Isolation of a novel<Emphasis Type="Italic"> Thermus thermophilus</Emphasis> metal efflux protein that improves<Emphasis Type="Italic"> Escherichia coli</Emphasis> growth under stress conditions

The mechanisms of metal ion transport in thermophilic organisms are poorly understood. Phage display-based screening of a Thermus thermophilus genomic library in Escherichia coli led to the identification of a novel metal cation efflux protein. The Thermus protein showed extensive sequence and putative structural conservation to Czr and Czc proteins in mesophilic bacterial and mammalian species. Expression of the gene in E. coli led to increased resistance to zinc and cadmium ions, but not to cobalt, in an effect that was apparently caused by increased efflux of metals from the cell. This increased resistance was inducible by zinc and cadmium and, to a lesser extent, by cobalt. Furthermore, E. coli cells containing the Thermus gene exhibited improved cell physiology and delayed cell lysis during recombinant protein production, leading to accumulation of higher levels of recombinant protein. The molecular basis and potential application of the findings are discussed.     

Karyotypic changes associated with spontaneous acquisition and loss of tumorigenicity in a human transformed bronchial epithelial cell line: Evidence for In vivo selection of transformed clones

Summary In this study, we describe the karyotypic changes associated with the spontaneous acquisition of tumorigenicity in an immortalized tumor bronchial cell line. Neoplastic transformation of the NL20 human bronchial epithelial cell line occurred after 3 yr in culture, and was associated with loss of chromosome 18 together with acquisition of multiple copies of 9q21.2→34. The nontumorigenic NL20 cell line had been established by transfection of human bronchial epithelial cells with the SV40 T antigen, and had retained a relatively stable karyotype after the first 32 passages in vitro. However, when cells from p184 were inoculated into nude mice, a transplantable tumor was obtained that was derived from a minor clone present in this otherwise stable line. Subsequent passaging of the NL20 cells in vitro did not yield further tumors, and the minor clone from which the tumorigenic NL20T cell line derived was no longer evident in NL20 cells by Passage 205. Furthermore, the original tumorigenic NL20T cells lost the neoplastic phenotype after 25 passages in vitro and reverted to the nontumorigenic karyotype observed at p189. In contrast to the loss of the tumorigenic phenotype and karyotype, which occurred with in vitro passaging of the original tumor, when the NL20T cells were passaged in other nude mice, they continued to give rise to tumors with sevenfold amplifications of 9q sequences and loss of chromosome 18, and cells from the secondary tumors (NL20T-A cells) have maintained a stable karyotype and remain tumorigenic even after 64 passages in vitro. A mixture of 10% tumorigenic NL20T-A and 90% nontumorigenic NL20 cells formed tumors in athymic nude mice when cultured in vitro on fibronectin, but not on plastic; cytogenetic analysis demonstrated that the tumors and cell cultures were composed of tumorigenic NL20T-A cells, whereas cytogenetic analysis of cells cultured on plastic were identical to the nontumorigenic NL20 cells. These data support the hypothesis that neoplastic transformation in our original cell line arose from in vivo selection of a small mutant clone, which had arisen in culture and was subsequently selected in vivo but was lost with in vitro culture.     

In situ localization of PCR-amplified DNA and cDNA

Combining the high sensitivity of PCR with the cell localizing ability ofin situ hybridization allows for the reproducible detection of low copy targets in intact cells. This article describes several key variables that include fixation, protease digestion, the hot start maneuver, stringency, and, for RNA analysis, DNase digestion that are important to successfulin situ PCR. Also stressed is the importance of performing and interpreting controls with each experiment. Important controls include omission of key components, use of samples known either to contain or lack the target of interest and, most importantly, the in-built controls invariably present in the heterogeneous component of any given tissue type.     

pH dependence of the enantioselective excited-state quenching of Λ,Δ-Tb(III) and Λ,Δ-Eu(III)tris(pyridine-2,6-dicarboxylate) chelates by ferricytochrome c from horse heart and ferricytochrome c-550 from Paracoccus versutus

 The pH dependence of the dynamic quenching of the luminescence from Tb(III) and Eu(III) tris(pyridine-2,6-dicarboxylate≡DPA) chelates by the title proteins is studied. For Tb(DPA)₃ ^3– also the quenching by the Lys 14→Glu and Lys99→Glu mutants of cytochrome c-550 (cytc-550) is investigated. The rate constants for quenching of the electronically excited Λ and Δ enantiomers of the luminophore by equine cytochrome c show a sharp decrease upon increasing the pH from 7 to 10, which can be described phenomenologically by deprotonation of a single acidic group with pK _a of 9.2±0.1 for Eu and 9.4±0.1 for Tb. These values are similar to that found for the alkaline transition of the protein. The alkaline conformer(s) of the protein at pH>10 is found to be a very inefficient quencher of the lanthanide luminescence. For Tb, but not for Eu, a significant lowering of the degree of enantioselectivity (E _q) in the quenching is found along with a reduction of the quenching rates. For cytc-550, the decrease of the quenching rate constants with increasing pH is described by pK _a=9.8±0.1 and for the two mutants the same value is obtained. For the cytc-550 proteins the change of the quenching rates does not correlate with the alkaline transition, for which a pK _a of 11.2 has been reported by other workers. For all proteins, the reduction of the quenching rates at high pH is ascribed to a reduction of the binding affinity of the excited lanthanide complex to the surface area of the protein near the exposed heme edge, caused by deprotonation of (presumably) several lysine residues. Received: 3 April 1998 / Accepted: 15 June 1998     

Juvenile three-spined sticklebacks avoid parasitized conspecifics

Synopsis Juvenile three-spined sticklebacks,Gasterosteus aculeatus, were given a series of four choice tests to determine whether they avoided schools of conspecifics in which individuals were parasitized with the ectoparasiteArgulus canadensis. Results from these tests indicate that juvenile sticklebacks can avoid schools of parasitized conspecifics. Furthermore, parasites alone did not elicit an avoidance response, suggesting that it is both the presence of the parasite and its effect on stickleback behavior that causes avoidance of parasitized individuals.     

The Bacillus subtilis addAB genes are fully functional in Escherichia coli

An Escherichia coli recBCD deletion mutant was transformed with plasmids containing the Bacillus subtilis add genes. The transformants had relatively high ATP-dependent exonuclease- and ATP-dependent helicase activities, and their viability, the ability to repair u.v.-damaged DNA and the recombination in conjugation were nearly completely restored. The B. subtilis Add enzyme did not show Chi-activity in phage lambda recombination. The individual B. subtilis Add proteins were not able to form an enzymatically active complex with the E. coli RecB,C,D proteins, and they could not complement the recB,C,D deficiency. Evidence is presented that only two subunits are involved in the B. subtilis ATP-dependent exonuclease. This is in contrast to E. coli in which the RecBCD enzyme consists of three subunits.     

Quantification of methane oxidation in the rhizosphere of emergent aquatic macrophytes: defining upper limits

Rates of rhizospheric methane oxidation were evaluated by aerobic incubations of subcores collected in flooded anoxic soils populated by emergent macrophytes, by greenhouse whole plant incubations, and by CH₄ stable isotopic analysis. Subcore incubations defined upper limits for rhizospheric methane oxidation on an areal basis which were equal to or greater than emission rates. These rates are considered upper limits because O₂ did not limit CH₄ uptake as is likely to occur in situ. The ratio of maximum potential methane oxidation (MO) to methane emission (ME) ranged from 0.7 to 1.9 in Louisiana rice (Oryza sativa), from 1.0 to 4.0 in a N. Florida Sagittaria lancifolia marsh, and from 5.6 to 51 in Everglades Typha domingensis and Cladium jamaicense areas. Methane oxidation/methane emission ratios determined in whole plant incubations of Sagittaria lancifolia under oxic and anoxic conditions ranged from 0.5 to 1.6. Methane oxidation activity associated with emergent aquatic macrophytes was found primarily in fine root material. A weak correlation was observed between live root biomass and CH₄ uptake in Typha. Rhizomes showed small or zero rates of methane uptake and no uptake was associated with plant stems. Methane stable isotope data from a S. lancifolia marsh were as follows: CH₄ emitted from plants: −61.6 ± 0.3%; CH₄ within stems: −42.0 ± 0.2%; CH₄ within sedimentary bubbles: −51.7 ± 0.3%). The ¹³C enrichment observed relative to emitted CH₄ could be due to preferential mobilization of CH₄ containing the lighter isotope and/or the action of methanotrophic bacteria.     

Survival of alginate-entrapped cells of Azospirillum lipoferum during dehydration and storage in relation to water properties

Survival of alginate-entrapped cells of Azospirillum lipoferum was studied during dehydration using a dry air stream and during prolonged storage at various constant water activity values (a_w). During the drying operation, the viability loss remained almost constant from the initial water content to 0.35 g water/g dry weight (DW) corresponding to a 98.5% water removal, strongly increased until a water content of 0.25 g/g DW and then stopped until the end of the drying operational (final a_w 0.18). A water content of 0.25 g/g DW (a_w=0.55) corresponded to the critical point of the moisture sorption isotherm curve from which water became restricted to the dry material. A high drying rate (5 g/g DW per hour) was shown to be more detrimental for cell viability than a low drying rate (1.18 g/g DW per hour). When the product was stored in a closed chamber with a regulated a_w (0.23), the number of living cells decreased during a short period (less than 15 days) corresponding to the product a_w stabilization, and then remained constant for more than 150 days. In addition, cell survival during storage was not affected by a_w values in the range 0–0.55. Above a_w=0.55, the higher the a_w and the storage duration, the lower the residual survival percentage. The influence of the drying and storage conditions on the cell death rate are discussed with regard to both the mechanisms generally involved in viability loss and the hydration properties of water. Correspondence to: A. Pareilleux     

Inhibition of growth and decreased survival of B104 rat neuroblastoma cells after exposure to hyperbaric oxygen

Summary The toxic effects of hyperbaric oxygen (HBO) on growth and survival of B104 rat neuroblastoma cells were investigated. Cells in log phase growth were incubated at 37°C with 10 atm O₂ for 1 to 4 h. After exposure to HBO, cells were monitored for their subsequent growth and survival. Two hours of exposure caused a slowing of growth, which returned to normaly by the end of the 7th d of the postexposure period. Exposures to O₂ of 3 h or longer caused a complete cessation of growth for 4 d after the exposure and very litle or no recovery after this period. Increased hydrostatic pressure for 6 h using helium as the inert gas had no effect on growth. A colony formation assay was used to quantitative the degree of cell death induced by HBO. The resulting survival curve was of the exponential type with a broad shoulder between 0 to 2.5 h of exposure to 10 atm O₂. The curve fell off sharply at 2.5 h with an exponential decrease in survival when the exposure to HBO was extended to 4 h. At 2 h about 50% of cells were killed, but at 4 h only 2% survived the treatment. These results show that the depression of the growth rate by HBO is related to the number of cells that are killed by the exposure. This system provides a model in which the molecular and cellular effects of HBO can be investigated. This work was supported by the National Institutes of Health, Bethesda, MD, Grants AM-21423 and CA-14489, and by grants from the W. W. Smith Foundation and the Philadelphia Foundation. The studies presented here are part of a dissertation submitted by G. J. Gendimenico in partial fulfillment of the requirements for the degree of Ph.D. in pharmacology from the University of Pennsylvania.