of regulatory mechanisms underlying normal flagellar movement are working with others being still suppressed. To address ourselves to the study of starting transients of flagella, we analyzed flagellar movement of sea urchin sperm whose motility initiation had been retarded in an experimental solution, so that we could capture the instance at which individual spermatozoa began their flagellar beating. Initially straight and immotile flagella began to shiver at low amplitude, then propagated exclusively the principal bend (P bend), and finally started stable flagellar beating. The site of generation of the P bend in the P-bend propagating stage varied in position in the basal region up to 10 microm from the base, indicating that the ability of autonomous bend generation is not exclusively possessed by the very basal region but can be unmasked throughout a wider region when the reverse bend (R bend) is suppressed. The rate of change in the shear angle, the curvature of the R bend and the frequency and regularity of beating substantially increased upon transition from P-bend propagating to full-beating, while the propagation velocity of bends remained unchanged. These findings indicate that artificially delayed motility initiation may accompany sequential modification of the motile system and that mechanisms underlying flagellar motility can be analyzed separately under experimentally retarded conditions.     

Evaluation of antioxidant and antibacterial activity of methanolic extracts of Gentiana kurroo royle

In this study our objective was to evaluate the antioxidant and antimicrobial activity of methanolic extracts of leaves and roots of Gentiana kurroo. The antioxidant activities of the extracts were examined using different biochemical assays namely diphenylpicrylhydrazyl (DPPH), nitroblue tetrazolium (NBT) and ferric reducing power (FRAP). In all the assays, root extract exhibited stronger antioxidant activity than that of leaves. The antibacterial activity of the extracts was also evaluated and MIC values were calculated by broth dilution method. Although, the extracts prevented the growth of both Gram positive and Gram negative bacteria, the MIC values of methanolic extract of the leaves were higher than those of the root extract. The antibacterial and antioxidant activity of the extracts was found to be positively associated with the total phenolic and flavonoid content of the extracts.     

Mycolic acids from Rhodococcus, Gordonia, and Dietzia

The mycolic acids from 11 species of Rhodococcus, seven species of Gordonia, and one species of Dietzia were analyzed using capillary gas chromatography and mass spectrometry (GLC/MS). All strains tested in this study were divided into three groups according to the degree of double bonds and the average carbon number (Av.Nc.) of their mycolic acids. The genus Gordonia belongs to the first group possessing an Av.Nc. in the upper 50s and 60s with 0 to 5 double bonds. Some Rhodococcus species possessed Av.Nc. in the 40s with a variety of distributions of polyunsaturated fatty acids from 0 to 4. The rest of the Rhodococcus species and the genus Dietzia possessed Av.Nc. in the 30s with saturated fatty acids. We previously reported on Nocardia strains whose Av.Nc. were in the 50s. Considering the identification of mycolic acid-containing Actinomycetales at the generic level, the Av.Nc. proved to be useful as a means of differentiating the genera Rhodococcus, Gordonia and Nocardia. The genus Dietzia was found to have its own characteristic constitution of mycolic acid molecular species. The mycolic acids from D. maris 58001T were characterized by an almost equal amount of constituents of even- and odd-numbered carbon chains, whereas the major components of mycolic acids in all other strains had even-numbered carbon chains. Another characteristic of Dietzia was some even-numbered mycolic acids which contained odd-numbered straight chains with odd-numbered alpha-branches. These characteristics indicated that Dietzia might possess a novel fatty acid biosynthesis system.     

Phosphorylation by protein kinase C of a synthetic heptapeptide bearing a lysine residue on the C terminal side of serine

A peptide, Ala-Ser-Gly-Ser-Phe-Lys-Leu, which corresponds to Ala103-Leu109 of Hl histone, was synthesized and tested as substrate for protein kinase C. The serine residue at position 4 was phosphorylated specifically. Another peptide lacking the lysine at position 6 was not phosphorylated by the same enzyme, indicating the importance of that basic residue as the recognition site for protein kinase C.     

Dual-tagging gene trap of novel genes in Drosophila melanogaster

A gene-trap system is established for Drosophila. Unlike the conventional enhancer-trap system, the gene-trap system allows the recovery only of fly lines whose genes are inactivated by a P-element insertion, i.e., mutants. In the gene-trap system, the reporter gene expression reflects precisely the spatial and temporal expression pattern of the trapped gene. Flies in which gene trap occurred are identified by a two-step screening process using two independent markers, mini-w and Gal4, each indicating the integration of the vector downstream of the promoter of a gene (dual tagging). mini-w has its own promoter but lacks a polyadenylation signal. Therefore, mini-w mRNA is transcribed from its own promoter regardless of the vector integration site in the genome. However, the eyes of flies are not orange or red unless the vector is incorporated into a gene enabling mini-w to be spliced to a downstream exon of the host gene and polyadenylated at the 3' end. The promoter-less Gal4 reporter is expressed as a fusion mRNA only when it is integrated downstream of the promoter of a host gene. The exons of trapped genes can be readily cloned by vectorette RT-PCR, followed by RACE and PCR using cDNA libraries. Thus, the dual-tagging gene-trap system provides a means for (i) efficient mutagenesis, (ii) unequivocal identification of genes responsible for mutant phenotypes, (iii) precise detection of expression patterns of trapped genes, and (iv) rapid cloning of trapped genes.     

Intravitreal injection or topical eye-drop application of a μ-calpain C2L domain peptide protects against photoreceptor cell death in Royal College of Surgeons' rats, a model of retinitis pigmentosa

Mitochondrial μ-calpain initiates apoptosis-inducing factor (AIF)-dependent apoptosis in retinal photoreceptor degeneration. Mitochondrial μ-calpain inhibitors may represent therapeutic targets for the disease. Therefore, we sought to identify inhibitors of mitochondrial calpains and determine their effects in Royal College of Surgeons' (RCS) rats, an animal model of retinitis pigmentosa (RP). We synthesized 20-mer peptides of the C2-like (C2L) domain of μ-calpain. Two μ-calpain peptides N2 and N9 inhibited mitochondrial μ-calpain activity (IC(50); 892 and 498nM, respectively), but not other proteases. Western blotting showed that 50μM of both μ-calpain peptides caused specific degradation of mitochondrial μ-calpain. Three-dimensional structure of calpains suggested that the peptides N2 and N9 corresponded to the regions forming salt bridges between the protease core domain 2 and the C2L domain. We determined the inhibitory regions of μ-calpain peptides N2 and N9 using 10-mers, and one peptide, N2-10-2, inhibited the activity of mitochondrial μ-calpain (IC(50); 112nM). We next conjugated the peptide N2-10-2 to the C-terminal of HIV-1 tat (HIV), a cell-penetrating peptide. Using isolated rat liver mitochondria, 50μM HIV-conjugated μ-calpain N2-10-2 peptide (HIV-Nμ, IC(50); 285nM) significantly inhibited AIF truncation. The intravitreal injection of 20mM HIV-Nμ also prevented retinal photoreceptor apoptosis determined by TUNEL staining, and preserved retinal function assessed by electroretinography in RCS rats. Topical application of 40mM HIV-Nμ also prevented apoptosis of retinal photoreceptors in RCS rats. Our results demonstrate that HIV-Nμ, a peptide inhibitor of mitochondrial μ-calpain, offers a new modality for treating RP.