Molecular evolution of rodent insulins

Several trees of amino acid sequences of rodent insulins were derived with the maximum-parsimony procedure. Possible orthologous and paralogous relationships were investigated. Except for a recent gene duplication in the ancestor of rat and mouse, there are no strong arguments for other paralogous relationships. Therefore, a tree in agreement with other biological data is the most reasonable one. According to this tree, the capacity to form zinc-binding hexamers was lost once in the ancestor of the hystricomorph rodents, followed by moderately increased evolutionary rates in the lineages to African porcupine and chinchilla but highly increased rates in at least three independent lines to other taxa of this suborder: guinea pig, cuis, and Octodontoidea (coypu and casiragua).      

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.     

The abundance of additional N-acetyllactosamine units in N-linked tetraantennary oligosaccharides of human Tamm-Horsfall glycoprotein is a donor-specific feature

Previously, treatment of Tamm-Horsfall glycoprotein (THp) from different donors with endo-beta-galactosidase has been shown to liberate a tetra- and a Sd(a)-active pentasaccharide, concluding the presence of N-linked carbohydrate chains containing additional N - acetyllactosamine units. These type of oligosaccharides were not found in a detailed structure elucidation of the carbohydrate moiety of THp of one male donor, suggesting a donor-specific feature for these type of structures. Therefore, THp was isolated from four healthy male donors and each subjected to endo-beta-galactosidase treatment in order to release these tetra- and Sd(a)-active pentasaccharide. Differences were observed in the total amount of released tetra- and Sda-active pentasaccharide of the used donors (42, 470, 478, 718 microg/100 mg THp), indicating that the presence of repeating N-acetyllactosamine units incorporated into the N-glycan moiety of THp is donor specific. Furthermore, a higher expression of the Sd(a) determinant on antennae which display N-acetyllactosamine elongation was observed, suggesting a better accessibility for the beta-N-acetylgalactosaminyltransferase. In order to characterize the N-glycans containing repeating N- acetyllactosamine units, carbohydrate chains were enzymatically released from THp and isolated. The tetraantennary fraction, which accounts for more than 33% of the total carbohydrate moiety of THp, was used to isolate oligosaccharides containing additional N - acetyllactosamine units. Five N-linked tetraantennary oligosaccharides containing a repeating N-acetyllactosamine unit were identified, varying from structures bearing four Sd(a) determinants to structures containing no Sd(a) determinant (see below). One compound was used in order to specify the branch location of the additional N- acetyllactosamine unit, and it appeared that only the Gal-6' and Gal-8' residues were occupied by a repeating N -acetyllactosamine unit.      

Wnt family proteins are secreted and associated with the cell surface.

Members of the Wnt gene family are proposed to function in both normal development and differentiation as well as in mammary tumorigenesis. To understand the function of Wnt proteins in these two processes, we present here a biochemical characterization of seven Wnt family members. For these studies, AtT-20 cells, a neuroendocrine cell line previously shown to efficiently process and secrete Wnt-1, was transfected with expression vectors encoding Wnt family members. All of the newly characterized Wnt proteins are glycosylated, secreted proteins that are tightly associated with the cell surface or extracellular matrix. We have also identified native Wnt proteins in retinoic acid-treated P19 embryonal carcinoma cells, and they exhibit the same biochemical characteristics as the recombinant proteins. These data suggest that Wnt family members function in cell to cell signaling in a fashion similar to Wnt-1.     

Uptake kinetics of 99Tc in common duckweed

The uptake of the nuclear waste product technetium-99 was studied in common duckweed (Lemna minor). In addition to measurements, a model involving two compartments in duckweed with different chemical forms of technetium was derived. The model was tested by chemical speciation, i.e. differentiating between reduced Tc-compounds and Tc(VII)O(4)(-). The TcO(4)(-) concentrations measured were in good agreement with those predicted by the model. Two processes determine technetium uptake: (1) transport of Tc(VII)O(4)(-) across the cell membrane, and (2) reduction of Tc(VII). The TcO(4)(-) concentration in duckweed reaches a steady state within 2 h while reduced Tc-compounds are stored, as a result of absence of release or re-oxidation processes. Bioaccumulation kinetic properties were derived by varying 99Tc concentration, temperature, nutrient concentrations, and light intensity. The reduction of technetium in duckweed was highly correlated with light intensity and temperature. At 25 degrees C the maximum reduction rate was observed at light intensities above 200 μmol m(-2) s(-1) while half of the maximum transformation rate was reached at 41 μmol m(-2) s(-1). Transport of TcO(4)(-) over the cell membrane requires about 9.4 kJ mol(-1), indicating an active transport mechanism. However, this mechanism behaved as first-order kinetics instead of Michaelis-Menten kinetics between 1x10(-14) and 2.5x10(-5) mol l(-1) TcO(4)(-). Tc uptake could not be inhibited by 10(-3) mol l(-1) nitrate, phosphate, sulphate or chloride.     

Long-term development of the radionuclide exposure of murine rodent populations in Belarus after the Chernobyl accident

As a determinant of the associated health risks, the behavior of radionuclides in natural ecosystems needs to be better understood. Therefore, the activity concentration of various long-lived radionuclides released due to the Chernobyl accident, and the corresponding contributions to the whole-body dose rate, was studied as a function of time in mammalian indicator species inhabiting the natural forest ecosystems of Belarus, the bank vole (Clethrionomys glareolus) and the yellow-necked mouse (Apodemus flavicollus). The activity concentrations of ¹³⁷Cs, ¹³⁴Cs, ⁹⁰Sr, ²³⁸Pu, ^239,240Pu, ²⁴¹Pu and ²⁴¹Am in soil and in animals were measured at five monitoring sites with different ground deposition of radionuclides at different distances from the destroyed reactor. The observed temporal pattern of the radionuclide activity concentration in the studied animal populations reflects the changes in biological availability of these isotopes for biota, mostly due to fuel particle destruction and appearance of dissolved and exchangeable forms of radionuclides. The time course of ^134+137Cs activity concentrations in animal populations appeared as a sequence of increase, peak and decrease. Maximal levels of radiocesium occurred 1–2 years after deposition, followed by an exponential decrease. Concentrations of incorporated ⁹⁰Sr increased up to the tenth year after deposition. The activity concentrations of transuranic elements (²³⁸Pu, ^239,240Pu, ²⁴¹Pu and ²⁴¹Am) were much lower than those of the other radionuclides, in the studied animals. A considerable activity of ²⁴¹Am in animals from areas with high levels of contamination was firstly detected 5 years after deposition, it increased up to the tenth year and is expected to increase further in the future. Maximal values of the whole-body absorbed dose rates occurred during the year of deposition, followed by a decrease in the subsequent period. Generally, this decrease was monotonic, mainly determined by the decrease of the external γ-ray dose rate, but there were exceptions due to the delayed maximum of internal exposure. The inter-individual distributions of radionuclide concentrations and lifetime whole-body absorbed doses were asymmetric and close to log-normal, including concentrations and doses considerably higher than the population mean values.