The Role of Overweight and Obesity in Calcium Oxalate Stone Formation

Objective: The aim of the study was to assess the influence of overweight and obesity on the risk of calcium oxalate stone formation. Research Methods and Procedures: BMI, 24‐hour urine, and serum parameters were evaluated in idiopathic calcium oxalate stone formers (363 men and 164 women) without medical or dietetic pretreatment. Results: Overweight and obesity were present in 59.2% of the men and in 43.9% of the women in the study population. Multiple linear regression analysis revealed a significant positive relationship between BMI and urinary uric acid, sodium, ammonium, and phosphate excretion and an inverse correlation between BMI and urinary pH in both men and women, whereas BMI was associated with urinary oxalate excretion only among women and with urinary calcium excretion only among men. Serum uric acid and creatinine concentrations were correlated with BMI in both genders. Because no association was established between BMI and urinary volume, magnesium, and citrate excretion, inhibitors of calcium oxalate stone formation, the risk of stone formation increased significantly with increasing BMI among both men and women with urolithiasis (p = 0.015). The risk of calcium oxalate stone formation, median number of stone episodes, and frequency of diet‐related diseases were highest in overweight and obese men. Discussion: Overweight and obesity are strongly associated with an elevated risk of stone formation in both genders due to an increased urinary excretion of promoters but not inhibitors of calcium oxalate stone formation. Overweight and obese men are more prone to stone formation than overweight women.     

Dissecting the localization and function of Atg18, Atg21 and Ygr223c

Atg18p and Atg21p are two highly homologous yeast autophagy proteins. Atg18p functions in both autophagy and the selective Cvt-pathway, while the function of Atg21p is restricted to the Cvt-pathway. The yeast genome encodes with Ygr223cp (Hsv2p), a third member of this protein family. So far no function has been assigned to Ygr223cp. By colocalization with the endosomal marker Snf7-RFP and an RFP-tagged FYVE domain, we here identify the localization of a pool of Atg18p, Atg21p and Ygr223cp at endosomes. Endosomal recruitment of all three proteins depends on PtdIns3P generated by the Vps34-complex II containing Vps38p, but not on the function of the Vps34-complex I. Since only the Vps34-complex I is essential for autophagy, we expect that at endosomes Atg18p, Atg21p and Ygr223cp have a function distinct from autophagy. Some Vps Class D mutants involved in Golgi-to-endosome transport are required for the endosomal recruitment of GFP-Atg18p, -Atg21p and -Ygr223cp. These include the Qa-SNARE Pep12p, its SM protein Vps45p, the Rab GTPase Vps21p and the Rab effector Vac1p. Deletion of ATG18, ATG21 and YGR223c, alone or simultaneously has no obvious function on the MVB-pathway and CPY-sorting. However, overexpression of ATG21 leads to CPY secretion. We further show, to our knowledge for the first time, that Ygr223cp affects an autophagic process, namely micronucleophagy.     

LeMAN4 endo-β-mannanase from ripe tomato fruit can act as a mannan transglycosylase or hydrolase

Mannan transglycosylases are cell wall enzymes able to transfer part of the mannan polysaccharide backbone to mannan-derived oligosaccharides (Schröder et al. in Planta 219:590–600, 2004). Mannan transglycosylase activity was purified to near homogeneity from ripe tomato fruit. N-terminal sequencing showed that the dominant band seen on SDS-PAGE was identical to LeMAN4a, a hydrolytic endo-β-mannanase found in ripe tomato fruit (Bewley et al. in J Exp Bot 51:529–538, 2000). Recombinant LeMAN4a protein expressed in Escherichia coli exhibited both mannan hydrolase and mannan transglycosylase activity. Western analysis of ripe tomato fruit tissue using an antibody raised against tomato seed endo-β-mannanase revealed four isoforms present after 2D-gel electrophoresis in the pH range 6–11. On separation by preparative liquid isoelectric focussing, these native isoforms exhibited different preferences for transglycosylation and hydrolysis. These results demonstrate that endo-β-mannanase has two activities: it can either hydrolyse mannan polysaccharides, or in the presence of mannan-derived oligosaccharides, carry out a transglycosylation reaction. We therefore propose that endo-β-mannanase should be renamed mannan transglycosylase/hydrolase, in accordance with the nomenclature established for xyloglucan endotransglucosylase/hydrolase. The role of endo-acting mannanases in modifying the structure of plant cell walls during cell expansion, seed germination and fruit ripening may need to be reinterpreted in light of their potential action as transglycosylating or hydrolysing enzymes.     

Magnetic orientation in birds: non-compass responses under monochromatic light of increased intensity

Migratory Australian silvereyes (Zosterops lateralis) were tested under monochromatic light at wavelengths of 424 nm blue and 565 nm green. At a low light level of 7 x 10(15) quanta m(-2) s(-1) in the local geomagnetic field, the birds preferred their seasonally appropriate southern migratory direction under both wavelengths. Their reversal of headings when the vertical component of the magnetic field was inverted indicated normal use of the avian inclination compass. A higher light intensity of 43 x 10(15) quanta m(-2) s(-1), however, caused a fundamental change in behaviour: under bright blue, the silvereyes showed an axial tendency along the east-west axis; under bright green, they showed a unimodal preference of a west-northwesterly direction that followed a shift in magnetic north, but was not reversed by inverting the vertical component of the magnetic field. Hence it is not based on the inclination compass. The change in behaviour at higher light intensities suggests a complex interaction between at least two receptors. The polar nature of the response under bright green cannot be explained by the current models of light-dependent magnetoreception and will lead to new considerations on these receptive processes.     

Evolution of trnF(GAA) pseudogenes in cruciferous plants

In several studies we used the 5′-trnL(UAA)–trnF(GAA) region of the chloroplast DNA for phylogeographic reconstructions, gene diversity calculations and phylogenetic analyses among the genera Arabidopsis and Boechera. Despite the fact that extensive gene duplications are rare within the chloroplast genome of higher plants, within several genera of the Brassicaceae the anticodon domain of the trnF(GAA) gene exhibit extensive gene duplications with 1–12 tandemly repeated copies in close 5′-proximity of the functional gene. A recent re-examination and additional analysis of trnL(UAA)–trnF(GAA) regions from numerous cruciferous taxa not only reveal extensive trnF gene duplications, but also favour the hypothesis that in cruciferous taxa at least four independent phylogenetic lineages are characterized by these pseudogenes. Among these lineages there is one major clade of taxa carrying pseudogenes indicating an ancient split in crucifer evolution. In two case studies, Boechera and Arabidopsis, intra- and inter-molecular recombinations have been shown to be the reason for the reciprocal exchange of several similar motifs. However, functional constraints might favour two to three or five to six copies as shown for Arabidopsis and Boechera. Herein, we compare the occurrence and distribution of pseudogene copy number in the framework of a comprehensive survey of cpDNA haplotype variation in Boechera, the former genus Cardaminopsis and Arabidopsis thaliana and comment on the value of such kind of mutations in phylogenetic and evolutionary reconstructions.     

Measuring piecemeal microautophagy of the nucleus in Saccharomyces cerevisiae

Piecemeal microautophagy of the nucleus (PMN) selectively removes and degrades small fragments of the Saccharomyces cerevisiae nucleus. Inter-organelle contact sites called nucleus-vacuole (NV) junctions determine the selectivity of PMN by establishing a platform for the biogenesis of PMN blebs and vesicles. PMN structures can be observed by fluorescence microscopy using GFP-tagged reporters; however, this approach is best supported with quantitative immunoblot assays of PMN-specific cargo degradation. Together, these assays should facilitate the further study of this fascinating but poorly understood autophagic process in different genetic backgrounds, physiological states, and environmental conditions.     

Expression of the Xylulose 5-Phosphate Phosphoketolase Gene, xpkA, from Lactobacillus pentosus MD363 Is Induced by Sugars That Are Fermented via the Phosphoketolase Pathway and Is Repressed by Glucose Mediated by CcpA and the Mannose Phosphoenolpyruvate Phosphotransferase System

Purification of xylulose 5-phosphate phosphoketolase (XpkA), the central enzyme of the phosphoketolase pathway (PKP) in lactic acid bacteria, and cloning and sequence analysis of the encoding gene, xpkA, from Lactobacillus pentosus MD363 are described. xpkA encodes a 788-amino-acid protein with a calculated mass of 88,705 Da. Expression of xpkA in Escherichia coli led to an increase in XpkA activity, while an xpkA knockout mutant of L. pentosus lost XpkA activity and was not able to grow on energy sources that are fermented via the PKP, indicating that xpkA encodes an enzyme with phosphoketolase activity. A database search revealed that there are high levels of similarity between XpkA and a phosphoketolase from Bifidobacterium lactis and between XpkA and a (putative) protein present in a number of evolutionarily distantly related organisms (up to 54% identical residues). Expression of xpkA in L. pentosus was induced by sugars that are fermented via the PKP and was repressed by glucose mediated by carbon catabolite protein A (CcpA) and by the mannose phosphoenolpyruvate phosphotransferase system. Most of the residues involved in correct binding of the cofactor thiamine pyrophosphate (TPP) that are conserved in transketolase, pyruvate decarboxylase, and pyruvate oxidase were also conserved at a similar position in XpkA, implying that there is a similar TPP-binding fold in XpkA.     

The reprolysin jararhagin,a snake venom metalloproteinase,functions as a fibrillar collagen agonist involved in fibroblast cell adhesion and signaling

The integrins alpha(2)beta(1) and alpha(1)beta(1) have been shown to modulate cellular activities of fibroblasts on contact with fibrillar collagen. Previously it has been shown that collagen binding to alpha(2)beta(1) regulates matrix metalloproteinase MMP-1 and membrane-type MT1-MMP expression. Jararhagin is a snake venom metalloproteinase of the Reprolysin family of zinc metalloproteinases, containing a metalloproteinase domain followed by disintegrin-like and cysteine-rich domains. Jararhagin blocks type I collagen-induced platelet aggregation by binding to the alpha(2)beta(1) integrin and inhibiting collagen-mediated intracellular signaling events. Here we present evidence that, in contrast to the observations in platelets, jararhagin binding to the integrin receptor alpha(2)beta(1) in fibroblasts produces collagen-like cell signaling events such as up-regulation of MMP-1 and MT1-MMP. Inactivation of the metalloproteinase domain had no effect on these properties of jararhagin. Thus, in fibroblasts the snake venom metalloproteinase jararhagin functions as a collagen-mimetic substrate that binds to and activates integrins. Given the homology between the metalloproteinase, disintegrin-like and cysteine-rich domains of jararhagin and those of the members of the ADAMs (a disintegrin-like and metalloproteinase) family of proteins, this work demonstrates the potential of the disintegrin-like/cysteine-rich domains in the ADAMs as cellular signaling agents to elicit responses relevant to the biological function of these proteins.     

PI3P binding by Atg21 organises Atg8 lipidation

Autophagosome biogenesis requires two ubiquitin‐like conjugation systems. One couples ubiquitin‐like Atg8 to phosphatidylethanolamine, and the other couples ubiquitin‐like Atg12 to Atg5. Atg12~Atg5 then forms a heterodimer with Atg16. Membrane recruitment of the Atg12~Atg5/Atg16 complex defines the Atg8 lipidation site. Lipidation requires a PI3P‐containing precursor. How PI3P is sensed and used to coordinate the conjugation systems remained unclear. Here, we show that Atg21, a WD40 β‐propeller, binds via PI3P to the preautophagosomal structure (PAS). Atg21 directly interacts with the coiled‐coil domain of Atg16 and with Atg8. This latter interaction requires the conserved F5K6‐motif in the N‐terminal helical domain of Atg8, but not its AIM‐binding site. Accordingly, the Atg8 AIM‐binding site remains free to mediate interaction with its E2 enzyme Atg3. Atg21 thus defines PI3P‐dependently the lipidation site by linking and organising the E3 ligase complex and Atg8 at the PAS.