Deficiency of X and Y chromosomal pairing at meiotic prophase in spermatocytes of sterile interspecific hybrids between laboratory mice (Mus domesticus) and Mus spretus

The normal association between the X and Y chromosomes at metaphase I of meiosis, as seen in air-dried light microscope preparations of mouse spermatocytes, is frequently lacking in the spermatocytes of the sterile interspecific hybrid between the laboratory mouse strains C57BL/6 and Mus spretus. The purpose of this work is to determine whether the separate X and Y chromosomes in the hybrid are asynaptic, caused by failure to pair, or desynaptic, caused by precocious dissociation. Unpaired X-Y chromosomes were observed in air-dried preparations at diakinesis, just prior to metaphase I. Furthermore, immunocytology and electron microscopy studies of surface-spread pachytene spermatocytes indicate that the X and Y chromosomes frequently fail to initiate synapsis as judged by the failure to form a synaptonemal complex between the pairing regions of the X and Y Chromosomes. Several additional chromosomal abnormalities were observed in the hybrid. These include fold-backs of the unpaired X or Y cores, associations between the autosome and sex chromosome cores, and autosomal univalents. The occurrence of abnormal autosomal and XY-autosomal associations was also correlated with cell degeneration during meiotic prophase. The primary breakdown in hybrid spermatogenesis occurs at metaphase I (MI), with the appearance of degenerated cells at late MI. In those cells, the X and Y are decondensed rather than condensed as they are in normal mouse MI spermatocytes. These results, in combination with the previous genetic analysis of spermatogenesis in hybrids and backcrosses with fertile female hybrids, suggest that the spermatogenic breakdown in the interspecific hybrid is primarily correlated with the failure of XY pairing at meiotic prophase, asynapsis, followed by the degeneration of spermatocytes at metaphase I. Secondarily, the failure of XY pairing can be accompanied by failure of autosomal pairing, which appears to involve an abnormal sex vesicle and degeneration at pachytene or diplotene.by C. Heyting     

Production and characterization of ligninolytic enzymes ofBjerkandera adusta grown on wood meal/wheat bran culture and production of these enzymes using a rotary-solid fermenter

Manganese peroxidase (MnP) and lignin peroxidase (LiP) were produced by growing a white-rot fungusBjerkandera adusta statically, on a wood meal/wheat bran culture in flasks. MnP and LiP reached their maximum activity after 6 and 19 days of inoculation, respectively. Both MnP and LiP are thought to be important enzymes in lignin biodegradation byB. adusta. Ion exchange chromatography showed thatB. adusta produced a single LiP and a single MnP enzyme in wood meal/wheat bran culture. These enzymes were separated and characterized. The molecular weight of MnP was 46,500 with a pl of 3.9. The molecular weight of LiP was estimated to be 47,000 with a pl of 3.5. Spectral analysis demonstrated that both enzymes are heme proteins. Production of these enzymes was also achieved using a rotarysolid culture fermenter. MnP, LiP and veratryl alcohol oxidase were produced byB. adusta in the fermenter.     

The interaction between importin-α and Nup153 promotes importin-α/β-mediated nuclear import

Nuclear transport is mediated by transport factors, including the importin β family members. The directionality of nuclear transport is governed by the asymmetrical distribution of the small GTPase Ran. Of note, importin α/β-mediated import of classical nuclear localization signal (cNLS)--containing cargo is more efficient than other Ran-dependent import pathways that do not require importin α. In this study, we characterized the role of importin α in nuclear transport by examining import efficiencies of cNLS-cargo/importin α/β complexes. We first depleted digitonin-permeabilized semi-intact cells of endogenous importin α and used the cells to show that the interaction between importin α and Nup153--a component of the nuclear pore complex (NPC)--is essential for efficient import of importin β-binding domain containing substrates, but not other cargoes that directly bind to importin β. Moreover, we found that the binding of importin α to Nup153 facilitates cNLS-mediated import, and demonstrated that importin α in import complexes and cargo-free importin α prebound to Nup153 promote efficient import of cNLS-containing proteins. This is the first in vitro study showing that in conjunction with Nup153, importin α contributes to directionally biased exit of cNLS-containing cargo to the nuclear side of NPCs.     

A novel human hepatoma cell line, FLC-4, exhibits highly enhanced liver differentiation functions through the three-dimensional cell shape

We characterized three-dimensional human hepatoma cell lines, functional liver cell (FLC) cell lines, to establish a highly differentiated hepatoma cell line. We investigated the effect of extracellular matrix and cell morphology on liver-specific gene expression in FLC cells. The hepatocyte nuclear factor-4α (HNF-4α) and other liver-specific gene expressions were enhanced in spherical FLC-4 cells on EHS-gel, but other human hepatoma cells such as HepG2 did not show the enhancement. Importantly, the liver-specific gene expression levels in spherical FLC-4 cells cultured on EHS-gel were comparable to those of human liver and were much higher than those of other human hepatoma cell lines. The major matrix components and growth factors in EHS-gel did not affect cell shape and liver functions. To exclude any effect of the extracellular matrix, we made spherical FLC-4 cells by actin filament disruption. The actin-disrupted spherical cells also showed an enhanced liver-specific gene expression. We concluded that three-dimensional cell shape per se is one of the most important determinants of liver differentiation functions in FLC-4 cells. Cell morphology-dependent induction of liver-specific gene expression was mediated through microtubule organization. In conclusion, differentiation of FLC-4 human hepatoma cell line can be enhanced to a human liver-like level through the three-dimensional cell shape in a microtubule-dependent manner.     

<Emphasis Type="Italic">Sporotomaculum syntrophicum</Emphasis> sp. nov., a novel anaerobic,syntrophic benzoate-degrading bacterium isolated from methanogenic sludge treating wastewater from terephthalate manufacturing

An anaerobic, mesophilic, syntrophic benzoate-degrading bacterium, designated strain FB(T), was isolated from methanogenic sludge which had been used to treat wastewater from the manufacture of terephthalic acid. Cells were non-motile gram-positive rods that formed spores. The optimum temperature for growth was 35-40 degrees C, and the optimum pH was 7.0-7.2. A co-culture with the hydrogenotrophic methanogen Methanospirillum hungatei converted benzoate to acetate, carbon dioxide, and methane. Butyrate transiently accumulated at a high concentration of 2.5 mM during degradation. Besides benzoate, no other compound tested supported growth of the co-culture. Crotonate supported growth of strain FB(T) in pure culture. Furthermore, the strain degraded benzoate in pure culture with crotonate as co-substrate to produce acetate and butyrate. The strain was not able to utilize sulfate, sulfite, thiosulfate, nitrate, fumarate, or Fe(III) as electron acceptor. The G+C content of the DNA was 46.8 mol%. Strain FB(T) contained MK-7 as the major quinone and C(16:1) as the major fatty acid. 16S rDNA sequence analysis revealed that the strain was a member of the genus Sporotomaculum, even though it exhibited significant differences, such as the capacity for syntrophic growth, to the known member of the genus. Hence, we propose the name Sporotomaculum syntrophicum sp. nov. for strain FB(T). The type strain is strain FB(T) (DSM 14795, JCM 11475).     

Sequence-Specific Cleavage of Small-Subunit (SSU) rRNA with Oligonucleotides and RNase H: a Rapid and Simple Approach to SSU rRNA-Based Quantitative Detection of Microorganisms

A rapid and simple approach to the small-subunit (SSU) rRNA-based quantitative detection of a specific group of microorganisms in complex ecosystems has been developed. The method employs sequence-specific cleavage of rRNA molecules with oligonucleotides and RNase H. Defined mixtures of SSU rRNAs were mixed with an oligonucleotide (referred to as a “scissor probe”) that was specifically designed to hybridize with a particular site of targeted rRNA and were subsequently digested with RNase H to proceed to sequence-dependent rRNA scission at the hybridization site. Under appropriate reaction conditions, the targeted rRNAs were correctly cut into two fragments, whereas nontargeted rRNAs remained intact under the same conditions. The specificity of the cleavage could be properly adjusted by controlling the hybridization stringency between the rRNA and the oligonucleotides, i.e., by controlling either the temperature of the reaction or the formamide concentration in the hybridization-digestion buffer used for the reaction. This enabled the reliable discrimination of completely matched rRNA sequences from single-base mismatched sequences. For the detection of targeted rRNAs, the resulting RNA fragment patterns were analyzed by gel electrophoresis with nucleotide-staining fluorescent dyes in order to separate cleaved and intact rRNA molecules. The relative abundance of the targeted SSU rRNA fragments in the total SSU rRNA could easily be calculated without the use of an external standard by determining the signal intensity of individual SSU rRNA bands in the electropherogram. This approach provides a fast and easy means of identification, detection, and quantification of a particular group of microbes in clinical and environmental specimens based on rRNA.     

Structural and biochemical characterization of glycoside hydrolase family 79 β-glucuronidase from Acidobacterium capsulatum

We present the first structure of a glycoside hydrolase family 79 β-glucuronidase from Acidobacterium capsulatum, both as a product complex with β-D-glucuronic acid (GlcA) and as its trapped covalent 2-fluoroglucuronyl intermediate. This enzyme consists of a catalytic (β/α)(8)-barrel domain and a β-domain with irregular Greek key motifs that is of unknown function. The enzyme showed β-glucuronidase activity and trace levels of β-glucosidase and β-xylosidase activities. In conjunction with mutagenesis studies, these structures identify the catalytic residues as Glu(173) (acid base) and Glu(287) (nucleophile), consistent with the retaining mechanism demonstrated by (1)H NMR analysis. Glu(45), Tyr(243), Tyr(292)-Gly(294), and Tyr(334) form the catalytic pocket and provide substrate discrimination. Consistent with this, the Y292A mutation, which affects the interaction between the main chains of Gln(293) and Gly(294) and the GlcA carboxyl group, resulted in significant loss of β-glucuronidase activity while retaining the side activities at wild-type levels. Likewise, although the β-glucuronidase activity of the Y334F mutant is ~200-fold lower (k(cat)/K(m)) than that of the wild-type enzyme, the β-glucosidase activity is actually 3 times higher and the β-xylosidase activity is only 2.5-fold lower than the equivalent parameters for wild type, consistent with a role for Tyr(334) in recognition of the C6 position of GlcA. The involvement of Glu(45) in discriminating against binding of the O-methyl group at the C4 position of GlcA is revealed in the fact that the E45D mutant hydrolyzes PNP-β-GlcA approximately 300-fold slower (k(cat)/K(m)) than does the wild-type enzyme, whereas 4-O-methyl-GlcA-containing oligosaccharides are hydrolyzed only 7-fold slower.     

Asymmetric Reduction of 4-Oxoisophorone Using Immobilized Thermophilic Bacteria Under Conditions of Controlled Cell Growth

Asymmetric reduction of 2,6,6,-trimethyl-2-cyclohexene-l,4-dione (4-oxoisophrone) to (6R)-2,2,6-trimethyl-1,4-cyclohexane-dione((3R)-dihydro-4-oxoisophorone) was catalysed by immobilized thermophilic bacteria, Thermomonospora curvata JTS 321. Because of leakage of entrapped cells from gel beads during reactions using culture medium, we optimized the medium to allow the microbial conversion under conditions of controlled cell growth. Of the media screened, liver infusion medium was found to be the most suitable and microbial conversion was achieved without cell leakage from the immobilized gels. Immobilized T. curvata cells were repeatedly used for the asymmetric reduction of 4-oxoisophorone, more than 15 times, with an extent of conversion of 50%.     

Isolation and Characterization of 2-Nitroimidazole Produced by Streptomyces Species as an Inhibitor of Both Carbonic Anhydrase and Shell Formation in the Barnacle Balanus amphitrite

Carbonic anhydrase is thought to be involved in the process of calcium carbonate deposition in calcified tissues of many organisms. Barnacles form hard calcified shells for protection against predation, and represent a class of marine-fouling animals. In order to inhibit barnacle growth by inhibiting shell formation, we searched for carbonic anhydrase inhibitors from microbial secondary metabolites. A simple assay for assessing carbonic-anhydrase-inhibiting activity was developed. Screening of many microorganisms isolated from soil with this assay resulted in a microbial strain that produced a carbonic anhydrase inhibitor. This strain was identified as Streptomyces eurocidicus mf294. The inhibitor was isolated through 4 purification steps and identified as 2-nitroimidazole on the basis of spectroscopic data. 2-Nitroimidazole inhibited barnacle carbonic anhydrase dose-dependently and complete inhibition was reached at the concentration of 1 x 10(-5) M. 2-Nitroimidazole did not affect settlement or metamorphosis of barnacle larvae, but inhibited shell formation at concentrations higher than 1 x 10(-4) M. These findings strongly support the idea that carbonic anhydrase is involved in calcification.