Differential use of two AP-3-mediated pathways by lysosomal membrane proteins

The adaptor protein complex AP-3 is involved in the sorting of lysosomal membrane proteins to late endosomes/lysosomes. It is unclear whether AP-3-containing vesicles form at the trans-Golgi network (TGN) or early endosomes. We have compared the trafficking routes of endolyn/CD164 and 'typical' lysosomal membrane glycoproteins (lgp120/lamp-1 and CD63/lamp-3) containing cytosolic YXXPhi-targeting motifs preceded by asparagine and glycine, respectively. Endolyn, which has a NYHTL-motif, is concentrated in lysosomes, but also occurs in endosomes and at the cell surface. We observed predominant interaction of the NYHTL-motif with the mu-subunits of AP-3 in the yeast two-hybrid system. Endolyn was mislocalized to the cell surface in AP-3-deficient pearl cells, confirming a major role of AP-3 in endolyn traffic. However, lysosomal delivery of endolyn (or a NYHTL-reporter), but not GYXXPhi-containing proteins, was practically abolished when AP-2-mediated endocytosis or traffic from early to late endosomes was inhibited in NRK and 3T3 cells. This indicates that endolyn is mostly transported along the indirect lysosomal pathway (via the cell surface), rather than directly from the TGN to late endosomes/lysosomes. Our results suggest that AP-3 mediates lysosomal sorting of some membrane proteins in early endosomes in addition to sorting of proteins with intrinsically strong AP-3-interacting lysosomal targeting motifs at the TGN.     

The PM2 virion has a novel organization with an internal membrane and pentameric receptor binding spikes

Biological membranes are notoriously resistant to structural analysis. Excellent candidates to tackle this problem in situ are membrane-containing viruses where the membrane is constrained by an icosahedral capsid. Cryo-EM and image reconstruction of bacteriophage PM2 revealed a membrane bilayer following the internal surface of the capsid. The viral genome closely interacts with the inner leaflet. The capsid, at a resolution of 8.4 A, reveals 200 trimeric capsomers with a pseudo T = 21 dextro organization. Pentameric receptor-binding spikes protrude from the surface. It is evident from the structure that the PM2 membrane has at least two important roles in the life cycle. First, it acts as a scaffold to nucleate capsid assembly. Second, after host recognition, it fuses with the host outer membrane to promote genome entry. The structure also sheds light on how the viral supercoiled circular double-stranded DNA genome might be packaged and released.     

Chlorophyll breakdown in tobacco: on the structure of two nonfluorescent chlorophyll catabolites

In extracts of senescent leaves of the tobacco plant Nicotiana rustica, two colorless compounds with UV/VIS characteristics of nonfluorescent chlorophyll catabolites (NCCs) were detected and tentatively identified as Nr-NCCs. These two polar NCCs were found in similar amounts in the fresh extracts, and their constitutions could be determined by spectroscopic analysis. The data showed both of the two Nr-NCCs to have the same tetrapyrrolic core structure, as reported previously for all other NCCs from senescent higher plants. In the less polar catabolite, named Nr-NCC-2, this core structure was conjugated with a glucopyranose unit, as similarly discovered earlier in Bn-NCC-2, an NCC from oilseed rape (Brassica napus). The more polar NCC from tobacco leaves, Nr-NCC-1, carried an additional malonyl substituent at the 6'-OH group of the glucopyranosyl moiety. Partial (enzyme-catalyzed) hydrolysis of Nr-NCC-1 gave Nr-NCC-2, while enzyme-catalyzed malonylation of Nr-NCC-2 gave Nr-NCC-1, establishing the identity of their basic tetrapyrrole structure. In earlier work (on the polar NCCs from oilseed rape), only separate glucopyranosyl and malonyl functionalities were detected. Nr-NCC-1, thus, represents a further variant of the structures of NCCs from senescent higher plants and exhibits an unprecedented peripheral refunctionalization in chlorophyll catabolites.     

The role of calcium in signal transduction processes in Sertoli cells

Sertoli cells play a pivotal role in regulation and maintenance of spermatogenesis. They are hormonally regulated predominantly by follicle-stimulating hormone (FSH) and testosterone (T). Although FSH and T have distinct mechanisms of action they act synergistically in promoting spermatogenesis. Stimulation of freshly isolated Sertoli cells with FSH evokes a prompt rise in cytosolic calcium which is quantitatively reproduced by cAMP. The cytosolic calcium response to FSH in Sertoli cells is predominantly attributable to serial signaling after the generation of endogenous cAMP. Calcium homeostasis of Sertoli cells may also be regulated by cAMP-independent metabolism. Vasoactive testicular paracrine hormones such as angiotensin II (AII) and vasopressin acting via inositol triphosphate generation induce cytosolic calcium rise predominantly derived from the thapsigargin-sensitive endoplasmic reticulum. Investigations involving androgens action on cytosolic calcium reveal a common mechanism of action between the peptide and steroid regulators of Sertoli cell function, indicating that cytosolic calcium ions may represent a unifying biochemical mechanism that could explain the synergism of FSH and T. Androgens rapidly and specifically increase cytosolic calcium, consistent with a plasma membrane site of action. This argues for the possible existence of a short term non-genomic signaling pathway in hormonal regulation of Sertoli cell function in addition to the classical longer term, slower genomic response.     

In vitro studies on the evaluation of mycotoxin detoxifying agents for their efficacy on deoxynivalenol and zearalenone

A simple in vitro system was developed to study the efficacy of commercially available mycotoxin detoxifying agents and adsorbing substances as feed additives to detoxify deoxynivalenol (DON) and zearalenone (ZON) in situ. The in vitro model simulates the conditions (pH, temperature and transit time) of the porcine gastrointestinal tract, as pigs react most sensitively to these mycotoxins. The commercially available products were not effective in detoxifying DON and ZON under the applied conditions, while activated carbon was able to bind both toxins and cholestyramine, and a modified aluminosilicate showed good adsorption abilities for ZON. Data obtained in dose dependency studies showed an estimated adsorption capacity of cholestyramine and the modified aluminosilicate of 11.7 and 5.7 g ZON/kg detoxifying agent. The in vitro system deployed in the present study was demonstrated to be a simple, helpful tool in screening substances for their ability to detoxify DON and ZON under the simulated conditions of the porcine gastrointestinal tract. Nonetheless in vivo experiments are indispensable to proof the efficacy.     

Effect of dehulling of rapeseed on feed value and nutrient digestibility of rape products in pigs

In the presented study the influence of dehulling rapeseed on the composition of rapeseed meal (RM) and rapeseed cake (RC) and on its feed value for piglets and growing-finishing pigs was investigated. Before withdrawal of oil, rapeseed (variety Express) was dehulled applying a procedure developed by SKET GmbH Magdeburg and the Section Food-Technology of the University Essen. The steps of the dehulling procedure were described. For RM the oil was removed by the prepress-solvent procedure till a crude fat content of 2.1% in DM. RC was produced by pressing only resulting approximately 13% crude fat in DM. The RM and RC from not dehulled (ND) and dehulled (D) rapeseed were examined analytically. Crude nutrients, sugar and fibre substances, amino acids, some minerals and trace elements, fatty acids, glucosinolates and sinapine, and phytate were determined. By dehulling the seed the crude fibre content was decreased in RM and RC by approximately 40%. The ADF content declined by 35 and 39%, and the NDF content by 28% and 40% in RM and RC, respectively. The decrease in ADL content amounted to 50% and 65% for RM and RC, respectively. On the other hand, the CP content of RM and RC was increased by 7% and 13%, respectively, by dehulling the seed while the amino acid content of rape protein increased only slightly. The contents of glucosinolates and sinapine were also increased by dehulling, while the contents of phytate and phytate P were decreased. In digestibility and balance experiments with piglets and intact hybrid breeds of growing-finishing pigs, the digestibility of organic matter and of crude nutrients and the contents of digestible energy and metabolizable energy were estimated. Furthermore, the precaecal digestibility of crude nutrients and amino acids was determined with fistulated mini-pigs. By dehulling the seeds the digestibility of organic matter from RM and RC was improved in piglets and adult pigs by approximately 10%, and the ME contents increased by 13-15%. The precaecal digestibility of the sum of amino acids was increased by approximately 3 and 6 units in RM and RC, respectively. The precaecal digestibility of lysine in RM and RC reached that of soybean oil meal from not dehulled beans.     

The complete genome sequence of Bacillus licheniformis DSM13, an organism with great industrial potential

The genome of Bacillus licheniformis DSM13 consists of a single chromosome that has a size of 4,222,748 base pairs. The average G+C ratio is 46.2%. 4,286 open reading frames, 72 tRNA genes, 7 rRNA operons and 20 transposase genes were identified. The genome shows a marked co-linearity with Bacillus subtilis but contains defined inserted regions that can be identified at the sequence as well as at the functional level. B. licheniformis DSM13 has a well-conserved secretory system, no polyketide biosynthesis, but is able to form the lipopeptide lichenysin. From the further analysis of the genome sequence, we identified conserved regulatory DNA motives, the occurrence of the glyoxylate bypass and the presence of anaerobic ribonucleotide reductase explaining that B. licheniformis is able to grow on acetate and 2,3-butanediol as well as anaerobically on glucose. Many new genes of potential interest for biotechnological applications were found in B. licheniformis; candidates include proteases, pectate lyases, lipases and various polysaccharide degrading enzymes.     

Real-time process/quality control for HPC processing

BACKGROUND: JACIE Standards (FACT Standards in the USA) have been implemented in Europe since 1999. An on-site accreditation inspection took place at our center in January 2004. The purpose of this work was to develop a real-time process/quality control system meeting the JACIE Standards for HPC release. METHODS: Data from 194 HPC processing procedures for autologous transplantation performed over a 5-year period were analyzed. The results of different processing methods applied at our facility were compared: (1) cryopreservation without washing cells (n=50), (2) washing cells (n=87), (3) cell-density separation (n=12) and (4) positive CD34 selection (n=45). RESULTS: Four critical control points were set for the validation of HPC processing: (a) number of lost CD34(+) cells during processing, (b) contamination, (c) viability of the cells after thawing and (d) ability to reconstitute hematopoiesis after transplantation. On the basis of statistical analysis, ranges of acceptable values were defined for each critical control point and for each processing method. Those acceptable values were used for cell release and real-time quality control. DISCUSSION: This study describes a model for the validation of HPC processing and for a real-time process/quality control system for HPC release. Optimization of processing techniques, standardization of methods and comparison between facilities will open the way towards external quality controls and quality improvement.     

Intraoperative isolation and processing of BM-derived stem cells

To improve tissue regeneration of ischemic myocardium, autologous bone marrow-derived stem cells have been injected intramyocardially in five patients undergoing coronary artery bypass grafting and transmyocardial laser revascularization. An innovative method for the intraoperative isolation of CD133(+)-stem cells in less than 3 hours has been established. After induction of general anesthesia, approx. 60-240 ml of bone marrow were harvested from the posterior iliac crest and processed in the operating room under GMP conditions using the automated cell selection device Clini-MACS. Following standard CABG surgery, LASER channels were shot in predefined areas within the hibernating myocardium. Subsequently, autologous CD133(+)-stem cells (1.9-9.7 x 10(6) cells; purity up to 97%) were injected in a predefined pattern around the laser channels. Through the intraoperative isolation of CD133(+)-cells, this effective treatment of ischemic myocardium can be applied to patients scheduled both for elective and for emergency revascularisation procedures.     

Calystegines in Calystegia sepium do not inhibit fungal growth and invertase activity but interact with plant invertase

Abstract: Calystegines are alkaloidal glycosidase inhibitors. They accumulate predominantly in young and meristemic parts of Calystegia sepium (Convolvulaceae). C. sepium, bindweed, infests meadows and cereal fields and is difficult to control chemically. Fungal pathogens against C. sepium are established as mycoherbicides. Stagonospora convolvuli LA39 attacks C. sepium and does not affect crop plants, but young plants of C. sepium are less susceptible to the fungus. The interaction of Stagonospora convolvuli with calystegines was investigated. Further, endophytic fungi of several classes were isolated from wild-grown Calystegia sepium leaves, and selected strains were tested for interaction with calystegines. Fungal growth on agar containing calystegines was not affected considerably. Plants in climate chambers were infected with an endophyte, Phomopsis, and with the fungal pathogen, Stagonospora convolvuli. Calystegine levels were measured in infected and non-infected plant tissues. Accumulation depended on developmental stage of the plant tissue and was not influenced by infection. Acid invertase was measured from fungal mycelia and from infected and non-infected plant tissues. Fungal acid invertase activity was not inhibited by 10 mM calystegine B₂, while invertase from C. sepium leaves was inhibited. It is concluded that calystegines do not inhibit fungal development and sucrose consumption under the conditions of the present investigation, but may act by redirection of plant carbohydrate metabolism.