Solution structure of the calmodulin‐like C‐terminal domain of Entamoeba α‐actinin2

Cell motility is dependent on a dynamic meshwork of actin filaments that is remodelled continuously. A large number of associated proteins that are severs, cross‐links, or caps the filament ends have been identified and the actin cross‐linker α‐actinin has been implied in several important cellular processes. In Entamoeba histolytica, the etiological agent of human amoebiasis, α‐actinin is believed to be required for infection. To better understand the role of α‐actinin in the infectious process we have determined the solution structure of the C‐terminal calmodulin‐like domain using NMR. The final structure ensemble of the apo form shows two lobes, that both resemble other pairs of calcium‐binding EF‐hand motifs, connected with a mobile linker. Proteins 2016; 84:461–466. © 2016 Wiley Periodicals, Inc.     

Advances in flavonoid glycosyltransferase research: integrating recent findings with long-term citrus studies

Flavonoid glycosides are required for a number of crucial roles in planta and have the potential for development in a variety of agricultural, medicinal, and biotechnological applications. A number of recent advancements have been made in characterizing glycosyltransferases, the enzymes that are responsible for the synthesis of these important molecules. In this review, glycosyltransferases are considered with regard to biochemical properties, expression patterns, levels of enzyme activity during development, and structure/function relationships. This is presented with historical context to highlight critical findings, particularly with regard to the innovative work that has come from research on citrus species. The plant glycosyltransferase crystal structures that have been solved over the past decade, either alone or in complex with sugar donor and/or acceptor molecules, are discussed. The application of results from these structures to inform current structure/function work as well as implications and goals for future crystallography and tertiary modeling studies are considered. A thorough understanding of the properties of glycosyltransferases will be a critical step in any future biotechnological application of these enzymes in areas such as crop improvement and custom design of enzymes to produce desired compounds for nutritional and/or medicinal usage.     

Sphingosine 1‐phosphate and its carrier apolipoprotein M in human sepsis and in Escherichia coli sepsis in baboons

Sphingosine 1‐phosphate (S1P) is an important regulator of vascular integrity and immune cell migration, carried in plasma by high‐density lipoprotein (HDL)‐associated apolipoprotein M (apoM) and by albumin. In sepsis, the protein and lipid composition of HDL changes dramatically. The aim of this study was to evaluate changes in S1P and its carrier protein apoM during sepsis. For this purpose, plasma samples from both human sepsis patients and from an experimental Escherichia coli sepsis model in baboons were used. In the human sepsis cohort, previously studied for apoM, plasma demonstrated disease‐severity correlated decreased S1P levels, the profile mimicking that of plasma apoM. In the baboons, a similar disease‐severity dependent decrease in plasma levels of S1P and apoM was observed. In the lethal E. coli baboon sepsis, S1P decreased already within 6–8 hrs, whereas the apoM decrease was seen later at 12–24 hrs. Gel filtration chromatography of plasma from severe human or baboon sepsis on Superose 6 demonstrated an almost complete loss of S1P and apoM in the HDL fractions. S1P plasma concentrations correlated with the platelet count but not with erythrocytes or white blood cells. The liver mRNA levels of apoM and apoA1 decreased strongly upon sepsis induction and after 12 hr both were almost completely lost. In conclusion, during septic challenge, the plasma levels of S1P drop to very low levels. Moreover, the liver synthesis of apoM decreases severely and the plasma levels of apoM are reduced. Possibly, the decrease in S1P contributes to the decreased endothelial barrier function observed in sepsis.     

Dp71Δ78‐79 dystrophin mutant stimulates neurite outgrowth in PC12 cells via upregulation and phosphorylation of HspB1

PC12 cells acquire a neuronal phenotype in response to nerve growth factor (NGF). However, this phenotype is more efficiently achieved when the Dp71Δ_78‐79 dystrophin mutant is stably expressed in PC12‐C11 cells. To investigate the effect of Dp71Δ_78‐79 overexpression on the protein profile of PC12‐C11 cells, we compared the expression profiles of undifferentiated and NGF‐differentiated PC12‐C11 and PC12 cells by 2DE. In undifferentiated cultures, one protein was downregulated, and five were upregulated. Dp71Δ_78‐79 overexpression had a greater effect on differentiated cultures, with ten proteins downregulated and seven upregulated. The protein with the highest upregulation was HspB1. Changes in HspB1 expression were validated by Western blot and immunofluorescence analyses. Interestingly, the neurite outgrowth in PC12‐C11 cells was affected by a polyclonal antibody against HspB1, and the level of HspB1 and HspB1Ser86 decreased, suggesting an important role for this protein in this cellular process. Our results show that Dp71Δ_78‐79 affects the expression level of some proteins and that the stimulated neurite outgrowth produced by this mutant is mainly through upregulation and phosphorylation of HspB1.     

Non-Classical Monocytes and Monocyte Chemoattractant Protein-1 (MCP-1) Correlate with Coronary Artery Calcium Progression in Chronically HIV-1 Infected Adults on Stable Antiretroviral Therapy

Background

Persistent inflammation and immune activation has been hypothesized to contribute to increased prevalence of subclinical atherosclerosis and cardiovascular disease (CVD) risk in patients with chronic HIV infection. In this study, we examined the correlation of peripheral monocyte subsets and soluble biomarkers of inflammation to coronary artery calcium (CAC) progression, as measured by cardiac computed tomography scan.

Methods

We conducted a longitudinal analysis utilizing baseline data of 78 participants with HIV infection on stable antiretroviral therapy (ART) in the Hawaii Aging with HIV-Cardiovascular study who had available baseline monocyte subset analysis as well as CAC measurement at baseline and at 2-year follow up. Monocyte phenotypes were assessed from cryopreserved blood by flow cytometry and plasma was assayed for soluble biomarkers using antibody-coated beads in a high sensitivity Milliplex Luminex platform. Change in CAC over 2 years was analyzed as the primary outcome variable.

Results

Of all monocyte subsets and biomarkers tested, higher non-classical monocyte percentage (ρ = 0.259, p = 0.022), interleukin (IL)-6 (ρ = 0.311, p = 0.012), and monocyte chemoattractant protein (MCP)-1 (ρ = 0.524, p = <0.001) were significantly correlated to higher 2-year CAC progression in unadjusted Spearman’s correlation. Non-classical monocyte percentage (ρ = 0.247, p = 0.039), and MCP-1 (ρ = 0.487, p = <0.001), remained significantly correlated to 2-year CAC progression, while IL-6 was not (ρ = 0.209, p = 0.120) after adjustment for age, hypertension, diabetes mellitus, total/HDL cholesterol ratio, smoking history, and BMI.

Conclusion

The percentage of non-classical monocytes and plasma MCP-1 levels were independently associated with CAC progression and may be related to the progression of atherosclerosis and increased CVD risk associated with chronic HIV infection on stable ART.     

Demographic and tumour prevalence data for juvenile green turtles at the Coastal-Marine Protected Area of Cerro Verde,Uruguay

Marine Protected Areas are increasingly considered in coastal areas as an instrument to preserve threatened fauna and fragile habitats from the detrimental effects of human activities. For this reason baseline data are of utmost importance for the evaluation of the outcomes of ongoing conservation efforts. Along the Uruguayan coast, the area of Cerro Verde (declared protected since 2011) represents the most important foraging and development area for green turtles (Chelonia mydas). Between 2002 and 2009, a long-term capture-mark-recapture programme for green turtles was developed to gather data on demography, ecology and status of the species in the area. Turtles captured were juveniles ranging from 28.8 to 64.3?cm in length over the curve of the carapace (n?=?514), and results indicated a size-based habitat segregation. Tumour prevalence was 5.3% (n?=?27) and was positively correlated with carapace length. The mean body condition index was 1.25?±?0.14 (n?=?494). From the total number of tagged turtles 10.6% were recaptured during the study period. Green turtles showed high site fidelity; 81% of the turtles were recaptured within the same season and 76% were recaptured in different seasons but were found at the original capture spot. Mean annual growth rate was 1.6?±?0.9?cm year^?1. The catch per unit effort of 2008 differed from 2009, higher in 2009, but also significantly different between capture spots. The present study constitutes a baseline dataset for future monitoring of green turtles in the area and provides valuable information for wider analyses of population dynamics in the Southwestern Atlantic Ocean.     

Kinetic and functional properties of human mitochondrial phosphoenolpyruvate carboxykinase

The cytosolic form of phosphoenolpyruvate carboxykinase (PCK1) plays a regulatory role in gluconeogenesis and glyceroneogenesis. The role of the mitochondrial isoform (PCK2) remains unclear. We report the partial purification and kinetic and functional characterization of human PCK2. Kinetic properties of the enzyme are very similar to those of the cytosolic enzyme. PCK2 has an absolute requirement for Mn²⁺ ions for activity; Mg²⁺ ions reduce the K_m for Mn²⁺ by about 60 fold. Its specificity constant is 100 fold larger for oxaloacetate than for phosphoenolpyruvate suggesting that oxaloacetate phosphorylation is the favored reaction in vivo. The enzyme possesses weak pyruvate kinase-like activity (k_cat=2.7 s^?1). When overexpressed in HEK293T cells it enhances strongly glucose and lipid production showing that it can play, as the cytosolic isoenzyme, an active role in glyceroneogenesis and gluconeogenesis.