Characterization of Disease-related 5β-Reductase (AKR1D1) Mutations Reveals Their Potential to Cause Bile Acid Deficiency

Bile acid deficiency is a serious syndrome in newborns that can result in death if untreated. 5β-Reductase deficiency is one form of bile acid deficiency and is characterized by dramatically decreased levels of physiologically active 5β-reduced bile acids. AKR1D1 (aldo-keto reductase 1D1) is the only known human enzyme that stereo-specifically reduces the Δ⁴ double bond in 3-keto steroids and sterols to yield the 5β-hydrogenated product. Analysis of the AKR1D1 gene in five patients with 5β-reductase deficiency revealed five different mutations resulting in an amino acid substitution in the protein. To investigate a causal role for these observed point mutations in AKR1D1 in 5β-reductase deficiency, we characterized their effect on enzymatic properties. Attempts to purify mutant enzymes by overexpression in Escherichia coli only yielded sufficient amounts of the P133R mutant for further characterization. This enzyme displayed a highly reduced K_m and V_max reminiscent of uncompetitive kinetics with 4-cholesten-7α-ol-3-one as substrate. In addition, this mutant displayed no change in cofactor affinity but was more thermolabile in the absence of NADPH as judged by CD spectroscopy. All mutants were compared following expression in HEK 293 cells. Although these enzymes were equally expressed based on mRNA levels, protein expression and functional activity were dramatically reduced. Cycloheximide treatment also revealed that several of the expressed mutants were less stable. Our findings show that the reported mutations in AKR1D1 in patients with 5β-reductase lead to significantly decreased levels of active enzyme and could be causal in the development of bile acid deficiency syndrome.     

Electrostatic interactions and complement activation on the surface of phospholipid vesicle containing acidic lipids: Effect of the structure of acidic groups

Anionic vesicles containing acidic phospholipids are known complement activators. To clarify which negative physicochemical electrostatic charges on vesicles and structural specificities of acidic lipids are critical to complement activation, the electrostatic properties and activity to complement of two anionic vesicles modified with a carboxylic acid derivative or a conventional acidic phospholipid were compared. Electrophoretic mobility measurements indicated that the negative zeta potential and the electrostatic interactivity of these two anionic vesicles were equal at pH 7.4. However, the infusion of vesicles containing acidic phospholipid induced significant complement activation, while vesicles containing the carboxylic acid derivative failed to activate complement. These results indicate that the negative charge on the surface of vesicles is not critical for the activation complement, suggesting that complement activation is specific to the structure of acidic groups. This finding is likely to be important to the design of anionic biointerfaces and may support the promising medical applications of this anionic vesicle modified with a carboxylic acid derivative.     

Blistering of supported lipid membranes induced by Phospholipase D, as observed by real-time atomic force microscopy

Phospholipase D from Streptomyces chromofuscus (PLDSc) is a soluble enzyme known to be activated by the phosphatidic acid (PA)-calcium complexes. Despite the vast body of literature that has accumulated on this enzyme, the exact mechanism of activation remains poorly understood. In this work, we report the first observation of PLDSc activity in real time and at nanometer resolution using atomic force microscopy (AFM). AFM images of continuous and patchy dipalmitoylphosphatidylcholine (DPPC) bilayers were recorded, prior and after incubation with PLDSc. For continuous bilayers, the enzyme induced important morphological alterations; holes corresponding to the bilayer thickness were created, while an additional elevated phase, about 2.5 nm high, was observed. This bilayer blistering is believed to be due to the production of the negatively charged lipid PA that would cause localized repulsions between the bilayer and the underlying mica surface. By contrast, these elevated domains were not seen on patchy bilayers incubated with the enzyme. Instead, the shapes of DPPC patches were strongly deformed by enzyme activity and evolved into melted morphologies. These results point to the importance of lipid packing on PLD activity and illustrate the potential of AFM for visualizing remodeling enzymatic activities.     

The Effect of Leaf Age on Gas Exchange and Malate Accumulation in C3-CAM Plant Marrubium Frivaldszkyanum (Lamiaceae)

For the first time the expression of C₃ and CAM in the leaves of different age of Marrubium frivaldszkyanum Boiss, is reported. With increasing leaf age a typical C₃ photosynthesis pattern and high transpiration rate were found. In older leaves a shift to CAM occurred and the 24-h transpiration water loss decreased. A correlation was established between leaf area and accumulation of malate. Water loss at early stages of leaf expansion may be connected with the shift to CAM and the water economy of the whole plant.     

Gradients in maize roots: local elongation and pH

The elongation rate, the gradient of the local elongation rate and the surface pH of maize roots were measured over 12 h. A data bank was constituted by storing these values. By sorting these results on the basis of different elongation rates, different classes of root were obtained. Two classes were chosen: the low-growth roots and the high-growth roots. The mean growth of these two root classes was stable with time and differed significantly from one another. The surface pH of the elongation zone was the same for the roots of these two classes, but the roots selected for their higher growth rate had a larger acid efflux in this zone.     

Interaction between the Msh2 and Msh6 Nucleotide-binding Sites in the Saccharomyces cerevisiae Msh2-Msh6 Complex

Indirect evidence has suggested that the Msh2-Msh6 mispair-binding complex undergoes conformational changes upon binding of ATP and mispairs, resulting in the formation of Msh2-Msh6 sliding clamps and licensing the formation of Msh2-Msh6-Mlh1-Pms1 ternary complexes. Here, we have studied eight mutant Msh2-Msh6 complexes with defective responses to nucleotide binding and/or mispair binding and used them to study the conformational changes required for sliding clamp formation and ternary complex assembly. ATP binding to the Msh6 nucleotide-binding site results in a conformational change that allows binding of ATP to the Msh2 nucleotide-binding site, although ATP binding to the two nucleotide-binding sites appears to be uncoupled in some mutant complexes. The formation of Msh2-Msh6-Mlh1-Pms1 ternary complexes requires ATP binding to only the Msh6 nucleotide-binding site, whereas the formation of Msh2-Msh6 sliding clamps requires ATP binding to both the Msh2 and Msh6 nucleotide-binding sites. In addition, the properties of the different mutant complexes suggest that distinct conformational states mediated by communication between the Msh2 and Msh6 nucleotide-binding sites are required for the formation of ternary complexes and sliding clamps.     

Quantitative and qualitative assessment on the suitability of seed oil from water plant (Trichilia emetica) for soap making

Despite widespread and its local available as a naturalized hedge and shade plant, the potential of Trichilia emetica was not utilized in soap making by the majority of local community in various parts of Dodoma, Tanzania. This study aimed to assess the quantity (yields) and quality (Acid Values (AVs), %Free Fatty Acids (%FFAs) and Saponification Values (SVs) of seed oil from water plant (T. emetica), suitable for soap making application. Solvent extraction method was used during oil extraction, where by 50gm of preheated and powdered seed materials were immersed in 250 ml of n-hexane in 1:5 (w/v) to dissolve the oil contained in the seed cake. The oil was collected by vaporizing solvent out through Rotary evaporation at 60 °C. Also standard titration methods were used to obtain SVs, AVs and %FFAs of the extracted oil. Results showed that T. emetica seeds contained higher quantity of oil (48.4%−50.2%) than many reported commercial plant seed oils. Also, the study found higher AV (7.4 mgKOH/g−7.8 mgKOH/g), %FFA (3.7% to 3.9%) and SVs (189.5 mgKOH/g − 191.4 mgKOH/g) than the maximum acceptable limits of 0.50 mg KOH/g, 0.020% and 175 mgKOH/g − 187 mgKOH/g prescribed by ASTM standards (2002). The obtained results showed that, T. emetica seeds yielded high oil quantity with low qualities due to higher levels of acidity. But high SVs guarantees the possibility of using T. emetica seed oil in soap making. However, the oil requires purification in order to bring levels of acidity to acceptable standards and guarantee its normal use in soap making.     

Enhancing native chemical ligation for challenging chemical protein syntheses

Native chemical ligation has enabled the chemical synthesis of proteins for a wide variety of applications (e.g., mirror-image proteins). However, inefficiencies of this chemoselective ligation in the context of large or otherwise challenging protein targets can limit the practical scope of chemical protein synthesis. In this review, we focus on recent developments aimed at enhancing and expanding native chemical ligation for challenging protein syntheses. Chemical auxiliaries, use of selenium chemistry, and templating all enable ligations at otherwise suboptimal junctions. The continuing development of these tools is making the chemical synthesis of large proteins increasingly accessible.