Combined genetic polymorphism and risk for development of lung cancer

Susceptibility to lung cancer has been shown to be modulated by host specific factors. Inheritance of different polymorphic cytochrome P450s (CYPs) and the glutathione S-transferases (GSTs) which affect metabolism of environmental toxicants may play a key role in individual susceptibility. Although individual polymorphic genes have been reported to be associated with development of lung cancer, little is known about the combined effects of several genes in carcinogenesis. From our study of 54 lung cancer patients and 50 matched controls, we observed that a combination of several versions of ‘unfavorable' metabolizing genes (CYP2D6, CYP2E1, GSTM1 and GSTT1) is strongly associated with lung cancer. The relative risk for the different combinations of these genotypes ranged between 1.3 and 14, with higher risk involving the activating genes. The duration and intensity of heavy smoking (expressed in pack-years) are the most important determinant for the development of lung cancer. For example, the estimated risk for development of lung cancer associated with smoking >30 pack-years is represented by an odds ratio=6.65; 95% CL=2.3–19.9 irrespective of an individual's genotype, whereas for smoking between >30 and <50 pack-years, odds ratio=4.5; 95% CL=1.37–15; and for smoking >50 pack-years, odds ratio=30; 95% CL=5.7–114. On the other hand, smoking of less than 30 pack-years is associated with an increased risk in the presence of the polymorphic genes (odds ratio=2.5; 95% CL=0.32–54). The results of our study indicate that the inheritance of multiple ‘unfavorable' genotypes, especially activating genes, is a crucial predisposing factor for the development of lung cancer from cigarette smoking. In addition, the genes may cause moderate smokers who would normally outlive the deleterious effects of smoking to develop lung cancer. The information can therefore be used to target individuals for prevention of health problems.     

Axenic aerobic biofilms inhibit corrosion of SAE 1018 steel through oxygen depletion

Corrosion inhibition of SAE 1018 steel by pure-culture biofilms of Pseudomonas fragi and Escheri-chia coli DH5α has been evaluated in complex Luria-Bertani medium, seawater-mimicking medium, and modified Baar's medium at 30 °C. In batch cultures, both bacteria inhibited corrosion three to six fold compared to sterile controls, and the corrosion was comparable to that observed in anaerobic sterile media. To corroborate this result, a continuous reactor and electrochemical impedance spectroscopy were used to show that both P. fragi K and E. coli DH5α decreased the corrosion rate by 4- to 40-fold as compared to sterile controls; this matched the decrease in corrosion found with sterile medium in the absence of oxygen and with E. coli DH5α grown anaerobically. In addition, the requirement for live respiring cells was demonstrated by the increase in the corrosion rate that was observed upon killing the P. fragi K biofilm in continuous cultures, and it was shown that fermentation products do not cause an increase in corrosion. Hence, pure-culture biofilms inhibit corrosion of SAE 1018 steel by depleting oxygen at the metal surface. Received: 16 December 1996 / Received revision: 18 March 1997 / Accepted: 27 March 1997     

Corrosion inhibition by aerobic biofilms on SAE 1018 steel

Carbon steel (SAE 1018) samples were exposed to complex liquid media containing either the aerobic bacterium Pseudomonas fragi or the facultative anaerobe Escherichia coli DH5α. Compared to sterile controls, mass loss was consistently 2- to 10-fold lower in the presence of these bacteria which produce a protective biofilm. Increasing the temperature from 23 °C to 30 °C resulted in a 2- to 5-fold decrease in corrosion inhibition with P. fragi whereas the same shift in temperature resulted in a 2-fold increase in corrosion inhibition with E. coli DH5α. Corrosion observed with non-biofilm-forming Streptomyces lividans TK24 was similar to that observed in sterile media. A dead biofilm, generated in situ by adding kanamycin to an established biofilm, did not protect the metal (corrosion rates were comparable to those in the sterile control), and mass loss in cell-free, spent Luria-Bertani (LB) medium was similar to that in sterile medium. Confocal laser scanning microscopy analysis confirmed the presence of a biofilm consisting of live and dead cells embedded in a sparse glycocalyx matrix. Mass-loss measurements were consistent with microscopic observations of the metal surface after 2 weeks of exposure, indicating that uniform corrosion occurred. The biofilm was also able to withstand mild agitation (60 rpm), provided that sufficient time was given for its development. Received: 3 May 1996 / Received revision: 8 August 1996 / Accepted: 24 August 1996     

A Kinetic and ESR Investigation of Iron(II) Oxalate Oxidation by Hydrogen Peroxide and Dioxygen as a Source of Hydroxyl Radicals

The reaction of Fe^II oxalate with hydrogen peroxide and dioxygen was studed for oxalate concentrations up to 20 mM and pH 2-5, under which conditions mono- and bis-oxalate comlexes (Fe^II(ox) and Fe^II(ox)₂^2-) and uncomplexed Fe²⁺ must be considered. The reaction of Fe^II oxalate with hydrogen peroxide (Fe²⁺ + H₂O₂ → Fe³⁺ + *OH + OH^-) was monitored in continuous flow by ESR with t-butanol as a radical trap. The reaction is much faster than for uncomplexed Fe²⁺ and a rate constant, k = 1 × 10⁴ M-¹ s^-1 is deduced for Fe^II(ox). The reaction of Fe^II oxalate with dioxygen is strongly pH dependent in a manner which indicates that the reactive species is Fe^II(ox)₂^2-, for which an apparent second order rate constant, k = 3.6 M^-1 s^-1, is deduced. Taken together, these results provide a mechanism for hydroxyl radical production in aqueous systems containing Fe^II complexed by oxalate. Further ESR studies with DMPO as spin trap reveal that reaction of Fe^II oxalate with hydrogen peroxide can also lead to formation of the carboxylate radical anion (CO₂^*-), an assignment confirmed by photolysis of Fe^III oxalate in the presence of DMPO.     

Recording of mitochondrial transmembrane potential and volume in cultured rat osteoclasts by confocal laser scanning microscopy

Osteoclasts are multinuclear bone-resorbing cells which contain abundant mitochondria. Morphological studies have suggested that a correlation may exist between mitochondrial concentration and bone resorption by osteoclasts. However, investigation of mitochondrial transmembrane potential (Delta rho) and volume has been hampered by the difficulty in obtaining a sufficient number of osteoclasts for assessing these characteristics by flow cytometric analysis. In this study, we have used confocal laser scanning microscopy after loading the cells with Rhodamine 123 and 10-nonyl Acridine Orange to record mitochondrial Delta rho and volume, respectively, in isolated rat osteoclasts cultured on bovine bone slices. Optimal staining conditions were found to be 10 mu g ml-1 for 40 min for Rhodamine, and 1 mu S mD for 10 min for the 10-nonyl Acridine Orange derivative. Two osteoclast populations, whose shape seemed to reflect bone resorption and migratory functions, were identified depending on their shape and on the distribution of the two dye probes. ‘Round-shaped’ osteoclasts had significantly higher mitochondrial Delta rho and volume in the apical regions than in the basolateral portions (p 0.00001). In contrast, mitochondrial Delta rho and volume in ‘irregular- shaped’ osteoclasts were rather evenly distributed in both these regions (p > 0.05). Our results indicate that there is an apical polarization of mitochondria in osteoclasts corresponding to the energy demands associated with bone resorption. This revised version was published online in November 2006 with corrections to the Cover Date.     

A one step PCR-based method for the detection of economically important soft rot Erwinia species on micropropagated potato plants

A simple, rapid and sensitive PCR-based method was developed for the detection of all five subspecies of Erwinia carotovora , including subsp. carotovora and subsp. atroseptica , and all pathovars/biovars of Erwinia chrysanthemi , on plant tissue culture material. Primers SR3F and SR1cR, based on a conserved region of the 16S rRNA gene, amplified a DNA fragment of 119 bp from all 65 such strains tested. Detection limits of the method in vitro were 2·0 × 10²–3·4 × 10³ cfu ml⁻¹ (equivalent to 1–17 cfu per PCR) and, following extraction of genomic DNA from plant extract, detection limits were 2·3 × 10²–1·9 × 10⁴ cfu per microplant sample (equivalent to 5 cfu – 3·8 × 10² cfu per PCR). To improve the sensitivity of the method in planta , to obviate the need for complex and laborious DNA extractions, and to remove inhibitory substances present in the plant extract, an enrichment step was included prior to PCR. Following enrichment, the sensitivity of detection was <10 cfu per microplant sample. This method provides the first sensitive means of detecting latent infection caused by several economically important soft rot erwinias simultaneously on potato tissue culture material.     

Expression of the amino-terminal domain of platelet glycoprotein Ib alpha: exploitation of a calmodulin tag for determination of its functional activity.

Platelet glycoprotein (GP) Ibalpha is a component of the GPIb-IX receptor complex, which is involved in multiple physiological and pathological processes, including platelet adhesion at sites of vascular injury, thrombin binding, Bernard-Soulier syndrome, platelet-type von Willebrand disease, and immune-mediated thrombocytopenias. The amino-terminal domain of approximately 300 residues of GPIbalpha mediates both normal biological function (by providing the sites for direct ligand interaction) and aberrant function (through amino acid substitutions). To investigate the molecular interactions mediated by this region of GPIbalpha, we have developed a recombinant baculovirus to facilitate its expression as a calmodulin fusion protein from insect cells. By employing the calmodulin tag, the fusion protein could be obtained at >90% purity after a single isolation step at yields of 8 mg/L of insect cell medium (purified fusion protein). The recombinant GPIbalpha fragment was shown to be posttranslationally sulfated and glycosylated, although its glycosylation differed from that of the equivalent GPIbalpha fragment isolated from human platelets. The differential glycosylation, however, did not affect the function of the recombinant GPIbalpha fragment in either von Willebrand factor (vWf) or thrombin binding as these were both found to be identical to those of the same-length GPIbalpha fragment derived from human platelets. The calmodulin tag was also exploited in the development of assays to measure directly vWf and thrombin binding, since it did not interfere with either, demonstrating the feasibility for the use of this soluble receptor fusion protein in detailed biophysical assays to investigate the molecular mode of binding of platelet glycoprotein Ibalpha to these ligands.