Metabolic channeling of carbamoyl phosphate, a thermolabile intermediate: evidence for physical interaction between carbamate kinase-like carbamoyl-phosphate synthetase and ornithine carbamoyltransferase from the hyperthermophile Pyrococcus furiosus

Two different approaches provided evidence for a physical interaction between the carbamate kinase-like carbamoyl-phosphate synthetase (CKase) and ornithine carbamoyltransferase (OTCase) from the hyperthermophilic archaeon Pyrococcus furiosus. Affinity electrophoresis indicated that CKase and OTCase associate into a multienzyme cluster. Further evidence for a biologically significant interaction between CKase and OTCase was obtained by co-immunoprecipitation combined with formaldehyde cross-linking experiments. These experiments support the hypothesis that CKase and OTCase form an efficient channeling cluster for carbamoyl phosphate, an extremely thermolabile and potentially toxic metabolic intermediate. Therefore, by physically interacting with each other, CKase and OTCase prevent the thermodenaturation of carbamoyl phosphate in the aqueous cytoplasmic environment.     

Cystatin C Is Not Causally Related to Coronary Artery Disease

Background

Strong and independent associations between plasma concentration of cystatin C and risk of cardiovascular disease (CVD) suggests causal involvement of cystatin C.

Aim

The aim of our study was to assess whether there is a causal relationship between plasma concentration of cystatin C and risk of coronary artery disease (CAD) using a Mendelian Randomization approach.

Methods

We estimated the strength of association of plasma cystatin C on CAD risk and the strength of association of the strongest GWAS derived cystatin C SNP (rs13038305) on plasma cystatin C in the population-based Malmö Diet and Cancer Study (MDC) and thereafter the association between rs13038305 and CAD in the MDC (3200 cases of CAD and 24418 controls) and CARDIOGRAM (22233 cases of CAD and 64762 controls).

Results

Each standard deviation (SD) increment of plasma cystatin C was associated with increased risk of CAD (OR = 1.20, 95% CI 1.07–1.34) after full adjustment. Each copy of the major allele of rs13038305 was associated with 0.34 SD higher plasma concentration of cystatin C (P<1 x 10^-35), resulting in a power of >98% to detect a significant relationship between rs13038305 and CAD in MDC and CARDIOGRAM pooled. The odds ratio for CAD (per copy of the major rs13038305 allele) was 1.00 (0.94–1.07); P = 0.92 in MDC, 0.99 (0.96–1.03); P = 0.84 in CARDIOGRAM and 1.00 (0.97–1.03); P = 0.83 in MDC and CARDIOGRAM pooled.

Conclusion

Genetic elevation of plasma cystatin C is not related to altered risk of CAD, suggesting that there is no causal relationship between plasma cystatin C and CAD. Rather, the association between cystatin C and CAD appears to be due to the association of eGFR and CAD.     

Loop migration in adult marsh harriers Circus aeruginosus,as revealed by satellite telemetry

Loop migration among birds is characterized by the spring route lying consistently west or east of the autumn route. The existence of loops has been explained by general wind conditions or seasonal differences in habitat distribution. Loop migration has predominantly been studied at the population level, for example by analysing ring recoveries. Here we study loop migration of individual marsh harriers Circus aeruginosus tracked by satellite telemetry. We show that despite a generally narrow migration corridor the harriers travelled in a distinct clockwise loop through Africa and southern Europe, following more westerly routes in spring than in autumn. We used the Normalized Difference Vegetation Index (NDVI) to identify potential feeding habitat in Africa. Suitable habitat seemed always more abundant along the western route, both in spring and autumn, and no important stopover site was found along the eastern route. Observed routes did thus not coincide with seasonal variation in habitat availability. However, favourable habitat might be more important during spring migration, when the crossing of the Sahara seems more challenging, and thus habitat availability might play an indirect role in the harriers’ route choice. Grid‐based wind data were used to reconstruct general wind patterns, and in qualitative agreement with the observed loop marsh harriers predominantly encountered westerly winds in Europe and easterly winds in Africa, both in autumn and in spring. By correlating tail‐ and crosswinds with forward and perpendicular movement rates, respectively, we show that marsh harriers are partially drifted by wind. Thus, we tentatively conclude that wind rather than habitat seems to have an overriding effect on the shape of the migration routes of marsh harriers. General wind conditions seem to play an important role also in the evolution of narrow migratory loops as demonstrated for individual marsh harriers.     

UGT73C6 and UGT78D1, glycosyltransferases involved in flavonol glycoside biosynthesis in Arabidopsis thaliana

Flavonol glycosides constitute one of the most prominent plant natural product classes that accumulate in the model plant Arabidopsis thaliana. To date there are no reports of functionally characterized flavonoid glycosyltransferases in Arabidopsis, despite intensive research efforts aimed at both flavonoids and Arabidopsis. In this study, flavonol glycosyltransferases were considered in a functional genomics approach aimed at revealing genes involved in determining the flavonol-glycoside profile. Candidate glycosyltransferase-encoding genes were selected based on homology to other known flavonoid glycosyltransferases and two T-DNA knockout lines lacking flavonol-3-O-rhamnoside-7-O-rhamnosides (ugt78D1) and quercetin-3-O-rhamnoside-7-O-glucoside (ugt73C6 and ugt78D1) were identified. To confirm the in planta results, cDNAs encoding both UGT78D1 and UGT73C6 were expressed in vitro and analyzed for their qualitative substrate specificity. UGT78D1 catalyzed the transfer of rhamnose from UDP-rhamnose to the 3-OH position of quercetin and kaempferol, whereas UGT73C6 catalyzed the transfer of glucose from UDP-glucose to the 7-OH position of kaempferol-3-O-rhamnoside and quercetin-3-O-rhamnoside, respectively. The present results suggest that UGT78D1 and UGT73C6 should be classified as UDP-rhamnose:flavonol-3-Orhamnosyltransferase and UDP-glucose:flavonol-3-O-glycoside-7-O-glucosyltransferase, respectively.     

Irreversible binding and activity control of the 1,2-diacylglycerol 3-glucosyltransferase from Acholeplasma laidlawii at an anionic lipid bilayer surface

1,2-Diacylglycerol 3-glucosyltransferase is associated with the membrane surface catalyzing the synthesis of the major nonbilayer-prone lipid alpha-monoglucosyl diacylglycerol (MGlcDAG) from 1,2-DAG in the cell wall-less Acholeplasma laidlawii. Phosphatidylglycerol (PG), but not neutral or zwitterionic lipids, seems to be essential for an active conformation and function of the enzyme. Surface plasmon resonance analysis was employed to study association of the enzyme with lipid bilayers. Binding kinetics could be well fitted only to a two-state model, implying also a (second) conformational step. The enzyme bound less efficiently to liposomes containing only zwitterionic lipids, whereas increasing molar fractions of the anionic PG or cardiolipin (CL) strongly promoted binding by improved association (k(a1)), and especially a decreased rate of return (k(d2)) from the second state. This yielded a very low overall dissociation constant (K(D)), corresponding to an essentially irreversible membrane association. Both liposome binding and consecutive activity of the enzyme correlated with the PG concentration. The importance of the electrostatic interactions with anionic lipids was shown by quenching of both binding and activity with increasing NaCl concentrations, and corroborated in vivo for an active enzyme-green fluorescent protein hybrid in Escherichia coli. Nonbilayer-prone lipids substantially enhanced enzyme-liposome binding by promoting a changed conformation (decreasing k(d2)), similar to the anionic lipids, indicating the importance of hydrophobic interactions and a curvature packing stress. For CL and the nonbilayer lipids, effects on enzyme binding and consecutive activity were not correlated, suggesting a separate lipid control of activity. Similar features were recorded with polylysine (cationic) and polyglutamate (anionic) peptides present, but here probably dependent on the selective charge interactions with the enzyme N- and C-domains, respectively. A lipid-dependent conformational change and PG association of the enzyme were verified by circular dichroism, intrinsic tryptophan, and pyrene-probe fluorescence analyses, respectively. It is concluded that an electrostatic association of the enzyme with the membrane surface is accompanied by hydrophobic interactions and a conformational change. However, specific lipids, the curvature packing stress, and proteins or small molecules bound to the enzyme can modulate the activity of the bound A. laidlawii MGlcDAG synthase.     

Automated quantification of steatosis: agreement with stereological point counting

Background

Steatosis is routinely assessed histologically in clinical practice and research. Automated image analysis can reduce the effort of quantifying steatosis. Since reproducibility is essential for practical use, we have evaluated different analysis methods in terms of their agreement with stereological point counting (SPC) performed by a hepatologist.

Methods

The evaluation was based on a large and representative data set of 970 histological images from human patients with different liver diseases. Three of the evaluated methods were built on previously published approaches. One method incorporated a new approach to improve the robustness to image variability.

Results

The new method showed the strongest agreement with the expert. At 20× resolution, it reproduced steatosis area fractions with a mean absolute error of 0.011 for absent or mild steatosis and 0.036 for moderate or severe steatosis. At 10× resolution, it was more accurate than and twice as fast as all other methods at 20× resolution. When compared with SPC performed by two additional human observers, its error was substantially lower than one and only slightly above the other observer.

Conclusions

The results suggest that the new method can be a suitable automated replacement for SPC. Before further improvements can be verified, it is necessary to thoroughly assess the variability of SPC between human observers.