Dynamic modulation of prohormone convertase 2 (PC2)-mediated precursor processing by 7B2 protein: preferential effect on glucagon synthesis

The small neuroendocrine protein 7B2 is required for the production of active prohormone convertase 2 (PC2), an enzyme involved in the synthesis of peptide hormones, such as glucagon and proopiomelanocortin-derived α-melanocyte-stimulating hormone. However, whether 7B2 can dynamically modulate peptide production through regulation of PC2 activity remains unclear. Infection of the pancreatic alpha cell line α-TC6 with 7B2-encoding adenovirus efficiently increased production of glucagon, whereas siRNA-mediated knockdown of 7B2 significantly decreased stored glucagon. Furthermore, rescue of 7B2 expression in primary pituitary cultures prepared from 7B2 null mice restored melanocyte-stimulating hormone production, substantiating the role of 7B2 as a regulatory factor in peptide biosynthesis. In anterior pituitary and pancreatic beta cell lines, however, overexpression of 7B2 affected neither production nor secretion of peptides despite increased release of active PC2. In direct contrast, 7B2 overexpression decreased the secretion and increased the activity of PC2 within α-TC6 cells; the increased intracellular concentration of active PC2 within these cells may therefore account for the enhanced production of glucagon. In line with these findings, we found elevated circulating glucagon levels in 7B2-overexpressing cast/cast mice in vivo. Surprisingly, when proopiomelanocortin and proglucagon were co-expressed in either pituitary or pancreatic alpha cell lines, proglucagon processing was preferentially decreased when 7B2 was knocked down. Taken together, these results suggest that proglucagon cleavage has a greater dependence on PC2 activity than other precursors and moreover that 7B2-dependent routing of PC2 to secretory granules is cell line-specific. The manipulation of 7B2 could therefore represent an effective way to selectively regulate synthesis of certain PC2-dependent peptides.     

Green fluorescent protein (GFP) as a marker during pollen development

The transient expression of three mutant forms of green fluorescent protein (GFP) genes, GFP4, GFP5ER, and GFP4S65C, under several constitutive and pollenspecific promoters throughout pollen development in Nicotianatabacum, thaliana and Antirrhinummajus is described. Immature pollen of tobacco, Arabidopsis and snapdragon, isolated at different developmental stages, were bombarded with plasmids containing the GFP and cultured in vitro for several days until maturity. The expression of GFP was monitored every day during in vitro maturation, germination and pollination, as well as after in situ pollination. The expression pattern of each construct was compared in parallel experiments to that of ßglucuronidase (GUS) constructs expressed by the same promoters. The results show that the expression level of all three GFP mutant forms was dependent on the strength of the promoter used. The strongest promoter was the DC3 promoter, and no notable differences in the intensity and brightness of all three versions of GFP were observed. GFPexpressing pollen from tobacco and snapdragon developed in vitro for several days until maturity and germinated in vitro as well as on the surface of stigmata, strongly suggesting that all three GFPs are not toxic for the development of functional pollen. Furthermore, stably transformed tobacco plants expressing GFP under the control of the strong pollenexpressed DC3 and LAT52 promoters were not impaired in reproductive function, confirming that GFP can be used as a nondestructive marker for plant reproductive biology and development.     

The plant nuclear envelope protein MAF1 has an additional location at the Golgi and binds to a novel Golgi-associated coiled-coil protein

Tomato MAF1 (LeMAF1) is a plant-specific, nuclear envelope (NE)-associated protein. It is the founding member of a group of WPP domain-containing, NE-associated proteins. This group includes the Arabidopsis WPP family, which is involved in cell division, as well as plant RanGAPs. In addition to its NE localization, LeMAF1 accumulates in speckles in the cytoplasm. Here, we show that the LeMAF1-containing speckles are components of the Golgi apparatus. A novel tomato coiled-coil protein was identified that specifically binds to LeMAF1. Tomato WPP domain-associated protein (LeWAP) interacts in yeast and in vitro through its coiled-coil domain with several WPP-domain containing proteins, including AtRanGAP1 and the WPP family (LeMAF, WPP1 and WPP2). Like LeMAF1, LeWAP is localized at the Golgi. Moreover, we present data showing that Arabidopsis WAP is necessary for the existence of a multi-protein complex containing WPP2. Electronic Supplementary Material Supplementary material is available for this article at .     

Cytochrome P450 CYP89A9 Is Involved in the Formation of Major Chlorophyll Catabolites during Leaf Senescence in Arabidopsis

Nonfluorescent chlorophyll catabolites (NCCs) were described as products of chlorophyll breakdown in Arabidopsis thaliana. NCCs are formyloxobilin-type catabolites derived from chlorophyll by oxygenolytic opening of the chlorin macrocycle. These linear tetrapyrroles are generated from their fluorescent chlorophyll catabolite (FCC) precursors by a nonenzymatic isomerization inside the vacuole of senescing cells. Here, we identified a group of distinct dioxobilin-type chlorophyll catabolites (DCCs) as the major breakdown products in wild-type Arabidopsis, representing more than 90% of the chlorophyll of green leaves. The molecular constitution of the most abundant nonfluorescent DCC (NDCC), At-NDCC-1, was determined. We further identified cytochrome P450 monooxygenase CYP89A9 as being responsible for NDCC accumulation in wild-type Arabidopsis; cyp89a9 mutants that are deficient in CYP89A9 function were devoid of NDCCs but accumulated proportionally higher amounts of NCCs. CYP89A9 localized outside the chloroplasts, implying that FCCs occurring in the cytosol might be its natural substrate. Using recombinant CYP89A9, we confirm FCC specificity and show that fluorescent DCCs are the products of the CYP89A9 reaction. Fluorescent DCCs, formed by this enzyme, isomerize to the respective NDCCs in weakly acidic medium, as found in vacuoles. We conclude that CYP89A9 is involved in the formation of dioxobilin-type catabolites of chlorophyll in Arabidopsis.     

Multi-Country Evaluation of the Sensitivity and Specificity of Two Commercially-Available NS1 ELISA Assays for Dengue Diagnosis

Background

Early diagnosis of dengue can assist patient triage and management and prevent unnecessary treatments and interventions. Commercially available assays that detect the dengue virus protein NS1 in the plasma/serum of patients offers the possibility of early and rapid diagnosis.

Methodology/Principal Findings

The sensitivity and specificity of the Pan-E Dengue Early ELISA and the Platelia™ Dengue NS1 Ag assays were compared against a reference diagnosis in 1385 patients in 6 countries in Asia and the Americas. Platelia was more sensitive (66%) than Pan-E (52%) in confirmed dengue cases. Sensitivity varied by geographic region, with both assays generally being more sensitive in patients from SE Asia than the Americas. Both kits were more sensitive for specimens collected within the first few days of illness onset relative to later time points. Pan-E and Platelia were both 100% specific in febrile patients without evidence of acute dengue. In patients with other confirmed diagnoses and healthy blood donors, Platelia was more specific (100%) than Pan-E (90%). For Platelia, when either the NS1 test or the IgM test on the acute sample was positive, the sensitivity versus the reference result was 82% in samples collected in the first four days of fever. NS1 sensitivity was not associated to disease severity (DF or DHF) in the Platelia test, whereas a trend for higher sensitivity in DHF cases was seen in the Pan-E test (however combined with lower overall sensitivity).

Conclusions/Significance

Collectively, this multi-country study suggests that the best performing NS1 assay (Platelia) had moderate sensitivity (median 64%, range 34–76%) and high specificity (100%) for the diagnosis of dengue. The poor sensitivity of the evaluated assays in some geographical regions suggests further assessments are needed. The combination of NS1 and IgM detection in samples collected in the first few days of fever increased the overall dengue diagnostic sensitivity.     

Novel loci for adiponectin levels and their influence on type 2 diabetes and metabolic traits: a multi-ethnic meta-analysis of 45,891 individuals

Circulating levels of adiponectin, a hormone produced predominantly by adipocytes, are highly heritable and are inversely associated with type 2 diabetes mellitus (T2D) and other metabolic traits. We conducted a meta-analysis of genome-wide association studies in 39,883 individuals of European ancestry to identify genes associated with metabolic disease. We identified 8 novel loci associated with adiponectin levels and confirmed 2 previously reported loci (P = 4.5×10⁻⁸–1.2×10⁻⁴³). Using a novel method to combine data across ethnicities (N = 4,232 African Americans, N = 1,776 Asians, and N = 29,347 Europeans), we identified two additional novel loci. Expression analyses of 436 human adipocyte samples revealed that mRNA levels of 18 genes at candidate regions were associated with adiponectin concentrations after accounting for multiple testing (p<3×10⁻⁴). We next developed a multi-SNP genotypic risk score to test the association of adiponectin decreasing risk alleles on metabolic traits and diseases using consortia-level meta-analytic data. This risk score was associated with increased risk of T2D (p = 4.3×10⁻³, n = 22,044), increased triglycerides (p = 2.6×10⁻¹⁴, n = 93,440), increased waist-to-hip ratio (p = 1.8×10⁻⁵, n = 77,167), increased glucose two hours post oral glucose tolerance testing (p = 4.4×10⁻³, n = 15,234), increased fasting insulin (p = 0.015, n = 48,238), but with lower in HDL-cholesterol concentrations (p = 4.5×10⁻¹³, n = 96,748) and decreased BMI (p = 1.4×10⁻⁴, n = 121,335). These findings identify novel genetic determinants of adiponectin levels, which, taken together, influence risk of T2D and markers of insulin resistance.     

Urodynamic parameters and plasma LH/FSH in spayed Beagle bitches before and 8 weeks after GnRH depot analogue treatment

The pathophysiology of urinary incontinence due to spaying remains unknown. Incontinent bitches can be treated successfully with depot preparations of GnRH-analogues and there are differences in plasma gonadotropin levels between continent and incontinent spayed bitches. It is therefore assumed that the supraordinated hormones, GnRH, FSH, and/or LH, have an effect on the urodynamic parameters. In this study, the potential influence of these hormones on the lower urinary tract was investigated by measuring urethral pressure profiles and cystometry. Simultaneously, plasma concentrations in 10 spayed Beagle bitches were determined 5 weeks prior to and 8 weeks after treatment with the GnRH analogue leuprolide. Within 1 week of GnRH analogue administration, plasma FSH and LH levels decreased from 72.5 and 7.7 to 7.75 and 0.72ng/mL, respectively. These plasma gonadotropin levels correspond with those of intact bitches during anoestrus. Urethral pressure profiles indicated that the treatment had no significant effect on maximum urethral closure pressure, functional and total length of the urethra, or area of the closure pressure curve. The data obtained by cystometry regarding mean bladder threshold volume showed a significant increase from 109 to 172mL. The improvement in bladder function after the application of GnRH-application is presumably a direct effect of the GnRH as a relationship between the plasma gonadotropin levels and the urodynamic parameters could not demonstrated.     

High-glucose-induced structural changes in the heparan sulfate proteoglycan, perlecan, of cultured human aortic endothelial cells

Hyperglycemia is an independent risk factor for diabetes-associated cardiovascular disease. One potential mechanism involves hyperglycemia-induced changes in arterial wall extracellular matrix components leading to increased atherosclerosis susceptibility. A decrease in heparan sulfate (HS) glycosaminoglycans (GAG) has been reported in diabetic arteries. The present studies examined the effects of high glucose on in vitro production of proteoglycans (PG) by aortic endothelial cells. Exposure of cells to high glucose (30 vs. 5 mM glucose) resulted in decreased [(35)S] sodium sulfate incorporation specifically into secreted HSPG. Differences were not due to hyperosmolar effects and no changes were observed in CS/DSPG. Enzymatic procedures, immunoprecipitation and Western analyses demonstrated that high glucose induced changes specifically in the HSPG, perlecan. In double-label experiments, lower sulfate incorporation in high-glucose-treated cells was accompanied by lower [(3)H] glucosamine incorporation into GAG but not lower [(3)H] serine incorporation into PG core proteins. Size exclusion chromatography demonstrated that GAG size was unchanged and GAG sulfation was not reduced. These results indicate that the level of regulation of perlecan by high glucose is posttranslational, involving a modification in molecular structure, possibly a decrease in the number of HS GAG chains on the core protein.     

Temporal and spatial habitat preferences and biotic interactions between mosquito larvae and antagonistic crustaceans in the field

Investigations on natural antagonists of mosquito larvae found that micro‐crustaceans (e.g., Cladocera) control mosquito populations under experimental conditions. However, their relevance for mosquito control under field situations remains widely unclear because important information about habitat preferences and time of occurrence of crustaceans and mosquito larvae are still missing. In order to fill this knowledge gap, a field study was undertaken in different wetland areas of Saxony, Germany, in different habitats (i.e., grassland, forest, and reed‐covered wetlands). We found negative interactions between larvae of Ae. vexans and predatory Cyclopoida (Crustacean: Copepoda), which both were dominant during the first two weeks of hydroperiod, at ponds located at grassland habitats. Larvae of Cx. pipiens were spatially associated with competing Cladocera, but they colonized ponds more rapidly. Populations of Cladocera established from the third week of hydroperiod and prevented Cx. pipiens colonization thereafter. Ostracoda were highly abundant during the whole hydroperiod, but their presence was restricted to habitats of reed‐covered wetland at one geographical area. Mosquito larvae hardly occurred at those ponds. In general, we found that ponds at the reed‐covered wetlands provided better conditions for the initial development of crustaceans and hence, mosquito larval colonization was strongly inhibited. Grassland habitat, in contrast, favored early development of mosquito larvae. This study showed that micro‐crustaceans are relevant for mosquito management but their impact on mosquito larvae varies between species and depends on environmental conditions.