Positive selection is a general phenomenon in the evolution of abalone sperm lysin

Lysin is a 16kDa acrosomal protein used by abalone sperm to create a hole in the egg vitelline envelope (VE). The interaction of lysin with the VE is species-selective and is one step in the multistep fertilization process that restricts heterospecific (cross-species) fertilization. For this reason, the evolution of lysin could play a role in establishing prezygotic reproductive isolation between species. Previously, we sequenced sperm lysin cDNAs from seven California abalone species and showed that positive Darwinian selection promotes their divergence. In this paper an additional 13 lysin sequences are presented representing species from Japan, Taiwan, Australia, New Zealand, South Africa, and Europe. The total of 20 sequences represents the most extensive analysis of a fertilization protein to date. The phylogenetic analysis divides the sequences into two major clades, one composed of species from the northern Pacific (California and Japan) and the other composed of species from other parts of the world. Analysis of nucleotide substitution demonstrates that positive selection is a general process in the evolution of this fertilization protein. Analysis of nucleotide and codon usage bias shows that neither parameter can account for the robust data supporting positive selection. The selection pressure responsible for the positive selection on lysin remains unknown.      

Functional interaction of diphenols with polyphenol oxidase. Molecular determinants of substrate/inhibitor specificity

Polyphenol oxidase (PPO) catalyzes the oxidation of o-diphenols to their respective quinones. The quinones autopolymerize to form dark pigments, an undesired effect. PPO is therefore the target for the development of antibrowning and antimelanization agents. A series of phenolic compounds experimentally evaluated for their binding affinity and inhibition constants were computationally docked to the active site of catechol oxidase. Docking studies suggested two distinct modes of binding, dividing the docked ligands into two groups. Remarkably, the first group corresponds to ligands determined to be substrates and the second group corresponds to reversible inhibitors. Analyses of the complexes provide structural explanations for correlating subtle changes in the position and nature of the substitutions on diphenols to their functional properties as substrates and inhibitors. Higher reaction rates and binding are reckoned by additional interactions of the substrates with key residues that line the hydrophobic cavity. The docking results suggest that inhibition of oxidation stems from an interaction between the aromatic carboxylic acid group and the apical His109 of the four coordinates of the trigonal pyramidal coordination polyhedron of CuA. The spatial orientation of the hydroxyl in relation to the carboxylic group either allows a perfect fit in the substrate cavity, leading to inhibition, or because of a steric clash flips the molecule vertically, facilitating oxidation. This is the first study to explain, at the molecular level, the determinants of substrate and inhibitor specificity of a catechol oxidase, thereby providing a platform for the design of selective inhibitors useful to both the food and pharmaceutical industries.     

Degradative potential of Stenotrophomonas strain HPC383 having genes homologous to dmp operon

A strain, Stenotrophomonas HPC383 is isolated from effluent treatment plant treating wastewater from pesticide industry; degrades various aromatic compounds (cresols, phenol, catechol, 4methyl-catechol and hydroquinone) and crude oil, as determined through HPLC and GC analysis. Culture HPC383 could degrade (%) various compounds (1 mM) from a mixture: phenol - 99, p-cresol - 100, 4-methylcatechol - 96 and hydroquinone - 43 within 48 h of incubation, whereas it took 7 days to degrade 94% of 0.5% crude oil. Gene locus dmpN, to identify phenol degrading capacity was determined by PCR followed by southern analysis. The sequenced DNA fragment exhibited 99% sequence similarity to phenol hydroxylase gene from Arthrobacter sp. W1 (FJ610336). Amino acid sequence analysis of phenol hydroxylase reveals it to belong to high-Ks (affinity constant) group. Application of HPC383 in bioremediation of aquatic and terrestrial sites contaminated with petrochemical has been suggested.     

Colonization and lignin decomposition of Camellia japonica leaf litter by endophytic fungi

Endophytic fungi occur on various types of leaf litter, but few studies have been done on their roles as saprophytes in decomposition. This study examined the succession of fungi in live, newly shed, and decomposing leaves at 2 months of decomposition of Camellia japonica and chemical changes in decomposing leaves colonized by endophytes. Coccomyces nipponicum, Lophodermium sp., Geniculosporium sp. 1, and Colletotrichum gloeosporioides were isolated from living leaves. Coccomyces nipponicum and Lophodermium sp. were also isolated frequently from newly shed and decomposing leaves. These two fungi caused a decrease of lignin content and bleaching in decomposing leaves under field and laboratory conditions. Total hyphal length in decomposing leaves was higher in bleached portions than in surrounding nonbleached portions, which probably reflected the early onset of hyphal growth of endophytes inside leaf tissue at leaf senescence or death. Incubation of newly shed leaves that were sterilized to exclude previously established endophytes resulted in no occurrence of bleached portions in decomposing leaves on the forest floor. This result indicated that these endophytes were incapable of colonizing leaves directly after litterfall and that the persistence of endophytes from live leaves was crucial for their colonization in decomposing leaves.     

Mechanical characterization of adult stem cells from bone marrow and perivascular niches

Therapies using adult stem cells often require mechanical manipulation such as injection or incorporation into scaffolds. However, force-induced rupture and mechanosensitivity of cells during manipulation is largely ignored. Here, we image cell mechanical structures and perform a biophysical characterization of three different types of human adult stem cells: bone marrow CD34+ hematopoietic, bone marrow mesenchymal and perivascular mesenchymal stem cells. We use micropipette aspiration to characterize cell mechanics and quantify deformation of subcellular structures under force and its contribution to global cell deformation. Our results suggest that CD34+ cells are mechanically suitable for injection systems since cells transition from solid- to fluid-like at constant aspiration pressure, probably due to a poorly developed actin cytoskeleton. Conversely, mesenchymal stem cells from the bone marrow and perivascular niches are more suitable for seeding into biomaterial scaffolds since they are mechanically robust and have developed cytoskeletal structures that may allow cellular stable attachment and motility through solid porous environments. Among these, perivascular stem cells cultured in 6% oxygen show a developed cytoskeleton but a more compliant nucleus, which can facilitate the penetration into pores of tissues or scaffolds. We confirm the relevance of our measurements using cell motility and migration assays and measure survival of injected cells. Since different types of adult stem cells can be used for similar applications, we suggest considering mechanical properties of stem cells to match optimal mechanical characteristics of therapies.     

Isolation and characterization of a phorate degrading bacterium

Aims:  To study the degradation of phorate by a bacterium isolated from phorate-contaminated sites.
Methods and Results:  Ralstonia eutropha strain AAJ1 isolated from soil was found to degrade phorate (supplied as sole carbon source) upto 85% in 10 days in liquid medium. Half-life ( t _½) of phorate in the liquid medium in control (uninoculated) and in experimental (inoculated with R. eutropha , strain AAJ1) samples was recorded as 36·49 and 6·29 days, respectively. Kinetics revealed that phorate degradation depends on time and the reaction follows the first order kinetics. Diethyl dithiophosphate was one of the degradation products, which is markedly less toxic than the parent compound; other degradation products included phorate sulfoxide and phorate sulfone. Release of inorganic phosphates and sulfates indicated the potential of the isolate to further degrade the above-mentioned metabolites to simpler forms. The strain was also found to posses phosphomonoesterase and phosphodiesterase enzymatic activity, which are involved in biodegradation of organophosphorus compounds.
Conclusions:  Ralstonia eutropha AAJ1 could degrade and detoxify phorate upto 85% in 10 days in laboratory conditions.
Significance and Impact of the Study:  The isolate has the potential to be utilized for remediation of phorate-contaminated water and soil.     

Chloroquine treatment increases detection of 5-fluorouracil-induced apoptosis index in vivo

Although apoptosis can be readily assessed in vitro with a variety of techniques, the detection of apoptosis in the in vivo setting poses a much more difficult proposition. Apoptosis in an organism is followed almost inevitably by rapid clearance of dying cells via phagocytosis, thus limiting the ability to analyze apoptosis in vivo using classical techniques. To address this issue, we developed a method to enhance in vivo apoptosis detection using pretreatment with chloroquine, an inhibitor of macrophage activity, in Swiss albino mice. This technique resulted in a significant increase in the accumulation of apoptotic cells induced by 5-fluorouracil, as detected by propidium iodide staining in solid and ascitic forms of Ehrlich ascitic tumors and in bone marrow cells. We further validated our technique using DNA fragmentation and endonuclease assays. Our results demonstrated that chloroquine pretreatment can significantly enhance accumulation of apoptotic cells in organisms, and we envision combining this method with modern imaging techniques to optimize in vivo detection of apoptosis.     

Mitochondrial DNA and bindin gene sequence evolution among allopatric species of the sea urchin genus Arbacia

Sea urchins of the genus Arbacia (order Stirodonta) have discontinuous allopatric distributions ranging over thousands of kilometers. Mitochondrial DNA (mtDNA) sequences were used to reconstruct phylogenetic relationships of four Arbacia species and their geographic populations. There is little evidence of genetic structuring of populations within species, except in two cases at range extremes. The mtDNA sequence differentiation between species suggests that divergence occurred about 4-9 MYA. Gene sequences encoding the sperm protein bindin and its intron were obtained and compared with the mtDNA phylogeny. Sea urchins among the well-studied echinoid order Camarodonta, with degrees of mtDNA divergence similar to those of Arbacia species, are known to have remarkable variation in bindin. However, in Arbacia, little variation in deduced amino acid sequences of bindin was found, indicating that purifying selection acts on the protein. In contrast, bindin intron sequences showed much differentiation, including numerous insertion/deletions. Fertilization experiments performed between a divergent pair of Arbacia species from the Atlantic and Pacific Oceans revealed no evidence of blocks to gamete recognition. In Arbacia, fertilization specificities may have evolved relatively slowly as a result of extensive gene flow within species, greater functional constraint on the bindin polypeptide, or reduced selective pressure for species recognition in singly occurring species.