<Emphasis Type="Italic">Pseudomonas extremaustralis</Emphasis> sp. nov., a Poly(3-hydroxybutyrate) Producer Isolated from an Antarctic Environment

A Gram-negative, mobile, rod-shaped, non-spore-forming bacterium (strain 14-3^T) was isolated from a temporary pond in Antarctica. On the basis of 16S rRNA gene sequence similarity, strain 14-3^T was shown to belong to the genus Pseudomonas sensu stricto. Physiological and biochemical tests supported the phylogenetic affiliation. Strain 14-3^T is closely related to Pseudomonas veronii DSM 11331^T, sharing 99.7% sequence similarity. DNA–DNA hybridization experiments between the two strains showed only moderate reassociation similarity (35.1%). Tests for arginine dihydrolase and nitrate reduction were positive, while those for denitrification, indol production, glucose acidification, urease, ß-galactosidase, esculin, caseine and gelatin hydrolysis were negative. Growth of this bacterium occurred in a range from 4 to 37°C but not at 42°C. It accumulated poly(3-hydroxybutyrate) when grown on sodium octanoate medium. Strain 14-3^T therefore represents the type strain of a new species, for which the name Pseudomonas extremaustralis sp. nov. is proposed. The type strain 14-3^T has been deposited as DSM 17835^T and as CIP 109839^T.     

Cytotoxic and Genotoxic Effects of <Emphasis Type="Italic">cis</Emphasis>-Tetraammine(oxalato)Ruthenium(III) Dithionate on the Root Meristem Cells of <Emphasis Type="Italic">Allium cepa</Emphasis>

Ruthenium complexes have attracted much attention as possible building blocks for new transition-metal-based antitumor agents. The present study examines the mitotoxic and clastogenic effects induced in the root tips of Allium cepa by cis-tetraammine(oxalato)ruthenium(III) dithionate {cis-[Ru(C₂O₂)(NH₃)₄]₂(S₂O₆)} at different exposure durations and concentrations. Correlation tests were performed to determine the effects of the time of exposure and concentration of ruthenium complex on mitotic index (MI) and mitotic aberration index. A comparison of MI results of cis-[Ru(C₂O₂)(NH₃)₄]₂(S₂O₆) to those of lead nitrate reveals that the ruthenium complex demonstrates an average mitotic inhibition eightfold higher than lead, with the frequency of cellular abnormalities almost fourfold lower and mitotic aberration threefold lower. A. cepa root cells exposed to a range of ruthenium complex concentrations did not display significant clastogenic effects. Cis-tetraammine(oxalato)ruthenium(III) dithionate therefore exhibits a remarkable capacity to inhibit mitosis, perhaps by inhibiting DNA synthesis or blocking the cell cycle in the G2 phase. Further investigation of the mechanisms of action of this ruthenium complex will be important to define its clinical potential and to contribute to a novel and rational approach to developing a new metal-based drug with antitumor properties complementary to those exhibited by the drugs already in clinical use.     

Evaluation of anesthetic protocol for the collection of semen from captive collared peccaries (Tayassu tajacu) by electroejaculation

The objective of this study is to verify and compare the effects of acepromazine–tiletamine–zolazepam and propofol used in anesthetic protocols for semen collection by electroejaculation from captive collared peccaries. Ten sexually mature animals were physically restrained and anesthetized by either intravenous administration of tiletamine–zolazepam (2 mg/kg) after acepromazine premedication, or a propofol dose of 5 mg/kg. The onset of anesthetic recovery was determined by the animals regaining consciousness and attempting to stand. Semen was collected by electroejaculation and evaluated for volume, pH, sperm concentration, progressive motility, morphology, percentage of live cells and functional membrane integrity. Six anesthetized animals with the acepromazine–tiletamine–zolazepam protocol showed erection, but semen could be collected in only four (40%) attempts. Of the animals anesthetized using propofol, nine showed erection, and the ejaculates were collected in eight (80%) attempts. Furthermore, propofol afforded rapid recovery of animals, and ejaculates with enhanced sperm motility and functional membrane integrity as compared with those collected by the other protocol (P < 0.05). In conclusion, use of propofol for anesthetic restraint of collared peccaries enhanced collection of semen by electroejaculation.     

The small GTPase RhoA is crucial for MC3T3‐E1 osteoblastic cell survival

Prolongation of cell survival through prevention of apoptosis is considered to be a significant factor leading to anabolic responses in bone. The current studies were carried out to determine the role of the small GTPase, RhoA, in osteoblast apoptosis, since RhoA has been found to be critical for cell survival in other tissues. We investigated the effects of inhibitors and activators of RhoA signaling on osteoblast apoptosis. In addition, we assessed the relationship of this pathway to parathyroid hormone (PTH) effects on apoptotic signaling and cell survival. RhoA is activated by geranylgeranylation, which promotes its membrane anchoring. In serum‐starved MC3T3‐E1 osteoblastic cells, inhibition of geranylgeranylation with geranylgeranyl transferase I inhibitors increased activity of caspase‐3, a component step in the apoptosis cascade, and increased cell death. Dominant negative RhoA and Y27632, an inhibitor of the RhoA effector Rho kinase, also increased caspase‐3 activity. A geranylgeranyl group donor, geranylgeraniol, antagonized the effect of the geranylgeranyl tranferase I inhibitor GGTI‐2166, but could not overcome the effect of the Rho kinase inhibitor. PTH 1‐34, a potent anti‐apoptotic agent, completely antagonized the stimulatory effects of GGTI‐2166, dominant negative RhoA, and Y27632, on caspase‐3 activity. The results suggest that RhoA signaling is essential for osteoblastic cell survival but that the survival effects of PTH 1‐34 are independent of this pathway. J. Cell. Biochem. 106: 896–902, 2009. © 2009 Wiley‐Liss, Inc.     

Phylogenetic analysis identifies many uncharacterized actin-like proteins (Alps) in bacteria: regulated polymerization, dynamic instability and treadmilling in Alp7A

Actin, one of the most abundant proteins in the eukaryotic cell, also has an abundance of relatives in the eukaryotic proteome. To date though, only five families of actins have been characterized in bacteria. We have conducted a phylogenetic search and uncovered more than 35 highly divergent families of actin-like proteins (Alps) in bacteria. Their genes are found primarily on phage genomes, on plasmids and on integrating conjugative elements, and are likely to be involved in a variety of functions. We characterize three Alps and find that all form filaments in the cell. The filaments of Alp7A, a plasmid partitioning protein and one of the most divergent of the Alps, display dynamic instability and also treadmill. Alp7A requires other elements from the plasmid to assemble into dynamic polymers in the cell. Our findings suggest that most if not all of the Alps are indeed actin relatives, and that actin is very well represented in bacteria.     

Disruption of Supv3L1 damages the skin and causes sarcopenia, loss of fat, and death

Supv3L1 is a conserved and ubiquitously expressed helicase found in numerous tissues and cell types of many species. In human cells, SUPV3L1 was shown to suppress apoptotic death and sister chromatid exchange, and impair mitochondrial RNA metabolism and protein synthesis. In vitro experiments revealed binding of SUPV3L1 to BLM and WRN proteins, suggesting a role in genome maintenance processes. Disruption of the Supv3L1 gene in the mouse has been reported to be embryonic lethal at early developmental stages. We generated a conditional mouse in which the phenotypes associated with the removal of exon 14 can be tested in a variety of tissues. Disruption mediated by a Mx1 promoter-driven Cre displayed a postnatal growth delay, reduced lifespan, loss of adipose tissue and muscle mass, and severe skin abnormalities manifesting as ichthyosis, thickening of the epidermis, and atrophy of the dermis and subcutaneous tissue. Using a tamoxifen-activatable Esr1/Cre driver, Supv3L1 disruption resulted in growth retardation and aging phenotypes, including loss of adipose tissue and muscle mass, kyphosis, cachexia, and premature death. Many of the abnormalities seen in the Mx1-Cre mice, such as hyperkeratosis characterized by profound scaling of feet and tail, could also be detected in tamoxifen-inducible Cre mice. Conditional ablation of Supv3L1 in keratinocytes confirmed atrophic changes in the skin and ichthyosis-like changes. Together, these data indicate that Supv3L1 is important for the maintenance of the skin barrier. In addition, loss of Supv3L1 function leads to accelerated aging-like phenotypes.     

A quinazoline-derivative DOTA-type gallium(III) complex for targeting epidermal growth factor receptors: synthesis, characterisation and biological studies

The novel DOTA-like chelator 1,4,7,10-tetraazacyclododecane-1-{4-[(3-chloro-4-fluorophenyl)amino]quinazoline-6-yl}propionamide-4,7,10-triacetic acid (H₃L) was synthesised by alkylation of 1,4,7,10-tetraazacyclododecane-1,4,7-tris(t-butyl acetate) with N-{4-[(3-chloro-4-fluorophenyl)amino]quinazoline-6-yl}-3-bromopropionamide, followed by hydrolysis of the ester groups with trifluoracetic acid. H₃L has been fully characterised by multinuclear NMR spectroscopy, mass spectrometry and high-performance liquid chromatography (HPLC). Five protonation constants, log K _Hi , of H₃L were determined by potentiometry and UV–vis spectrophotometry and the values found are 10.47, 9.18, 5.24, 4.00 and 2.23. These methods, complemented with variable-pH ⁷¹Ga NMR studies, allowed us to ascertain the stability constant of the Ga(III) complex of L. GaL has a remarkably high thermodynamic stability constant (log K _ML = 24.5). The radioactive complex ⁶⁷GaL was prepared in high yield and high radiochemical purity. Its HPLC chromatogram is identical to that obtained for the GaL complex prepared at the macroscopic level. At pH 7.4, ⁶⁷GaL has an overall neutral charge, is highly hydrophilic (log D = −1.02 ± 0.03) and presents high in vitro stability in physiological media and in the presence of an excess of diethylenetriaminepentaethanoic acid . In vitro studies indicated that H₃L and GaL do not inhibit the cell growth of epidermal growth factor receptor expressing cell lines, such as A431 cervical carcinoma cells, a result which agrees with the very low cell internalisation found for ⁶⁷GaL in the same cell line. Biodistribution studies in mice indicated high in vivo stability for ⁶⁷GaL, a high total excretion rate and a relatively slow blood clearance, in full accordance with its hydrophilic character and the relatively important protein binding. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The potential of Metarhizium anisopliae and Beauveria bassiana isolates for the control of Aedes aegypti (Diptera: Culicidae) larvae

Fungal isolates were screened against Aedes aegypti larvae. Exposure of larvae to conidial suspensions resulted in 6–90% mortality. An inoculum persistence assay using one of the most virulent isolates showed an approximate half-life of 10 days in water containers. Not all larvae surviving to form pupae resulted in adult emergence.     

Comparative analysis of leaf-type ferredoxin-NADP+ oxidoreductase isoforms in Arabidopsis thaliana

Physiological roles of the two distinct chloroplast-targeted ferredoxin-NADP⁺ oxidoreductase (FNR) isoforms in Arabidopsis thaliana were studied using T-DNA insertion line fnr1 and RNAi line fnr2 . In fnr2 FNR1 was present both as a thylakoid membrane-bound form and as a soluble protein, whereas in fnr1 the FNR2 protein existed solely in soluble form in the stroma. The fnr2 plants resembled fnr1 in having downregulated photosynthetic properties, expressed as low chlorophyll content, low accumulation of photosynthetic thylakoid proteins and reduced carbon fixation rate when compared with wild type (WT). Under standard growth conditions the level of F₀'rise' and the amplitude of the thermoluminescence afterglow (AG) band, shown to correlate with cyclic electron transfer (CET), were reduced in both fnr mutants. In contrast, when plants were grown under low temperatures, both fnr mutants showed an enhanced rate of CET when compared with the WT. These data exclude the possibility that distinct FNR isoforms feed electrons to specific CET pathways. Nevertheless, the fnr2 mutants had a distinct phenotype upon growth at low temperature. The fnr2 plants grown at low temperature were more tolerant against methyl viologen (MV)-induced cell death than fnr1 and WT. The unique tolerance of fnr2 plants grown at low temperature to oxidative stress correlated with an increased level of reduced ascorbate and reactive oxygen species (ROS) scavenging enzymes, as well as with a scarcity in the accumulation of thylakoid membrane protein complexes, as compared with fnr1 and WT. These results emphasize a critical role for FNR2 in the redistribution of electrons to various reducing pathways, upon conditions that modify the photosynthetic capacity of the plant.     

Isomaltulose production from sucrose by Protaminobacter rubrum immobilized in calcium alginate

Different culture conditions for Protaminobacter rubrum and enzymatic reaction parameters were evaluated with the goal of improving isomaltulose production. P. rubrum was grown in a medium with 1% (w/v) cane molasses and 0.5% yeast extract and achieved a maximum cell yield Y_x/s of 0.295 g of cells/g sucrose and a specific growth rate (μ) of 0.192 h⁻¹. The immobilization of P. rubrum cells was carried out with calcium alginate, glutaraldehyde and polyethyleneimine. Stabile immobilized cell pellets were obtained and used 24 times in batch processes. Enzymatic conversion was carried out at different sucrose concentrations and in pH 6 medium with 70% (w/v) sucrose at 30 °C an isomaltulose yield of 89–94% (w/v) was obtained. The specific activity of the P. rubrum immobilized pellets in calcium alginate at 30 °C ranged from 1.6 to 4.0 g isomaltulose g⁻¹ pellet h⁻¹, respectively with 70% and 65% sucrose solution, while in lower sucrose concentration had higher specific activities presumably due to substrate inhibition of the isomaltulose synthase in higher sucrose concentrations.