Acidithiobacillus ferrivorans, sp. nov.; facultatively anaerobic, psychrotolerant iron-, and sulfur-oxidizing acidophiles isolated from metal mine-impacted environments

Phenotypic and genotypic analysis was carried out on four iron- and sulfur-oxidizing acidophilic bacteria (the “NO-37 group”) isolated from different parts of the world. 16S rRNA phylogeny showed that they are highly related to each other, but are less related to the type strain of Acidithiobacillus ferrooxidans. The NO-37 group isolates are obligate chemolithoautotrophs, facultative anaerobes, diazotrophic, and psychrotolerant. They are less tolerant of extremely low pH, and in contrast to At. ferrooxidans ^T, all of the NO-37 group isolates are motile. The GC contents of genomic DNA of the NO-37 group isolates were around 56 mol% and the DNA–DNA hybridization value between genomic DNA of isolate NO-37 and At. ferrooxidans ^T was 37%. It also appears that the bacteria of the NO-37 group have a different biochemical mechanism for oxidizing ferrous iron than At. ferrooxidans ^T; the gene coding for the archetypal rusticyanin (RusA) was not detected in any of the NO-37 group isolates, rather a gene coding for a homologous protein (RusB) was amplified from three of the four novel isolates. Isolates of the NO-37 group clearly belong to a species that is different to those already recognized in the genus Acidithiobacillus, for which the name Acidithiobacillus ferrivorans is proposed.     

Separation of anti-angiogenic and cytotoxic activities of borrelidin by modification at the C17 side chain

A set of novel borrelidin analogues have been prepared by precursor-directed biosynthesis. Structure-activity relationship analysis suggests that steric structural arrangement within the C17 side chain is important for differentiating cytotoxic and anti-angiogenic activities. A C17-cyclobutyl analogue 3 was found to have markedly increased selectivity for in vitro angiogenesis inhibition over cytotoxicity and is therefore potentially useful as an anticancer agent.     

Novel human cell expression method reveals the role and prevalence of posttranslational modification in nonmuscle tropomyosins

Biochemical studies require large quantities of proteins, which are typically obtained using bacterial overexpression. However, the folding machinery in bacteria is inadequate for expressing many mammalian proteins, which additionally undergo posttranslational modifications (PTMs) that bacteria, yeast, or insect cells cannot perform. Many proteins also require native N- and C-termini and cannot tolerate extra tag amino acids for proper function. Tropomyosin (Tpm), a coiled coil protein that decorates most actin filaments in cells, requires both native N- and C-termini and PTMs, specifically N-terminal acetylation (Nt-acetylation), to polymerize along actin filaments. Here, we describe a new method that combines native protein expression in human cells with an intein-based purification tag that can be precisely removed after purification. Using this method, we expressed several nonmuscle Tpm isoforms (Tpm1.6, Tpm1.7, Tpm2.1, Tpm3.1, Tpm3.2, and Tpm4.2) and the muscle isoform Tpm1.1. Proteomics analysis revealed that human-cell-expressed Tpms present various PTMs, including Nt-acetylation, Ser/Thr phosphorylation, Tyr phosphorylation, and Lys acetylation. Depending on the Tpm isoform (humans express up to 40 Tpm isoforms), Nt-acetylation occurs on either the initiator methionine or on the second residue after removal of the initiator methionine. Human-cell-expressed Tpms bind F-actin differently than their Escherichia coli-expressed counterparts, with or without N-terminal extensions intended to mimic Nt-acetylation, and they can form heterodimers in cells and in vitro. The expression method described here reveals previously unknown features of nonmuscle Tpms and can be used in future structural and biochemical studies with Tpms and other proteins, as shown here for α-synuclein.     

Paraspermatogenesis in Littoraria (Palustorina) articulata, with reference to other Littorinidae (Littorinoidea, Caenogastropoda)

Abstract. The ultrastructure of paraspermatogenesis is examined in the littorinid subfamily Littorininae, with special emphasis on Littoraria (Palustorina) articulata (PHILIPPI 1846). In particular the study focuses on the fate of the nucleus and origin of the rod bodies during parasperm development. Parasperm of the Littorininae are rounded or oblong cells, which undergo an abortive meiosis and eliminate part of the nucleus but often retain a nuclear remnant. The cytoplasm is filled with numerous spherical vesicles in all Littorininae, but in Littoraria (and in certain species of Nodilittorina, Tectarius and Cenchritis) dense 'rod-bodies' also occur. Littoraria (Palustorina) are unique in possessing a flagellum-like structure termed the 'pseudotrich", which lacks an axoneme but contains microtubules during its development. Paraspermatogonia differ from euspermatogonia in the structure of the nucleus and in the extensive rough endoplasmic reticulum (RER) and swollen cytoplasm. Two types of secretions develop in Littoraria : (1) numerous, spherical granules (composed of putative glycoprotein, also seen in other Littorininae) and (2) rhomboid granules (composition uncertain but reacting positively to RNA stains; these granules arising within RER cisternae close to the nucleus). As the rhomboid granules fuse to form the larger, rod-bodies (polygonal in cross section), the RER membrane enclosing the rod-bodies becomes confluent with the outer nuclear membrane, thereby forming a common compartment. Results of this study clearly show that the rod-bodies are secretions of the RER cisternae and not, as claimed in some light microscopic accounts, the product of fusion of eusperm nuclei which have entered the parasperm cytoplasm (either by active eusperm penetration or by phagocytosis). Developmental characteristics of littorinid parasperm show differences between species and may, in some cases, provide characters diagnostic of subgenera.     

Immobilization of alpha-chymotrypsin on poly(urethane-graft-acrylic acid)

A study was made of the immobilization of alpha-chymotrypsin (alpha-CT) onto a previously well characterized synthetic polyurethane grafted with acrylic acid P(U-g-AA). The P(U-g-AA) had previously been prepared using 2,2'-azo-bis-isobutyronitrile (AIBN) as a radical initiator and acrylic acid as monomer in the presence of an unsaturated polyurethane in solution at 60 degrees C. Some kinetic parameters of both the native enzyme and the enzyme immobilized on the P(U-g-AA) were evaluated. Using a Lineweaver-Burk plot (double reciprocal), it was found that the Michaelis-Menten constant (Km(for the immobilized enzyme was (4.0 +/- 0.9) x 10(-3) M and that of the free enzyme was (3.0 +/- 0.2) x 10(-3) M. The enzyme alpha-chymotrypsin was immobilized on the grafted polyurethane micelles/aggregates with about 45% retention of activity. Also the immobilized alpha-CT retained this activity for at least 6 weeks. The immobilized enzyme was found to have a maximum stability at 43 degrees C compared with 36 degrees C in the case of free enzyme, and the pH optimum was shifted from pH 6.6 to pH 8.2. The long-term operational stability of the enzyme was investigated and this is of interest since the enzyme is probably trapped physically in a micellar environment. The assay of the enzyme was carried out in 0.01 M phosphate buffer, pH 7.5, using p-nitrophenyl acetate as a substrate. No inhibition of alpha-CT in the presence of the synthetic ungrafted and grafted polyurethane was observed.     

Localisation of mRNA and co-expression and molecular forms of GRP gene products in endocrine cells of fetal human lung

Summary The presence of bombesin (gastrin-releasing peptide, GRP)-like immunoreactivity in mucosal endocrine cells of human fetal lung is well established. In this study we have investigated the localisation of pro-GRP mRNA and GRP gene products and compared the distribution and levels of extractable GRP-and C-terminal flanking peptide of human pro-GRP-like immunoreactivity in order to verify synthesis and to investigate their coexistence and molecular forms. Human fetal lungs (14 to 23 weeks gestation) were immunostained, and extracts were assayed using regionspecific antisera to pro-GRP. Additional antisera to chromogranin and protein gene product 9.5 (PGP 9.5) were used for immunostaining by the peroxidase anti-peroxidase technique and for double immunofluorescence staining using antisera raised in two species. Immunoreactivity for both bombesin (GRP) and flanking peptide was seen mainly in the same endocrine cells, but more cells were stained with antisera to flanking peptide than with antiserum to bombesin (GRP). In situ hybridisation showed that pro-GRP mRNA was present and thus synthesis of the peptides was taking place. Endocrine cells and nerve fibres were PGP 9.5-immunoreactive, and a subset of cells was immunoreactive for bombesin gene products. Radioimmunoassay and chromatography show that pro-GRP is present in both the uncleaved and cleaved forms, and, in agreement with immunocytochemistry results, that an excess of C-terminal peptide of pro-GRP is detectable. It is therefore concluded that GRP-like peptides and flanking peptide are co-local-ised in human pulmonary endocrine cells, but the latter is found in larger concentrations than free GRP. Thus GRP-like peptides may be secreted separately from the flanking peptide(s) of pro-GRP. Furthermore PGP 9.5 appears to be a useful marker for endocrine cells in the respiratory epithelium of human fetal lung.     

Metabolism of Pyridine Compounds by Phthalate-Degrading Bacteria

Bacteria were isolated from marine sediments that grew aerobically on m-phthalate, p-phthalate, or dipicolinate (2,6-pyridine dicarboxylate [2,6-PDCA]). Strain OP-1, which grew on o-phthalate and was previously obtained from a marine source, was also studied. Intact cells of each organism demonstrated Na⁺-dependent oxidation of their growth substrates. Strain PCC5M grew on dipicolinate but did not metabolize m-phthalate. The phthalate degraders, however, demonstrated Na⁺-dependent metabolism of the appropriate PDCA analogs. 2,6-PDCA was transformed by strain CC9M when this strain was grown on m-phthalate, 2,5-PDCA was metabolized by strain PP-1 grown on p-phthalate, and 2,3-PDCA (quinolinate) was oxidized by strain OP-1 grown on o-phthalate. Spectral changes accompanying the Na⁺-dependent transformations of the PDCA analogs suggest the formation of hydroxylated compounds. Metabolism probably occurred via phthalate hydroxylases; this is a previously unrecognized route for the environmental transformation of pyridine compounds. Hydroxylated products may feed into known pathways for the catabolism of pyridines or be photochemically degraded because of their absorbance in the solar actinic range (wavelengths > 300 nm). The results reinforce recent evidence for the broad potential of aromatic hydroxylase systems for the destruction of pollutants.     

New Routes for Aerobic Biodegradation of Dimethylsulfoniopropionate

Dimethylsulfoniopropionate (DMSP), an osmolyte in marine plants, is biodegraded by cleavage of dimethyl sulfide (DMS) or by demethylation to 3-methiolpropionate (MMPA) and 3-mercaptopropionate (MPA). Sequential demethylation has been observed only with anoxic slurries of coastal sediments. Bacteria that grew aerobically on MMPA and DMSP were isolated from marine environments and phytoplankton cultures. Enrichments with DMSP selected for bacteria that generated DMS, whereas MMPA enrichments selected organisms that produced methanethiol (CH₃SH) from either DMSP or MMPA. A bacterium isolated on MMPA grew on MMPA and DMSP, but rapid production of CH₃SH from DMSP occurred only with DMSP-grown cells. Low levels of MPA accumulated during growth on MMPA, indicating demethylation as well as demethiolation of MMPA. The alternative routes for DMSP biodegradation via MMPA probably impact on net DMS fluxes to the marine atmosphere.     

The Earth’s Magnetic Field and Visual Landmarks Steer Migratory Flight Behavior in the Nocturnal Australian Bogong Moth

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Metabolism of methylated osmolytes by aerobic bacteria from Mono Lake, a moderately hypersaline, alkaline environment

Abstract: Three strains of aerobic bacteria were isolated from water and sediment samples of Mono Lake, a moderately hypersaline (90 ppt), alkaline (pH 9.7) lake in California. The organisms, Gram-negative rods, grew fastest at about pH 9.7 with no growth or much slower growth at pH 7.0. All three isolates grew on glycine betaine (GB) and respirometric experiments indicated that catabolism was by sequential demethylation with dimethyl glycine and sarcosine as intermediates. Two of the isolates also grew on dimethylsulfoniopropionate (DMSP), one with cleavage of the DMSP to yield dimethyl sulfide (DMS) and acrylate, and the other by demethylation with 3-methiolpropionate (MMPA) as an intermediate and the production of methanethiol from MMPA. The methylated osmolytes supported growth at salinities similar to those in Mono Lake, but, at higher salinities, catabolism was suppressed and GB and DMSP functioned as osmolytes. GB and DMSP probably originate from cyanobacteria and/or phytoplankton in Mono Lake and this report is the first indication of both the DMS and demethylation/methanethiol-producing pathways for DMSP degradation in a nonmarine environment.