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.     

Single nucleotide polymorphisms in the apolipoprotein B and low density lipoprotein receptor genes affect response to antihypertensive treatment

Background

Dyslipidemia has been associated with hypertension. The present study explored if polymorphisms in genes encoding proteins in lipid metabolism could be used as predictors for the individual response to antihypertensive treatment.

Methods

Ten single nucleotide polymorphisms (SNP) in genes related to lipid metabolism were analysed by a microarray based minisequencing system in DNA samples from ninety-seven hypertensive subjects randomised to treatment with either 150 mg of the angiotensin II type 1 receptor blocker irbesartan or 50 mg of the β₁-adrenergic receptor blocker atenolol for twelve weeks.

Results

The reduction in blood pressure was similar in both treatment groups. The SNP C711T in the apolipoprotein B gene was associated with the blood pressure response to irbesartan with an average reduction of 19 mmHg in the individuals carrying the C-allele, but not to atenolol. The C16730T polymorphism in the low density lipoprotein receptor gene predicted the change in systolic blood pressure in the atenolol group with an average reduction of 14 mmHg in the individuals carrying the C-allele.

Conclusions

Polymorphisms in genes encoding proteins in the lipid metabolism are associated with the response to antihypertensive treatment in a drug specific pattern. These results highlight the potential use of pharmacogenetics as a guide for individualised antihypertensive treatment, and also the role of lipids in blood pressure control.     

CD25<Superscript>bright</Superscript>CD4<Superscript>+</Superscript>regulatory T cells are enriched in inflamed joints of patients with chronic rheumatic disease

CD25⁺CD4⁺ regulatory T cells participate in the regulation of immune responses. We recently demonstrated the presence of CD25^brightCD4⁺ regulatory T cells with a capacity to control T cell proliferation in the joints of patients with rheumatoid arthritis. Here, we investigate a possible accumulation of these regulatory T cells in the inflamed joint of different rheumatic diseases including rheumatoid arthritis. The studies are also extended to analyze whether cytokine production can be suppressed by the regulatory T cells. Synovial fluid and peripheral blood samples were obtained during relapse from 36 patients with spondyloarthropathies, 21 adults with juvenile idiopathic arthritis and 135 patients with rheumatoid arthritis, and the frequency of CD25^brightCD4⁺ T cells was determined. Of 192 patients, 182 demonstrated a higher frequency of CD25^brightCD4⁺ T cells in synovial fluid than in peripheral blood. In comparison with healthy subjects, the patients had significantly fewer CD25^brightCD4⁺ T cells in peripheral blood. For functional studies, synovial fluid cells from eight patients were sorted by flow cytometry, and the suppressive capacity of the CD25^brightCD4⁺ T cells was determined in in vitro cocultures. The CD25^brightCD4⁺ T cells suppressed the production of both type 1 and 2 cytokines including interleukin-17, as well as proliferation, independently of diagnosis. Thus, irrespective of the inflammatory joint disease investigated, CD25^brightCD4⁺ T cells were reduced in peripheral blood and enriched in the joint, suggesting an active recruitment of regulatory T cells to the affected joint. Their capacity to suppress both proliferation and cytokine secretion might contribute to a dampening of local inflammatory processes.