A novel chimeric MOMP antigen expressed in Escherichia coli, Arabidopsis thaliana, and Daucus carota as a potential Chlamydia trachomatis vaccine candidate

The major outer membrane protein (MOMP) of Chlamydia trachomatis is a highly antigenic and hydrophobic transmembrane protein. Our attempts to express the full-length protein in a soluble form in Escherichia coli and in transgenic plants failed. A chimeric gene construct of C. trachomatis serovar E MOMP was designed in order to increase solubility of the MOMP protein but with retained antigenicity. The designed construct was successfully expressed in E. coli, in Arabidopsis thaliana, and in Daucus carota. The chimeric MOMP expressed in and purified from E. coli was used as antigen for production of antibodies in rabbits. The anti-chimeric MOMP antibodies recognized the corresponding protein in both E. coli and in transgenic plants, as well as in inactivated C. trachomatis elementary bodies. Transgenic Arabidopsis and carrots were characterized for the number of MOMP chimeric genetic inserts and for protein expression. Stable integration of the transgene and the corresponding protein expression were demonstrated in Arabidopsis plants over at least six generations. Transgenic carrots showed a high level of expression of the chimeric MOMP – up to 3% of TSP.     

A Pisum sativum glyoxysomal malate dehydrogenase induced by cadmium exposure.

The glyoxysomal malate dehydrogenase (gMDH) catalyses the formation of oxaloacetate from malate during beta-oxidation of fatty acids in the glyoxysome. A partial Pisum sativum L. (cv. Greenfeast) cDNA was first isolated from a suppression subtractive hybridisation cDNA library obtained from heavy metal stressed plants. The full length cDNA was then isolated by rapid amplification of cDNA ends. The translated sequence showed strong similarity to Cucumis sativus and Citrullus lanatus gMDH including a typical glyoxysome-targeting presequence comprising the PTS2 motif and a cleavage site for a cystein-directed protease. Exposure of pea plants to Cd2+ induced expression of the gMDH gene in mature pea leaves indicating that the enzyme is under environmental control in addition to the normal developmental regulation pattern.     

Leukotriene C4 synthase promoter driven expression of GFP reveals cell specificity

Leukotriene C₄ synthase is a key enzyme in leukotriene biosynthesis. Its gene has been cloned and mapped to mouse chromosome 11. Expression occurs in cells of myeloid origin and also in the choroid plexus, the hypothalamus and the medial eminence of mouse brain. In this study a vector that expresses enhanced green fluorescent protein (eGFP) under the control of the mouse leukotriene C₄ synthase promoter was constructed and used to study promoter activity in different cell lines. Specific eGFP expression was observed in human monocytic leukemia (THP-1) and rat basophilic leukemia (RBL-1) myeloid cells which both express leukotriene C₄ synthase, but not in human embryonic kidney (HEK293/T) epithelial cells which do not express this enzyme. In the myeloid cells, but not in the epithelial cells, we observed that the leukotriene C₄ synthase promoter activity was stimulated by 12-O-tetradecanoylphorbol-13-acetate and all-trans-retinoic acid. In contrast dimethyl sulfoxide did not affect promoter activity.     

Supplementary ultraviolet-B irradiation reveals differences in stress responses between Arabidopsis thaliana ecotypes

Irradiation of Arabidopsis thaliana ecotypes C24, Wassilewskija (Ws) and Columbia-0 (Col-0) with supplementary ultraviolet-A+B (UV-A+B) radiation revealed ecotype-specific differences in expression of the gene for the pathogenesis-related protein PR-5. C24 showed an increased expression level of PR-5 (5- and 20-fold higher compared with Col-0 and Ws, respectively). Expression of other molecular markers such as CHS (encoding chalcone synthase), MEB5.2 [encoding a gene strongly up-regulated by ultraviolet-B (UV-B)] and PYROA [encoding a pyridoxine (Vitamin B6) biosynthesis enzyme] only showed slight differences between ecotypes. Oxidative stress during UVA+B exposure was monitored by staining for H2O2. This analysis also revealed important ecotype-specific differences. 'H2O2 hot spots' were found in C24, whereas an even distribution of H2O2 was found in Ws and Col-0. Necrotic lesions also appeared on C24 leaves after prolonged UV-B exposure. There was a reverse correlation between the H2O2 steady-state concentration and the PR-5 gene expression; Ws showed the highest level of H2O2 accumulation but the lowest expression level of the PR-5 gene. Furthermore, application of paraquat on the rosettes led to similar PR-5 expression and H2O2 accumulation patterns as were found after UV-A+B irradiation. The observed ecotypic differences were also reflected in a statistically significant UV-B-dependent decrease in biomass, rosette size and leaf area for Ws, but not for C24 and Col-0. Our results show that a significant ecotype-specific genetic variability in general UV-B responses in Arabidopsis exists. Moreover, the signal transduction or gene regulation pathway for PR-5 differs from the other molecular markers used in this study.     

Division of divalent cations into two groups in relation to their effect on the coupling of the F0F1-ATPase of Rhodospirillum rubrum to the protonmotive force

Divalent cations are divided into two groups in relation to their ability to promote ATP synthase catalyzed reactions. In the presence of Mg2+, the following pattern rules: (i) uncoupler-stimulated ATP hydrolysis of Rhodospirillum rubrum chromatophores which shows an optimum concentration of the divalent cation; (ii) ATP-induced proton pumping in chromatophores; (iii) light-induced ATP synthesis in chromatophores; (iv) no or very low ATPase activity of purified F1-ATPase unmasked by diethylstilbestrol or n-octyl beta-D-glucopyranoside. In the presence of Ca2+, the following pattern occurs: (i) no stimulation of the ATP hydrolysis in chromatophores by carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone; (ii) no ATP-induced proton pumping; (iii) no light-induced ATP synthesis; (iv) a high ATPase activity of the purified F1-ATPase which is inhibited by diethylstilbestrol and n-octyl beta-D-glucopyranoside. Co2+, Mn2+, and Zn2+ are members of the "Mg2+-group", whereas Cd2+ is suggested to fall between the two groups. Intrinsic uncoupling of the membrane-bound ATP synthase has been suggested to account for the effect caused by Ca2+ in chloroplasts [Pick, U., & Weiss, M. (1988) Eur. J. Biochem. 173, 623-628]. Such an interpretation is consistent with our results on chromatophores. The uncoupling cannot occur at the level of the membrane since neither light-induced nor Mg-ATP-induced proton pumping is affected by Ca2+. A conformational change is suggested to be the reason for this intrinsic uncoupling, and it is proposed to be controlled by the diameters of the divalent cations (Ca2+ greater than Cd2+ greater than Mn2+ greater than Co2+ greater than Zn2+ greater than Mg2+).(ABSTRACT TRUNCATED AT 250 WORDS)     

A taxonomic review of the genus Hypocalymma (Myrtaceae)

The genus Hypocalymma endemic to Western Australia and comprising 22 species species including the widely cultivated Swan River Myrtle and Swamp Myrtle, is revised. Nine new species ( H. connatum, H. hirsutum, H, gardneri, H. jessicae, H. melaleucoides, H. serrulatum, H. sylvestris, H. tenuatum and H. uncinatum ), two new subspecies ( H. cordifolium subsp. minus and H. strictum subsp. elongatum ) and one new combination ( H. angustifolium subsp. longifolium ) are described or made. A key to all species is presented for this horticulturally significant genus.     

Rapid decay of Salmonella flagella antibodies during human gastroenteritis: a follow up study

An indirect enzyme-linked immunosorbent assay (ELISA) based on Salmonella re-polymerized flagella was employed to measure levels of immunoglobulin (Ig) G, IgM and IgA antibodies in sera from 303 Danish patients diagnosed with either Salmonella enteritidis or Salmonella typhimurium. The antibody-levels were assessed at one, three and six months after onset of salmonellosis, and sera from a control-group of 170 healthy blood donors were additionally analysed in order to establish cut-off values for the analysis. Cross-reactions to other Salmonella serotypes, as well as to Escherichia coli, Yersinia enterocolitica, Campylobacter jejuni, Campylobacter coli and Helicobacter pylori were observed. At one month after onset of symptoms, 70% of the patients recovering from a S. enteritidis infection carried detectable levels of anti-flagella antibodies, as did 77% of the patients recovering from S. typhimurium infection. Three months after onset of symptoms these detection rates had decreased to 46% and 40%; and six months after onset of symptoms the detection rates were 34% and 38%. This rapid decrease in the serum levels of flagella antibodies is in conflict with the "common knowledge" statement of a long-lasting anti-flagella immunoresponse. The present study suggests that such a tenacious statement is (or may be) inaccurate.     

Homogenization of detrital leachate in an old-growth coniferous forest,OR: DOC fluorescence signatures in soils undergoing long-term litter manipulations

Aims

Characterizing the relationship between plant detrital inputs and the resulting dissolved organic carbon (DOC) leachate is vital to understanding the ultimate fate of root carbon, fallen wood and needles in forested watersheds. Similarly, elucidating chemical differences in the soil DOC pool may help to explain which DOC fractions are sorbed to mineral surfaces and contribute to accumulation of soil organic carbon, are respired as CO₂, or are exported to nearby catchments.

Methods

In order to test the hypothesis that soils with different detrital inputs impart a detectable signal on DOC in mineral soil, soil solution DOC was sampled from the Detrital Input and Removal Treatment (DIRT) plots located in the H.J. Andrews Experimental Forest, OR. Multiple types of fresh litter extracts, along with lysimeter and soil extracts from DIRT treatment plots were characterized using UV-Vis and fluorescence spectroscopy coupled with the Cory and McKnight (Environ Sci Technol 39:8142–8149, 2005) parallel factor analysis (PARAFAC) model.

Results

Principal component analysis of 13 unique fluorophores distinguished using PARAFAC show that litter and soil extracts (Douglas-fir needles, wood of decomposition Class 2, Class 3 and Class 5, O-horizon, and 0–5 cm A-horizon) each have distinct fluorescence signatures. However, while litter-leached DOC chemistry varies by litter type, neither lysimeter-collected DOC or soil extracts in the DIRT plots show statistically significant differences in fluorescence signatures among treatments, even after 17 years of litter manipulations. The lack of observed differences among DIRT treatments suggests that both abiotic interactions and microbial activity effectively homogenize organic carbon constituents within the dissolved pool. Minor but observable changes in PARAFAC components and optical indices during a 1-month biodegradation incubation of litter and soil extracts indicate that while biodegradation significantly alters DOC chemistry, abiotic mechanisms are also critical to DOC transformation in these soils with high sorption capacity.

Conclusions

Although leachates from different plant detrital sources have distinct carbon chemical signatures, these DOC signatures are effectively homogenized after passage through mineral soil. These results highlight the dominant role of both biotic and abiotic interactions in controlling the chemistry of DOC in shallow soils.

     

Doctors' drinking habits and consumption of alcohol.

Alcohol consumption and drinking habits among Finnish doctors were studied as part of a survey of stress and burnout. A questionnaire containing 99 questions or groups of questions was sent to all 3496 practising doctors aged under 66 randomly selected from the registry of the Finnish Medical Association. Altogether 2671 doctors (76%) responded; this sample was representative of the Finnish medical profession. The average weekly consumption of alcohol during the past year and various aspects of drinking behaviour were assessed, and the presence or absence of symptoms and diseases often encountered among heavy drinkers and addicts was determined. The data were analysed separately for men and women, for those aged less than or equal to 40 and greater than 40, and for the men with high and low alcohol consumption and with high and low scores on the index of drinking habits. Selected variables related to work, stress, and coping were correlated with alcohol consumption and drinking behaviour. The median consumption of alcohol among male doctors was 4876 g (6.2 litres) and among female doctors 2226 g (2.8 litres) of absolute alcohol per person per year and was higher in those aged over 40. Beer was most commonly drunk by men and wine by women. Increased alcohol consumption was associated with older age, disappointment with career, heavy smoking, use of benzodiazepines, stress and burnout symptoms, suicidal thoughts, general dissatisfaction, and diseases related to alcohol. Drinking habits were heavier among doctors working in community health centres, those taking long sick leaves, younger doctors disappointed with their careers or the atmosphere at work, and older doctors immersed in their work. Alcohol consumption among doctors seems to be higher than that of the general population in Finland, and heavy drinking seems to be associated with stress and burnout.     

Conversion of coupling factor 1 of Rhodospirillum rubrum from a Ca2+-ATPase into a Mg2+-ATPase

Isolation of F1-ATPase from Rhodospirillum rubrum by chloroform extraction of chromatophores, followed by purification on a glycerol gradient, results in a very pure enzyme preparation containing five subunits with high Ca2+-ATPase activity (15 mumol per min per mg protein). Furthermore, conditions are reported under which the purified F1 exhibits Mg2+-dependent ATPase activity of about 35 mumol per min per mg protein. NaHCO3 stimulates the Mg2+-activity from 1.5 mumol per min per mg protein to 5 mumol per min per mg protein giving a maximal activity at a concentration of about 60 mM NaHCO3. Lauryl dimethylamine oxide (LDAO), octyl glucoside and nonanoyl N-methylglucamide enhance the Mg2+-ATPase activity from 1.5 to 14, 22 and 35 mumol per min per mg protein, respectively, in the absence of NaHCO3, and from 5 to 34, 30 and 37 mumol per min per mg protein, respectively, in the presence of 50 mM NaHCO3. The Vmax is increased, but the Km for ATP remains the same, about 0.22 mM, both in the absence of activators and in the presence of NaHCO3, LDAO or NaHCO3 plus LDAO. Ca2+-dependent ATPase activity is slightly stimulated by NaHCO3 but strongly inhibited by octyl glucoside.