Analysis of protein synthesis in morphologically classified bovine follicular oocytes before and after maturation in vitro

Bovine cumulus oocyte complexes (COCs) were isolated from antral ovarian follicles (4-8 mm). Immature COCs were classified into four categories, based on the homogeneity and clearness of the ooplasm and the transparency and compactness of the cumulus investment. In this study, the incorporation of TCA-precipitable 35S-methionine and the protein synthesis patterns of oocytes of these four categories were examined. Before maturation in vitro, similar incorporation rates and identical protein synthesis patterns were observed between oocytes of categories 1-3. Immature oocytes of category 4 showed reduced incorporation rates and exhibited aberrant protein synthesis patterns. After maturation in vitro, the patterns of category 4 oocytes were identical with the patterns of those in categories 1-3. The incorporation of 35S-methionine into in vitro matured oocytes was lower (P less than .001) in all categories. Based on these results, it is concluded that the initial classification of oocytes into four categories can be reduced to two categories.     

Critical nature of a specific uridine O2-carbonyl for cleavage by the hammerhead ribozyme.

Three modified hammerhead ribozyme/substrate complexes have been prepared in which individual uridine O2-carbonyls have been eliminated. The modified complexes were chemically synthesized with the substitution of a single 2-pyridone (2P) base analogue for residues U4, U7, and U16.1. Steady-state kinetic analyses indicate that the cleavage efficiencies for the U7 and U16.1 complexes were not significantly reduced relative to the native complex as measured by kcat/KM. The cleavage efficiency for the 2P4 complex, with the analogue present within the uridine loop, was reduced by greater than 2 orders of magnitude. This significant reduction in catalytic efficiency was due primarily to a decrease in kcat. The pH vs cleavage rate profile suggests that the O2-carbonyl of the U4 residue of the hammerhead complex is critical for transition state stabilization and efficient cleavage activity. The results of a Mg2+ rescue assay do not implicate the O2-carbonyl of U4 in an interaction with a divalent metal ion. In addition, the results of a ribozyme folding assay suggest that the presence of the 2P4 within the uridine loop does not alter the folding pathway (relative to the native sequence) both in the absence and in the presence of Mg2+. The O2-carbonyl of U4 appears oriented toward the interior of the catalytic pocket where it may be involved in a critical hydrogen bonding interaction necessary for transition state stabilization.     

Dissociation of yeast ribosomes; The effects of hydrostatic pressure and KCl

The dissociation of free ribosomes at elevated concentrations of KCl is dependent on hydrostatic pressure. The pressure necessary for the dissociation is determined for KCl concentrations ranging from 0.1–0.4 m. It varies between 425 kg cm^-2 at 0.1 m and 10 kg cm^-2 at 0.4 m. The partial dissociation of complex ribosomes in KCl is dependent on hydrostatic pressure in the same way as the complete dissociation of free ribosomes. Therefore, it is concluded that mRNA and peptidyl-tRNA do not contribute to the stability of the ribosome under these conditions.     

Tungsten transport protein A (WtpA) in Pyrococcus furiosus: the first member of a new class of tungstate and molybdate transporters

A novel tungstate and molybdate binding protein has been discovered from the hyperthermophilic archaeon Pyrococcus furiosus. This tungstate transport protein A (WtpA) is part of a new ABC transporter system selective for tungstate and molybdate. WtpA has very low sequence similarity with the earlier-characterized transport proteins ModA for molybdate and TupA for tungstate. Its structural gene is present in the genome of numerous archaea and some bacteria. The identification of this new tungstate and molybdate binding protein clarifies the mechanism of tungstate and molybdate transport in organisms that lack the known uptake systems associated with the ModA and TupA proteins, like many archaea. The periplasmic protein of this ABC transporter, WtpA (PF0080), was cloned and expressed in Escherichia coli. Using isothermal titration calorimetry, WtpA was observed to bind tungstate (dissociation constant [K(D)] of 17 +/- 7 pM) and molybdate (K(D) of 11 +/- 5 nM) with a stoichiometry of 1.0 mol oxoanion per mole of protein. These low K(D) values indicate that WtpA has a higher affinity for tungstate than do ModA and TupA and an affinity for molybdate similar to that of ModA. A displacement titration of molybdate-saturated WtpA with tungstate showed that the tungstate effectively replaced the molybdate in the binding site of the protein.     

Weekly self-monitoring and treatment adjustment benefit patients with partly controlled and uncontrolled asthma: an analysis of the SMASHING study

Background

Internet-based self-management has shown to improve asthma control and asthma related quality of life, but the improvements were only marginally clinically relevant for the group as a whole. We hypothesized that self-management guided by weekly monitoring of asthma control tailors pharmacological therapy to individual needs and improves asthma control for patients with partly controlled or uncontrolled asthma.

Methods

In a 1-year randomised controlled trial involving 200 adults (18-50 years) with mild to moderate persistent asthma we evaluated the adherence with weekly monitoring and effect on asthma control and pharmacological treatment of a self-management algorithm based on the Asthma Control Questionnaire (ACQ). Participants were assigned either to the Internet group (n = 101) that monitored asthma control weekly with the ACQ on the Internet and adjusted treatment using a self-management algorithm supervised by an asthma nurse specialist or to the usual care group (UC) (n = 99). We analysed 3 subgroups: patients with well controlled (ACQ ≤ 0.75), partly controlled (0.75>ACQ ≤ 1.5) or uncontrolled (ACQ>1.5) asthma at baseline.

Results

Overall monitoring adherence was 67% (95% CI, 60% to 74%). Improvements in ACQ score after 12 months were -0.14 (p = 0.23), -0.52 (p < 0.001) and -0.82 (p < 0.001) in the Internet group compared to usual care for patients with well, partly and uncontrolled asthma at baseline, respectively. Daily inhaled corticosteroid dose significantly increased in the Internet group compared to usual care in the first 3 months in patients with uncontrolled asthma (+278 μg, p = 0.001), but not in patients with well or partly controlled asthma. After one year there were no differences in daily inhaled corticosteroid use or long-acting β₂-agonists between the Internet group and usual care.

Conclusions

Weekly self-monitoring and subsequent treatment adjustment leads to improved asthma control in patients with partly and uncontrolled asthma at baseline and tailors asthma medication to individual patients'' needs.

Trial registration

Current Controlled Trials ISRCTN79864465     

Engineering the cell surface display of cohesins for assembly of cellulosome-inspired enzyme complexes on <Emphasis Type="Italic">Lactococcus lactis</Emphasis>

Background  

The assembly and spatial organization of enzymes in naturally occurring multi-protein complexes is of paramount importance for the efficient degradation of complex polymers and biosynthesis of valuable products. The degradation of cellulose into fermentable sugars by Clostridium thermocellum is achieved by means of a multi-protein "cellulosome" complex. Assembled via dockerin-cohesin interactions, the cellulosome is associated with the cell surface during cellulose hydrolysis, forming ternary cellulose-enzyme-microbe complexes for enhanced activity and synergy. The assembly of recombinant cell surface displayed cellulosome-inspired complexes in surrogate microbes is highly desirable. The model organism Lactococcus lactis is of particular interest as it has been metabolically engineered to produce a variety of commodity chemicals including lactic acid and bioactive compounds, and can efficiently secrete an array of recombinant proteins and enzymes of varying sizes.     

Bromocriptine-induced premature oestrus is associated with changes in the pulsatile secretion pattern of follicle-stimulating hormone in beagle bitches

The secretory profiles of LH and FSH were investigated before and during the administration of bromocriptine in six beagle bitches. Plasma samples were obtained via jugular venepuncture at 10 min intervals for 6 h every 2 weeks until the next ovulation. Bromocriptine treatment was started 100 days after ovulation. Both before and after bromocriptine treatment, LH and FSH pulses occurred together. The mean duration of the FSH pulse (120 min) was significantly longer than that of the LH pulse (80 min). The interoestrous interval in the bitches treated with bromocriptine was significantly shorter than that of the preceding cycle (160 +/- 3 versus 206 +/- 24 days). The mean basal plasma FSH concentration (7.4 +/- 0.6 versus 6.1 +/- 0.7 iu l-1) and the mean area under the curve for FSH (46.6 +/- 4.7 versus 40.4 +/- 4.4 iu l-1 in 6 h) increased significantly after the start of the bromocriptine treatment. In contrast, the differences in mean basal plasma LH concentration (2.1 +/- 0.2 versus 2.0 +/- 0.2 micrograms l-1) and the mean area under the curve for LH (19.0 +/- 3.1 versus 19.5 +/- 2.5 micrograms l-1 in 6 h) between the day before and 14 days after the start of the bromocriptine treatment were not significant. Bromocriptine administration also lowered the mean amplitude of the FSH pulse and shortened the mean duration of the FSH pulse, without influencing the LH pulse. In addition to demonstrating the concurrent pulsatile secretion of LH and FSH, the results of the present study demonstrate that the bromocriptine-induced shortening of the interoestrous interval in the bitch is associated with an increase in plasma FSH concentration without a concomitant increase in plasma LH concentration. This finding indicates that treatment with the dopamine agonist bromocriptine increase plasma FSH to a concentration that results in the enhancement of follicle development.     

Scott syndrome, a bleeding disorder caused by defective scrambling of membrane phospholipids

Normal quescent cells maintain membrane lipid asymmetry by ATP-dependent membrane lipid transporters, which shuttle different phospholipids from one leaflet to the other against their respective concentration gradients. When cells are challenged, membrane lipid asymmetry can be perturbed resulting in exposure of phosphatidylserine [PS] at the outer cell surface. Translocation of PS from the inner to outer membrane leaflet of activated blood platelets and platelet-derived microvesicles provides a catalytic surface for interacting coagulation factors. This process is dramatically impaired in Scott syndrome, a rare congenital bleeding disorder, underscoring the indispensible role of PS in hemostasis. This also testifies to a defect of a protein-catalyzed scrambling of membrane phospholipids. The Scott phenotype is not restricted to platelets, but can be demonstrated in other blood cells as well. The functional aberrations observed in Scott syndrome have increased our understanding of transmembrane lipid movements, and may help to identify the molecular elements that promote the collapse of phospholipid asymmetry during cell activation and apoptosis.     

The effect of oviductal epithelial cell co-culture during in vitro maturation on sow oocyte morphology,fertilization and embryo development

In vitro embryo production in the sow is challenged by poor cytoplasmic maturation, low sperm penetration and low normal fertilization, leading to the development of poor quality blastocysts containing a small number of nuclei. In prepubertal gilt oocytes, the presence of porcine oviductal epithelial cells (pOECs) during maturation increases cytoplasmic maturation and blastocyst development. These aspects, as well as blastocyst quality, may be improved when adult sow oocytes are matured with pOEC. Therefore, the effect of the presence of pOEC on sow oocyte morphology, fertilization and the progression of embryo development was evaluated. The pOEC were cultured in M199 for 18 h, then cultured in NCSU23 for 4 h before the oocytes were added. Oocytes from 2 to 6 mm follicles were matured in 500 microl NCSU23, with eCG and hCG, for 24 h, and then cultured with or without pOEC, in NCSU23 without hormones, for 18 h. In vitro fertilization took place in modified Tris-buffered medium, for 6 h, and the presumptive zygotes were then cultured for 162 h in NCSU23. Morphology of the IVM oocytes was compared to that of immature oocytes and in vivo matured MII oocytes from slaughtered sows in estrus. The in vitro matured oocytes had a greater diameter and a wider perivitelline space than the immature and in vivo matured MII oocytes (P < 0.01). Penetration, polyspermy and pronucleus formation did not differ between the pOEC and Control groups, although the total penetration rate was higher for the Control oocytes (26% versus 39%; P < 0.01). Fewer blastocysts developed in the pOEC group than in the Control group (19% versus 27%; P < 0.01), but blastocyst growth was accelerated, leading to a higher percentage of hatched blastocysts (3% versus 10%; P < 0.01). Finally, the average blastocyst cell number was higher in the pOEC group (47 versus 40; P < 0.05) and a greater percentage of blastocysts contained a superior number of nuclei. In conclusion, the addition of pOEC during the second half of in vitro maturation resulted in fewer blastocysts formed, but of those blastocysts that did form the quality was improved.