Carotenoid biosynthesis in the primitive red alga Cyanidioschyzon merolae

Cyanidioschyzon merolae is considered to be one of the most primitive of eukaryotic photosynthetic organisms. To obtain insights into the origin and evolution of the pathway of carotenoid biosynthesis in eukaryotic plants, the carotenoid content of C. merolae was ascertained, genes encoding enzymes of carotenoid biosynthesis in this unicellular red alga were identified, and the activities of two candidate pathway enzymes of particular interest, lycopene cyclase and beta-carotene hydroxylase, were examined. C. merolae contains perhaps the simplest assortment of chlorophylls and carotenoids found in any eukaryotic photosynthetic organism: chlorophyll a, beta-carotene, and zeaxanthin. Carotenoids with epsilon-rings (e.g., lutein), found in many other red algae and in green algae and land plants, were not detected, and the lycopene cyclase of C. merolae quite specifically produced only beta-ringed carotenoids when provided with lycopene as the substrate in Escherichia coli. Lycopene beta-ring cyclases from several bacteria, cyanobacteria, and land plants also proved to be high-fidelity enzymes, whereas the structurally related epsilon-ring cyclases from several plant species were found to be less specific, yielding products with beta-rings as well as epsilon-rings. C. merolae lacks orthologs of genes that encode the two types of beta-carotene hydroxylase found in land plants, one a nonheme diiron oxygenase and the other a cytochrome P450. A C. merolae chloroplast gene specifies a polypeptide similar to members of a third class of beta-carotene hydroxylases, common in cyanobacteria, but this gene did not produce an active enzyme when expressed in E. coli. The identity of the C. merolae beta-carotene hydroxylase therefore remains uncertain.     

An oxygen imaging system for medical applications: preliminary results.

The examination of functional processes in tissue is gaining importance in medical research. As a result the imaging and monitoring of biochemical parameters in vivo is the goal of many imaging methods. One key parameter in photodynamic therapy (PDT) is the molecular oxygen concentration. Two-dimensional monitoring of oxygen is demanded for PDT but has not yet been achieved. The use of optical methods provides a possible means of measuring molecular oxygen. The basis of this method is the measurement of the luminescence lifetime of a dye that is quenched by molecular oxygen. The molecular oxygen concentration can be monitored two-dimensionally by pixel-wise determination of the luminescence lifetime with a CCD-camera. A new O(2)-imaging system based on this principle is presented in this article. The dye Ru(bpy)(3)(2+) is quenched by molecular oxygen and was used in the first experiments with the system.     

The effect of α-mating factor secretion signal mutations on recombinant protein expression in Pichia pastoris

The methylotrophic yeast, Pichia pastoris, has been genetically engineered to produce many heterologous proteins for industrial and research purposes. In order to secrete proteins for easier purification from the extracellular medium, the coding sequence of recombinant proteins is initially fused to the Saccharomyces cerevisiae α-mating factor secretion signal leader. Extensive site-directed mutagenesis of the prepro-region of the α-mating factor secretion signal sequence was performed in order to determine the effects of various deletions and substitutions on expression. Though some mutations clearly dampened protein expression, deletion of amino acids 57–70, corresponding to the predicted 3rd alpha helix of α-mating factor secretion signal, increased secretion of reporter proteins horseradish peroxidase and lipase at least 50% in small-scale cultures. These findings raise the possibility that the secretory efficiency of the leader can be further enhanced in the future.     

Effect of unilateral ovariectomy, gonadotropin stimulation and immunization against a synthetic peptide of the inhibin alpha-subunit on follicular development and oocyte recovery in cattle

Nulliparous Holstein cows were randomly distributed among 4 treatment groups to test the effects of treatments, including unilateral ovariectomy, anti-inhibin immunization and gonadotropin stimulation on ovarian follicle population and oocyte recovery. The Control treatment consisted of intact cows (I-Control). Unilaterally ovariectomized cows were included in the 3 remaining treatments consisting of ovariectomy alone (U-Control), cows immunized against a synthetic peptide of the alpha(c)-subunit of bovine inhibin (alpha(c)I; U-IH), and cows stimulated with FSH (Super-Ov; 75 units/female/week) and also immunized with alpha(c)I as in the previous treatment (U-IH/FSH). Oocytes were collected by transvaginal ultrasound-guided aspiration on a weekly basis from cows in each treatment for 5 consecutive weeks. Intact Control cows had a greater (P<0.05) number of follicles > or = 3 mm per female (4.7) than the U-Control and U-IH cows (2.6 and 2.9, respectively), and had a similar number of follicles as the U-IH/FSH treatment group (3.5). The numbers of follicles aspirated (2.7 to 3.6) and oocytes recovered/cow (1.6 to 2.6) were similar for cows in the I-Control, U-IH and U-IH/FSH treatment groups. Cows in the U-Control treatment group had a lower (P<0.05) number of aspirated follicles (2.0) and recovered oocytes (1.1) than the I-Control cows. Cows in the U-IH/FSH and U-IH treatments had follicles with larger (P<0.01) diameters (8.7 and 8.2 mm, respectively) than cows in the I-Control (6.6 mm) and U-Control (5.7 mm) treatments. In conclusion, unilateral ovariectomy did not result in compensatory increase of follicle number or size in the intact ovary; cows in the U-IH/FSH treatment group had a greater number of follicles aspirated than the U-Control cows. In addition, the anti-alpha(c)I immunization may have played a role in increasing the number and diameter of the follicles. None of the treatments evaluated in this study improved oocyte retrieval over that of the intact, nontreated cows.     

Inhibitors of HIV-1 attachment. Part 9: An assessment of oral prodrug approaches to improve the plasma exposure of a tetrazole-containing derivative

7-(2H-Tetrazol-5-yl)-1H-indole 3 was found to be a potent inhibitor of HIV-1 attachment but the compound lacked oral bioavailability in rats. The cause of the low exposure was believed to be poor absorption attributed to the acidic nature of the tetrazole moiety and, in an effort to address this liability, three more lipohilic tetrazole analogs, N-acetoxymethyl 4, N-pivaloyloxymethyl 5, and N-methyl 6, were evaluated as potential oral prodrugs in rats. Prodrug 5 was ineffective in improving the plasma concentration of 3 in vivo but compound 4 provided a 15-fold enhancement of the plasma concentration of 3. Most interestingly, oral dosing of analog 6 afforded a substantial increase in the plasma concentration of the parent in rats when compared to dosing of parent. This represents a novel example of a methyl tetrazole that acts as a prodrug for a free NH tetrazole-containing compound.     

A Simplified Score to Quantify Comorbidity in COPD

Importance

Comorbidities are common in COPD, but quantifying their burden is difficult. Currently there is a COPD-specific comorbidity index to predict mortality and another to predict general quality of life. We sought to develop and validate a COPD-specific comorbidity score that reflects comorbidity burden on patient-centered outcomes.

Materials and Methods

Using the COPDGene study (GOLD II-IV COPD), we developed comorbidity scores to describe patient-centered outcomes employing three techniques: 1) simple count, 2) weighted score, and 3) weighted score based upon statistical selection procedure. We tested associations, area under the Curve (AUC) and calibration statistics to validate scores internally with outcomes of respiratory disease-specific quality of life (St. George''s Respiratory Questionnaire, SGRQ), six minute walk distance (6MWD), modified Medical Research Council (mMRC) dyspnea score and exacerbation risk, ultimately choosing one score for external validation in SPIROMICS.

Results

Associations between comorbidities and all outcomes were comparable across the three scores. All scores added predictive ability to models including age, gender, race, current smoking status, pack-years smoked and FEV₁ (p<0.001 for all comparisons). Area under the curve (AUC) was similar between all three scores across outcomes: SGRQ (range 0·7624–0·7676), MMRC (0·7590–0·7644), 6MWD (0·7531–0·7560) and exacerbation risk (0·6831–0·6919). Because of similar performance, the comorbidity count was used for external validation. In the SPIROMICS cohort, the comorbidity count performed well to predict SGRQ (AUC 0·7891), MMRC (AUC 0·7611), 6MWD (AUC 0·7086), and exacerbation risk (AUC 0·7341).

Conclusions

Quantifying comorbidity provides a more thorough understanding of the risk for patient-centered outcomes in COPD. A comorbidity count performs well to quantify comorbidity in a diverse population with COPD.     

Role of exhaled nitric oxide as a predictor of atopy

Background

The fractional exhaled nitric oxide (FeNO) is a quantitative, noninvasive and safe measure of airways inflammation that may complement the assessment of asthma. Elevations of FeNO have recently been found to correlate with allergic sensitization. Therefore, FeNO may be a useful predictor of atopy in the general population. We sought to determine the diagnostic accuracy of FeNO in predicting atopy in a population-based study.

Methods

We conducted a cross-sectional study in an age- and sex- stratified random sample of 13 to 15 year-olds in two communities in Peru. We asked participants about asthma symptoms, environmental exposures and sociodemographics, and underwent spirometry, assessment of FeNO and an allergy skin test. We used multivariable logistic regression to model the odds of atopy as a function of FeNO, and calculated area-under-the-curves (AUC) to determine the diagnostic accuracy of FeNO as a predictor of atopy.

Results

Of 1441 recruited participants, 1119 (83%) completed all evaluations. Mean FeNO was 17.6 ppb (SD=0.6) in atopics and 11.6 ppb (SD=0.8) in non-atopics (p<0.001). In multivariable analyses, a FeNO>20 ppb was associated with an increase in the odds of atopy in non-asthmatics (OR=5.3, 95% CI 3.3 to 8.5) and asthmatics (OR=16.2, 95% CI 3.4 to 77.5). A FeNO>20 ppb was the best predictor for atopy with an AUC of 68% (95% CI 64% to 69%). Stratified by asthma, the AUC was 65% (95% CI 61% to 69%) in non-asthmatics and 82% (95% CI 71% to 91%) in asthmatics.

Conclusions

FeNO had limited accuracy to identify atopy among the general population; however, it may be a useful indicator of atopic phenotype among asthmatics.     

Practical approaches to plant volatile analysis

Plants emit volatile organic compounds (VOCs) that play important roles in their interaction with the environment and have a major impact on atmospheric chemistry. The development of static and dynamic techniques for headspace collection of volatiles in combination with gas chromatography-mass spectrometry analysis has significantly improved our understanding of the biosynthesis and ecology of plant VOCs. Advances in automated analysis of VOCs have allowed the monitoring of fast changes in VOC emissions and facilitated in vivo studies of VOC biosynthesis. This review presents an overview of methods for the analysis of plant VOCs, including their advantages and disadvantages, with a focus on the latest technical developments. It provides guidance on how to select appropriate instrumentation and protocols for biochemical, physiological and ecologically relevant applications. These include headspace analyses of plant VOCs emitted by the whole organism, organs or enzymes as well as advanced on-line analysis methods for simultaneous measurements of VOC emissions with other physiological parameters.     

Development of bovine IVF oocytes cultured in medium supplemented with a nitric oxide scavenger or inhibitor in a co-culture system

Bovine IVF oocytes were cultured in modified bovine embryo culture medium (mBECM) supplemented with either a nitric oxide (NO) scavenger, hemoglobin (Hb, 1 microg/mL) and/or a NO synthesis inhibitor, L(omega)-nitro-L-arginine methyl ester (L-NAME, 1 or 1000 nM) in a cumulus-granulosa cell co-culture system. In Experiment 1, a total of 1,675 cumulus-oocytes complexes was collected for 7 mo and cultured to the blastocyst stage in mBECM with or without Hb after IVM and IVF. There were significant (P<0.0024) model effects of Hb addition and month of oocyte collection on embryo development. A significant (P<0.0023) monthly variation was detected in all developmental stages. However, addition of Hb to mBECM consistently enhanced embryo development to the blastocyst stage over all months. No statistical differences were found in the interaction between Hb addition and month except for the cleavage rate. Overall, a greater percentage of oocytes developed to the 8-cell (P<0.0459), 16-cell (P<0.001), morula (P<0.0013) and blastocyst (P<0.0024) stages after the addition of Hb. In Experiment 2, addition of L-NAME to mBECM supplemented with Hb did not further stimulate prehatched development. In conclusion, the promoting effect of Hb on in vitro development of embryos is highly repeatable over an extended period of time.