Plant growth-promoting Pseudomonas bearing catabolic plasmids: Naphthalene degradation and effect on plants

Two IncP-9 naphthalene degradative plasmids pOV17 and pBS216 were transferred into plant growth-promoting Pseudomonas which were represented by species P. aureofaciens, P. chlororaphis, P. fluorescens, and P. putida. The strains with the same plasmid differed significantly by their growth parameters, stability of the plasmid and plant protective effect from naphthalene action. Strains P. putida 53a(pOV17) and P. chlororaphis PCL1391(pOV17) demonstrated higher population number in the rhizosphere. Moreover, they protected the mustard plants from naphthalene toxic influence more effectively than the wild type strain P. aureofaciens OV17(pOV17). The activity of catechol-2,3-dioxygenase in the strains with the plasmid pOV17 was higher than that in strains with the plasmid pBS216. The strain P. putida 53a(pOV17) with high catechol-2,3-dioxygenase activity has been demonstrated to have the best protective effect. The strain P. putida 53a(pBS216) without catechol dioxygenases activities did not have protective effect but suppressed the plant germination.     

Hyalomma aegyptium as dominant tick in tortoises of the genus Testudo in Balkan countries, with notes on its host preferences

Collection of 1327 ticks sampled throughout Greece, Bulgaria, Romania and Croatia, from 211 tortoises belonging to three species, Testudo marginata Schoepff, T. graeca Linnaeus, and T. hermanni Gmelin, revealed the presence of four species of ixodid ticks, namely Hyalomma aegyptium (Linnaeus), Haemaphysalis sulcata Canestrini and Fanzago, H. inermis Birula and Rhipicephalus sanguineus (Latreille). Study confirmed the strong dominance of all life stages of H. aegyptium among ticks parasitizing west Palaearctic tortoises of genus Testudo Linnaeus. Furthermore, a considerable portion of ticks collected from tortoises in southwestern Bulgaria represent larvae and nymphs of H. sulcata. At the same area we collected as exception one larva and one nymph of H. inermis from a single specimen of T. hermanni. Our findings of four adults of R. sanguineus is the first record of this species from reptilian host. According to our results achieved on localities with syntopic occurrence of two tortoise species, T. marginata and T. graeca represent in the Balkans the principal hosts of H. aegyptium, whereas T. hermanni serves only as an alternative host in the areas close to range of either T. marginata or T. graeca.     

Oxygen enhances lethal effect of high-intensity, ultrashort electrical pulses

The study explored the effect of ambient oxygen on mammalian cell survival after exposure to 10 ns duration, high voltage electrical pulses (nsEP, 80-90 or 120-130 kV/cm; 200-400 pulses per exposure). Cell samples were equilibrated with pure nitrogen, atmospheric air, or pure oxygen prior to the nsEP treatment and were returned to the incubator (air + 5% CO2) shortly after the exposure. The experiments established that survival of hypoxic Jurkat and U937 cells exceeded that of air-equilibrated controls about twofold (P < .01). Conversely, saturation of the medium with oxygen prior to exposure decreased Jurkat cell survival about 1.5 times, P < .01. Attenuation of the cytotoxic effect under hypoxic conditions resembled a well-known effect of oxygen on cell killing by sparsely ionizing radiations and may be indicative of the similarity of underlying cell damage mechanisms.     

Mammalian Rad9 plays a role in telomere stability, S- and G2-phase-specific cell survival, and homologous recombinational repair

The protein products of several rad checkpoint genes of Schizosaccharomyces pombe (rad1+, rad3+, rad9+, rad17+, rad26+, and hus1+) play crucial roles in sensing changes in DNA structure, and several function in the maintenance of telomeres. When the mammalian homologue of S. pombe Rad9 was inactivated, increases in chromosome end-to-end associations and frequency of telomere loss were observed. This telomere instability correlated with enhanced S- and G2-phase-specific cell killing, delayed kinetics of gamma-H2AX focus appearance and disappearance, and reduced chromosomal repair after ionizing radiation (IR) exposure, suggesting that Rad9 plays a role in cell cycle phase-specific DNA damage repair. Furthermore, mammalian Rad9 interacted with Rad51, and inactivation of mammalian Rad9 also resulted in decreased homologous recombinational (HR) repair, which occurs predominantly in the S and G2 phases of the cell cycle. Together, these findings provide evidence of roles for mammalian Rad9 in telomere stability and HR repair as a mechanism for promoting cell survival after IR exposure.     

Comparative anatomical study of internal brooding in three anascan bryozoans (Cheilostomata) and its taxonomic and evolutionary implications

The anatomical structure of internal sacs for embryonic incubation was studied using SEM and light microscopy in three cheilostome bryozoans-Nematoflustra flagellata (Waters,1904), Gontarella sp., and Biflustra perfragilis MacGillivray, 1881. In all these species the brood sac is located in the distal half of the maternal (egg-producing) autozooid, being a conspicuous invagination of the body wall. It consists of the main chamber and a passage (neck) to the outside that opens independently of the introvert. There are several groups of muscles attached to the thin walls of the brood sac and possibly expanding it during oviposition and larval release. Polypide recycling begins after oviposition in Gontarella sp., and the new polypide bud is formed by the beginning of incubation. Similarly, polypides in brooding zooids degenerate in N. flagellata and, sometimes, in B. perfragilis. In the evolution of brood chambers in the Cheilostomata, such internal sacs for embryonic incubation are considered a final step, being the result of immersion of the brooding cavity into the maternal zooid and reduction of the protecting fold (ooecium). Possible reasons for this transformation are discussed, and the hypothesis of Santagata and Banta (Santagata and Banta1996) that internal brooding evolved prior to incubation in ovicells is rejected.     

Quantification of cerebrospinal fluid transport across the cribriform plate into lymphatics in rats

A major pathway by which cerebrospinal fluid (CSF) is removed from the cranium is transport through the cribriform plate in association with the olfactory nerves. CSF is then absorbed into lymphatics located in the submucosa of the olfactory epithelium (olfactory turbinates). In an attempt to provide a quantitative measure of this transport, 125I-human serum albumin (HSA) was injected into the lateral ventricles of adult Fisher 344 rats. The animals were killed at 10, 20, 30, 40, and 60 min after injection, and tissue samples, including blood (from heart puncture), skeletal muscle, spleen, liver, kidney, and tail were excised for radioactive assessment. The remains were frozen. To sample the olfactory turbinates, angled coronal tissue sections anterior to the cribriform plate were prepared from the frozen heads. The average concentration of 125I-HSA was higher in the middle olfactory turbinates than in any other tissue with peak concentrations achieved 30 min after injection. At this point, the recoveries of injected tracer (percent injected dose/g tissue) were 9.4% middle turbinates, 1.6% blood, 0.04% skeletal muscle, 0.2% spleen, 0.3% liver, 0.3% kidney, and 0.09% tail. The current belief that arachnoid projections are responsible for CSF drainage fails to explain some important issues related to the pathogenesis of CSF disorders. The rapid movement of the CSF tracer into the olfactory turbinates further supports a role for lymphatics in CSF absorption and provides the basis of a method to investigate the novel concept that diseases associated with the CSF system may involve impaired lymphatic CSF transport.     

A Rich1/Amot complex regulates the Cdc42 GTPase and apical-polarity proteins in epithelial cells

Using functional and proteomic screens of proteins that regulate the Cdc42 GTPase, we have identified a network of protein interactions that center around the Cdc42 RhoGAP Rich1 and organize apical polarity in MDCK epithelial cells. Rich1 binds the scaffolding protein angiomotin (Amot) and is thereby targeted to a protein complex at tight junctions (TJs) containing the PDZ-domain proteins Pals1, Patj, and Par-3. Regulation of Cdc42 by Rich1 is necessary for maintenance of TJs, and Rich1 is therefore an important mediator of this polarity complex. Furthermore, the coiled-coil domain of Amot, with which it binds Rich1, is necessary for localization to apical membranes and is required for Amot to relocalize Pals1 and Par-3 to internal puncta. We propose that Rich1 and Amot maintain TJ integrity by the coordinate regulation of Cdc42 and by linking specific components of the TJ to intracellular protein trafficking.     

Immunogenicity of a novel, bivalent, plant-based oral vaccine against hepatitis B and human immunodeficiency viruses

A synthetic chimeric gene, TBI-HBS, encoding the immunogenic ENV and GAG epitopes of human immunodeficiency virus (HIV-1) and the surface protein antigen (HBsAg) of hepatitis B virus (HBV), was expressed in tomato plants. Tomato fruits containing the TBI-HBS antigen were fed to experimental mice and, on days 14 and 28 post-feeding, high levels of HIV- and HBV-specific antibodies were present in the serum and feces of the test animals. Intraperitoneal injection of a DNA vaccine directing synthesis of the same TBI-HBsAg antigen boosted the antibody response to HIV in the blood serum; however, it had no effect on the high level of antibodies produced to HBV.Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.     

Purification of the YadA membrane anchor for secondary structure analysis and crystallization

Non-fimbrial adhesins, such as Yersinia YadA, Moraxella UspA1 and A2, Haemophilus Hia and Hsf, or Bartonella BadA, represent an important class of molecules by which pathogenic proteobacteria adhere to their hosts. They form trimeric surface structures with a head-rod-anchor architecture. Whereas their head and rod domains may be of heterologous origin, their anchor domains are homologous and display the properties of autotransporters. Conflicting topology models exist for these membrane anchors. Here, we describe the expression and purification of the membrane anchor of YadA from Yersinia enterocolitica for structural biology experiments. We expressed YadA-M in the Escherichia coli outer membrane. After solubilization and purification, it is a trimer of extreme stability. Using protein FTIR and secondary structure analysis, we show that the anchor is a beta-barrel, but contains a helical part at its N-terminus. We have crystallized the protein under various conditions and present X-ray data to 3.8 A resolution.