Processing of pro-vitellogenin in insect fat body: a role for high-mannose oligosaccharide

Several discrete events were resolved in the processing of vitellogenin in Blattella germanica. Using tunicamycin to inhibit the synthesis of high-mannose oligosaccharide, a high molecular weight pro-vitellogenin peptide (apo-proVG, Mr 215,000) was identified in fat body. Dosages of tunicamycin which inhibited glycosylation of vitellogenin by 98% inhibited its synthesis by as much as 59%, yet led to an intracellular accumulation of apo-proVG. Reversibility and dose dependency of these effects on vitellogenin synthesis, glycosylation, proteolytic processing, and secretion were demonstrated. In control insects, glycosylation of apo-proVG yielded a Mr 240,000 pro-vitellogenin peptide (proVG). FITC-Concanavalin A bound to purified proVG but not to apo-proVG, thus confirming an absence of high-mannose oligosaccharide in the apo-protein. Following its glycosylation, proVG was processed rapidly in fat body to Mr 160,000 (VG160) and Mr 102,000 (VG102) peptides which subsequently were secreted into hemolymph. After uptake into developing oocytes, the VG160 peptide was processed further prior to chorionation, yielding subunits of Mr 95,000 and 50,000. Uniqueness of the peptides of mature vitellin (Mr 102,000, 95,000, and 50,000) was indicated by comparison of the CNBr fragments of each purified subunit. Staining of CNBr fragments with FITC-Concanavalin A also indicated that high-mannose oligosaccharides are attached at one or more sites within each vitellin subunit. Resolution of the substructure of this insect vitellin and identification of events involved in the processing and secretion of its fat body apo-protein provide a basis for further study of the assembly and transport of vitellogenin, its packaging in eggs, and utilization during embryogenesis.     

The closed structure of presequence protease PreP forms a unique 10,000 Angstroms3 chamber for proteolysis

Presequence protease PreP is a novel protease that degrades targeting peptides as well as other unstructured peptides in both mitochondria and chloroplasts. The first structure of PreP from Arabidopsis thaliana refined at 2.1 Angstroms resolution shows how the 995-residue polypeptide forms a unique proteolytic chamber of more than 10,000 Angstroms(3) in which the active site resides. Although there is no visible opening to the chamber, a peptide is bound to the active site. The closed conformation places previously unidentified residues from the C-terminal domain at the active site, separated by almost 800 residues in sequence to active site residues located in the N-terminal domain. Based on the structure, a novel mechanism for proteolysis is proposed involving hinge-bending motions that cause the protease to open and close in response to substrate binding. In support of this model, cysteine double mutants designed to keep the chamber covalently locked show no activity under oxidizing conditions. The manner in which substrates are processed inside the chamber is reminiscent of the proteasome; therefore, we refer to this protein as a peptidasome.     

Ecophysiological adjustment of two Sphagnum species in response to anthropogenic nitrogen deposition

Here, it was investigated whether Sphagnum species have adjusted their nitrogen (N) uptake in response to the anthropogenic N deposition that has drastically altered N-limited ecosystems, including peatlands, worldwide. A lawn species, Sphagnum balticum, and a hummock species, Sphagnum fuscum, were collected from three peatlands along a gradient of N deposition (2, 8 and 12 kg N ha(-1) yr(-1)). The mosses were subjected to solutions containing a mixture of four N forms. In each solution one of these N forms was labeled with (15)N (namely (15)NH(+)(4), (15)NO(-)(3) and the amino acids [(15)N]alanine (Ala) and [(15)N]glutamic acid (Glu)). It was found that for both species most of the N taken up was from , followed by Ala, Glu, and very small amounts from NO(-)(3). At the highest N deposition site N uptake was reduced, but this did not prevent N accumulation as free amino acids in the Sphagnum tissues. The reduced N uptake may have been genetically selected for under the relatively short period with elevated N exposure from anthropogenic sources, or may have been the result of plasticity in the Sphagnum physiological response. The negligible Sphagnum NO(-)(3) uptake may make any NO(-)(3) deposited readily available to co-occurring vascular plants.     

The Properties of Amyloid-β Fibrils Are Determined by their Path of Formation

Fibril formation of the amyloid-β peptide (Aβ) follows a nucleation-dependent polymerization process and is associated with Alzheimer's disease. Several different lengths of Aβ are observed in vivo, but Aβ1–40 and Aβ1–42 are the dominant forms. The fibril architectures of Aβ1–40 and Aβ1–42 differ and Aβ1–42 assemblies are generally considered more pathogenic. We show here that monomeric Aβ1–42 can be cross-templated and incorporated into the ends of Aβ1–40 fibrils, while incorporation of Aβ1–40 monomers into Aβ1–42 fibrils is very poor. We also show that via cross-templating incorporated Aβ monomers acquire the properties of the parental fibrils. The suppressed ability of Aβ1–40 to incorporate into the ends of Aβ1–42 fibrils and the capacity of Aβ1–42 monomers to adopt the properties of Aβ1–40 fibrils may thus represent two mechanisms reducing the total load of fibrils having the intrinsic, and possibly pathogenic, features of Aβ1–42 fibrils in vivo. We also show that the transfer of fibrillar properties is restricted to fibril-end templating and does not apply to cross-nucleation via the recently described path of surface-catalyzed secondary nucleation, which instead generates similar structures to those acquired via de novo primary nucleation in the absence of catalyzing seeds. Taken together these results uncover an intrinsic barrier that prevents Aβ1–40 from adopting the fibrillar properties of Aβ1–42 and exposes that the transfer of properties between amyloid-β fibrils are determined by their path of formation.     

Management framework for sustainable development indicators in the State of Selangor, Malaysia

The perception that better information on environment and development is the determinant of effective rational decision- and policy-making processes provide the impetus for global interest in the use of sustainable development indicators (SDIs). Accordingly, proposals for SDIs are framed either on organisational goals or on disciplinary and multidisciplinary theories—aiming to reduce uncertainties in choosing the best alternative among a set of options concerning sustainability. Despite the fact that many SDI initiatives are explicitly aimed at improving policy-making, it is not apparent that political settings and organisational realities are taken into consideration in designing the framework for sustainability assessment. Ignoring the realities of policy-making dynamics can result in poor institutionalisation of the SDI development process, and therefore reduced impact of indicators. Linkage of SDIs to policy processes must also take into account the complex role of information in policy processes. The importance of societal values, cultural contexts and behaviour of bureaucracies must be understood and used to assist the assessment of progress towards sustainability using SDIs. Essentially, objective knowledge must be tampered with pragmatism in governance. This paper highlights the case of SDI development in the state of Selangor where the notion of instrumental rationality is balanced with the ‘incrementalism’ of the policy process that provided the foundation for institutionalising the reporting and use of SDIs. The ideals and paradoxes of participatory decision-making, the principles of the rational model and decision-making processes within a state government are critically examined.     

pH is decreased in transplanted rat pancreatic islets

Recent studies of transplanted pancreatic islets have indicated incomplete revascularization. We investigated the pH, in relation to oxygen tension (Po(2)), in endogenous islets and islets syngeneically transplanted to the renal subcapsular site of nondiabetic and streptozotocin-diabetic recipients. Tissue pH and Po(2) were measured using microelectrodes. In the endogenous islets, tissue pH was similar to that in arterial blood. In the transplanted islets, tissue pH was 0.11-0.15 pH units lower. No differences in islet graft pH were seen between nondiabetic and diabetic animals, and none if the islet grafts were investigated 1 day or 1 mo posttransplantation. The Po(2) in the endogenous islets was approximately 35 mmHg. Transplanted islets had a markedly lower tissue Po(2) both 1 day and 1 mo after transplantation. A negative correlation between the tissue Po(2) and the hydrogen ion concentration was seen in the 1-mo-old islet transplants in diabetic animals. In conclusion, decreased Po(2) in transplanted islets is associated with a decreased tissue pH, suggesting a shift toward more anaerobic glucose metabolism after transplantation.     

Galactosaminogalactan from cell walls of Aspergillus niger.

A new heteropolysaccharide has been isolated by alkaline extraction of hyphal walls of Aspergillus niger NRRL 326 grown in surface culture. Its composition by weight, as determined by paper and gas chromatography and colorimetric analyses, is 70% galactose, 20% galactosamine, 6% glucose, and 1% acetyl. Two independent experiments have been used to ascertain copolymer structure: permeation chromatography in 6 M guanidinium hydrochloride, with controlled-pore glass columns of two fractionation ranges, and nitrous acid deaminative cleavage of galactosaminogalactan followed by reduction of fragments with [3H]borohydride and gel filtration chromatography. One of the tritiated fragments is tentatively identified as the disaccharide derivative galactopyranosyl 2,5-anhydrotalitol, on the basis of chromatographic properties and by kinetics of its acid hydrolysis. Smith degradation, methylation, deamination, and optical rotation studies indicate that the galactosaminogalactan consists of a linear array of hexopyranosyl units joined almost exclusively by alpha-(1 leads to 4) linkages. Hexosaminyl moieties are distributed randomly along the chains, which have an average degree of polymerization of about 100. The possible significance of this macromolecule in hyphal structure is considered.     

Quantification of dissimilatory (bi)sulphite reductase gene expression in Desulfobacterium autotrophicum using real-time RT-PCR

We developed a real-time RT-PCR method for the quantification of dissimilatory (bi)sulphite reductase (DSR) mRNA in Desulfobacterium autotrophicum cells. The amount of DSR mRNA was determined relative to the amount of 16S rRNA at different growth conditions during transition from exponential to stationary phase: sulphate respiration with lactate, thiosulphate respiration with lactate, sulphate respiration with H2 and pyruvate fermentation. The dsr gene was expressed constitutively, although DSR mRNA content per-cell varied under different growth conditions. The maximum DSR mRNA per-cell content was 2.0 to 4.1-fold higher during sulphate or thiosulphate respiration than during pyruvate fermentation. After transfer of a pyruvate-fermenting culture into sulphate-rich medium, upregulation of the DSR mRNA content was observed. Irrespective of the mode of metabolism the per-cell DSR mRNA content changed significantly during growth (up to 310-fold from the early to the late exponential phase during respiration with thiosulphate). The maximum DSR mRNA per-cell contents correlated with cell-specific sulphate reduction rates for all experiments. Environmental applications for the quantification of DSR mRNA are discussed.     

Hepatocyte Growth Factor Enables Enhanced Integrin–Cytoskeleton Linkage by Affecting Integrin Expression in Subconfluent Epithelial Cells

Hepatocyte growth factor (HGF) exerts mitogenic and motogenic effects in different cell types. In the epithelial cell line mHepR1 we found that HGF induced pronounced alterations in cell morphology and promoted cell adhesion and spreading. To analyze the mechanisms how HGF affects these integrin mediated functions we studied the physical linkage of integrins with the cytoskeleton. First we found that HGF increased the expression of different integrin subunits in subconfluent cells and influenced the distribution of integrins on the cell surface. To address the physical association of integrins with the cytoskeleton we analyzed Triton X-100-extracted cell fractions using flow cytometry. Here we show that cultivation of the cells with HGF for 24 h prior to integrin cross-linking significantly enhanced the cytoskeletal anchorage of integrins. To further find out whether HGF directly induces an integrin–cytoskeleton link without subsequent cross-linking we added HGF to suspended cells but failed to detect cytoskeletally immobilized integrins in the detergent-insoluble cell fraction which could be related to the absence of a calcium response induced by HGF. Overall, the results indicate that HGF promotes the physical linkage of integrins to the cytoskeleton which requires additional stimulation of integrins.