Formation of coumarines during the degradation of alkyl substituted aromatic oil components by the yeast <Emphasis Type="Italic">Trichosporon asahii</Emphasis>

In this study, we investigated the ability of the yeast Trichosporon asahii SBUG-Y 833 to assimilate phenylalkanes with alkyl chain lengths from 7 to 12 carbon atoms, and we describe for the first time the formation of coumarines via a novel degradation pathway other than the normal terminal and ß-oxidation pathway of the alkyl residues. Besides benzoic acid and its further oxidation products, six new metabolites were identified. These were the three coumarines—4-hydroxycoumarin, 4,6-dihydroxycoumarin, 4,8-dihydroxycoumarin—and the three alkyl substituted aromatic acids—7-phenylheptanoic acid, 2-hydroxyphenylheptanoic acid, and 2-hydroxyphenylpropanoic acid. 4-Hydroxycoumarin was the main extracellular metabolite during the degradation of both odd- and even-chain phenylalkanes and was also produced during further biotransformation of 2-hydroxyphenylpropanoic acid and trans-2-hydroxycinnamic acid. Due to the ability of T. asahii to form hydroxylated coumarines, the transformation of 7-hydroxycoumarin and 2,4-dihydroxyphenylpropanoic acid was investigated. Yeast cells supplemented with 7-hydroxycoumarin formed 6,7-dihydroxycoumarin and 4,7-dihydroxycoumarin. The transformation of 2,4-dihydroxyphenylpropanoic acid yielded to 4,7-dihydroxycoumarin as the main product. All hydroxylated coumarines were continuously accumulated and are very resistant to further oxidation. The high potential of the yeast T. asahii SBUG-Y 833 to form different hydroxylated coumarines from alkylaromatics suggests possible applications in the biotechnological production of coumarine structures with medical potential as anticoagulative and antitumor pharmaceutical.     

The Caenorhabditis elegans HNF4α Homolog, NHR-31, Mediates Excretory Tube Growth and Function through Coordinate Regulation of the Vacuolar ATPase

Nuclear receptors of the Hepatocyte Nuclear Factor-4 (HNF4) subtype have been linked to a host of developmental and metabolic functions in animals ranging from worms to humans; however, the full spectrum of physiological activities carried out by this nuclear receptor subfamily is far from established. We have found that the Caenorhabditis elegans nuclear receptor NHR-31, a homolog of mammalian HNF4 receptors, is required for controlling the growth and function of the nematode excretory cell, a multi-branched tubular cell that acts as the C. elegans renal system. Larval specific RNAi knockdown of nhr-31 led to significant structural abnormalities along the length of the excretory cell canal, including numerous regions of uncontrolled growth at sites near to and distant from the cell nucleus. nhr-31 RNAi animals were sensitive to acute challenge with ionic stress, implying that the osmoregulatory function of the excretory cell was also compromised. Gene expression profiling revealed a surprisingly specific role for nhr-31 in the control of multiple genes that encode subunits of the vacuolar ATPase (vATPase). RNAi of these vATPase genes resulted in excretory cell defects similar to those observed in nhr-31 RNAi animals, demonstrating that the influence of nhr-31 on excretory cell growth is mediated, at least in part, through coordinate regulation of the vATPase. Sequence analysis revealed a stunning enrichment of HNF4α type binding sites in the promoters of both C. elegans and mouse vATPase genes, arguing that coordinate regulation of the vATPase by HNF4 receptors is likely to be conserved in mammals. Our study establishes a new pathway for regulation of excretory cell growth and reveals a novel role for HNF4-type nuclear receptors in the development and function of a renal system.     

Swimming of Xenopus laevis sperm exhibits multiple gears and its duration is extended by egg jelly constituents

The motility of Xenopus sperm is initiated by the osmotic shock experienced when these cells are ejaculated into low-salinity pond water. Motility is brief and is required for the sperm to penetrate the jelly layers and fertilize the egg. In this study we demonstrate that extracts of egg jelly contain factors that extend the period of sperm motility as well as providing a chemoattractant activity as previously reported. Both activities are partially dependent on extracellular calcium. Time-lapse and video microscopy show that after activation of motility the number of motile sperm decreases rapidly, with a half-time of about 2 min. Addition of 10% v/v egg jelly extract ("egg water") increased the number of motile sperm 2-fold over controls at 20 s and about 4- to 10-fold over controls at 10 min after initiation of motility. Extension of motility lifetime was not mediated by a nonspecific protein or by allurin, the egg-water protein that has chemoattractant activity. The helical path of Xenopus sperm exhibited tight coupling between rotational and forward velocities in egg jelly, but coupling changed rapidly from moment to moment in low-salinity buffer. Our observations suggest that jelly-derived factors regulate both the longevity and directionality of sperm propulsion.     

Mouse sperm exhibit chemotaxis to allurin, a truncated member of the cysteine-rich secretory protein family

Allurin, a 21 kDa protein isolated from egg jelly of the frog Xenopus laevis, has previously been demonstrated to attract frog sperm in two-chamber and microscopic assays. cDNA cloning and sequencing has shown that allurin is a truncated member of the Cysteine-Rich Secretory Protein (CRISP) family, whose members include mammalian sperm-binding proteins that have been postulated to play roles in spermatogenesis, sperm capacitation and sperm–egg binding in mammals. Here, we show that allurin is a chemoattractant for mouse sperm, as determined by a 2.5-fold stimulation of sperm passage across a porous membrane and by analysis of sperm trajectories within an allurin gradient as observed by time-lapse microscopy. Chemotaxis was accompanied by an overall change in trajectory from circular to linear thereby increasing sperm movement along the gradient axis. Allurin did not increase sperm velocity although it did produce a modest increase in flagellar beat frequency. Oregon Green 488-conjugated allurin was observed to bind to the sub-equatorial region of the mouse sperm head and to the midpiece of the flagellum. These findings demonstrate that sperm have retained the ability to bind and respond to truncated Crisp proteins over 300 million years of vertebrate evolution.     

A new phenol oxidase produced during melanogenesis and encystment stage in the nitrogen-fixing soil bacterium Azotobacter chroococcum

Laccases are copper-containing phenol oxidases that are commonly found in many types of plant, insect, fungi and bacteria. Whilst phenol oxidases have been well characterized in fungal species, laccase-type enzymes originating from bacteria have been much less well defined. Bacteria belonging to the family Azotobacteraceae share many morphological characteristics with strains already known to exhibit polyphenol and phenol oxidase activity; and hence the aim of this work was to identify and characterize a novel laccase from the isolated strain Azotobacter chroococcum SBUG 1484 in an attempt to provide further understanding of the roles such enzymes play in physiological development. Laccase activity was clearly observed through oxidation of 2,6-dimethoxyphenol, other typical substrates including: methoxy-monophenols, ortho- and para-diphenols, 4-hydroxyindole, and the non-phenolic compound para-phenylenediamine. A. chroococcum SBUG 1484 showed production of a cell-associated phenol oxidase when grown under nitrogen-fixing conditions, and was also observed when cells enter the melanogenic and encystment stages of growth. Catechol which is structurally related to melanin compounds was also released from Azotobacter cells into the surrounding culture medium during nitrogen-fixing growth. From our results we propose that a membrane-bound laccase plays an important role in the formation of melanin, which was monitored to correlate with progression of A. chroococcum SBUG 1484 cells into the encystment stage of growth.     

Cleavage and synthesis function of high and low redox potential laccases towards 4-morpholinoaniline and aminated as well as chlorinated phenols

Laccases are able to mediate both cleavage and synthesis processes. The basis for this dual reaction capability lies in the property of the enzyme laccase to oxidize phenolic, and to some extent non-phenolic substances, to reactive radicals which can undergo on the one hand separations of small substitutents or large molecule parts from the parent compound and on the other hand coupling reactions with other radicals or molecules which are not themselves oxidizable by laccase. The cleavage of the non-phenolic compound 4-morpholinoaniline as well as the deamination of 4-aminophenol and the dechlorination of 4-chlorophenol resulted in the formation of 1,4-hydroquinone which is immediately oxidized by laccase to 1,4-benzoquinone. The formation of the 1,4-hydroquinone/1,4-benzoquinone is the rate limiting step for the synthesis of the heteromolecular dimers and trimers composed of 1,4-benzoquinone and one or two molecules of morpholine. In addition to the synthesis of new compounds from the cleavage products, 4-morpholinoaniline polymerized probably via azo groups and C-N bonds to a homomolecular dimer and trimer. Similarities and differences in cleavage and synthesis reactions catalyzed by the low redox potential laccase of Myceliophthora thermophila (0.46 V) and the high redox potential laccase of Pycnoporus cinnabarinus (0.79 V) were determined. In addition, the dependency of the cleavage and synthesis efficiencies on the (a) structure and redox potential of the laccase, (b) structure and redox potential of the substrate, (c) pH value of the buffer used, (d) incubation temperature, (e) solvent concentration, and (f) laccase activity is discussed in general.     

Population Genomic Analyses Based on 1 Million SNPs in Commercial Egg Layers

Identifying signatures of selection can provide valuable insight about the genes or genomic regions that are or have been under selective pressure, which can lead to a better understanding of genotype-phenotype relationships. A common strategy for selection signature detection is to compare samples from several populations and search for genomic regions with outstanding genetic differentiation. Wright''s fixation index, F_ST, is a useful index for evaluation of genetic differentiation between populations. The aim of this study was to detect selective signatures between different chicken groups based on SNP-wise F_ST calculation. A total of 96 individuals of three commercial layer breeds and 14 non-commercial fancy breeds were genotyped with three different 600K SNP-chips. After filtering a total of 1 million SNPs were available for F_ST calculation. Averages of F_ST values were calculated for overlapping windows. Comparisons of these were then conducted between commercial egg layers and non-commercial fancy breeds, as well as between white egg layers and brown egg layers. Comparing non-commercial and commercial breeds resulted in the detection of 630 selective signatures, while 656 selective signatures were detected in the comparison between the commercial egg-layer breeds. Annotation of selection signature regions revealed various genes corresponding to productions traits, for which layer breeds were selected. Among them were NCOA1, SREBF2 and RALGAPA1 associated with reproductive traits, broodiness and egg production. Furthermore, several of the detected genes were associated with growth and carcass traits, including POMC, PRKAB2, SPP1, IGF2, CAPN1, TGFb2 and IGFBP2. Our approach demonstrates that including different populations with a specific breeding history can provide a unique opportunity for a better understanding of farm animal selection.