Comparative immunological characterization of various photosynthetic cytochromes c from pro- and eukaryotic algae

Photosynthetic c-type cytochromes isolated from various pro- and eukaryotic algae have been compared by an immunochemical method. Thereby the extent of cross-reactivity of several cytochromes with antisera to cytochrome c from Spirulina platensis, Bumilleriopsis filiformis, and Scenedesmus acutus was quantitatively determined by antigen-binding tests. When immunological relationship is taken as a measure of structural relationship, the following conclusions can be drawn: (1) c-type cytochromes from Anabaena variabilis, Nostoc muscorum, Calothrix membranacea, and Spirulina platensis show large differences in cross-reactivity. (2) The acidic Spirulina cytochrome c is fairly closely related to the two eukaryotic cytochromes assayed here.Abbreviations SAUG Sammlung von Algenkulturen am Pflanzenphysiologischen Institut der Universität Göttingen, FRG - PCC Pasteur Culture Collection     

Determination of human thrombin-antithrombin III complex by enzyme immunoassay

We have developed a specific and sensitive ELISA for the measurement of the TAT in human plasma. The assay follows the sandwich principle and uses two different antibodies directed against human thrombin and human antithrombin III, respectively. The anti-thrombin antibody population used for coating was purified by immunoadsorption on immobilized prothrombin and thrombin, respectively. Antithrombin III antibodies were conjugated with peroxidase. Plasma samples containing TAT were incubated in polystyrene tubes coated with anti-thrombin antibodies; after washing, peroxidase-conjugated antithrombin III antibodies were added and bound enzyme activity was subsequently measured using o-phenylenediamine. The assay was calibrated with definite concentrations (2.0 to 60 micrograms/l) of preformed purified TAT added to TAT-poor plasma. Plots of absorbance at 492 nm against TAT concentrations revealed a linear correlation (r = 0.98). A reference range from 0.85 to 3.0 micrograms/l was calculated from TAT concentration in plasma samples from 88 healthy donors (mean value +/- SD: 1.45 +/- 0.4 micrograms/l). In patients with deep vein thrombosis confirmed by phlebography (n = 15), TAT was found up to 7-13 micrograms/l. Patients with septicemia associated with a consumption coagulopathy (n = 10) showed markedly increased TAT values (greater than or equal to 10 micrograms/l). From these data it can be concluded that measurement of TAT might be a parameter for detection of a latent clotting pathway activation.     

Evaluation of an on-farm blood progesterone test for predicting the day of parturition in cattle

An on-farm blood progesterone enzymeimmunoassay (EIA) was evaluated as a diagnostic test to predict the time of calving within a 24-hour period in near-term dairy cows. Blood samples were taken daily from 45 cows beginning 5 days prior to their expected due dates until calving, and plasma was stored at -20 degrees C until all cows had calved. The EIA test was performed on frozen-thawed plasma samples, and progesterone concentrations were determined to be low (positive test for calving within 24 hours) or high (negative test for calving within 24 hours). Sensitivity, specificity and predictive value of the EIA to accurately determine parturition within 24 hours were 86.7, 90.8 and 75.0%, respectively. The EIA correctly predicted the day of parturition in 168 of 187 (89.8%) plasma samples. Ten additional cows were similarly monitored except the EIA was performed on whole blood immediately after collection, and the sensitivity, specificity and predictive value of the test were 80.0, 97.6 and 88.9%, respectively. The day of parturition was correctly predicted in 49 of 52 (94.2%) whole blood samples. More than 95% of the cows calved within 24 hours when their plasma progesterone reached < 1.3 ng/ml. When results of the EIA were compared with those of a radioimmunoassay (RIA), the EIA findings were used to correctly classify 190 of 232 (81.9%) plasma samples as having low (< 2.0 ng/ml) or high (>/= 2.0 ng/ml) concentrations of progesterone. The EIA test was found to be a quick, practical means of estimating progesterone concentrations in bovine plasma or whole blood and was a useful test for predicting the day of parturition in cows.     

UBE1L2, a novel E1 enzyme specific for ubiquitin

UBE1 is known as the human ubiquitin-activating enzyme (E1), which activates ubiquitin in an ATP-dependent manner. Here, we identified a novel human ubiquitin-activating enzyme referred to as UBE1L2, which also shows specificity for ubiquitin. The UBE1L2 sequence displays a 40% identity to UBE1 and also contains an ATP-binding domain and an active site cysteine conserved among E1 family proteins. UBE1L2 forms a covalent link with ubiquitin in vitro and in vivo, which is sensitive to reducing conditions. In an in vitro polyubiquitylation assay, recombinant UBE1L2 could activate ubiquitin and transfer it onto the ubiquitin-conjugating enzyme UbcH5b. Ubiquitin activated by UBE1L2 could be used for ubiquitylation of p53 by MDM2 and supported the autoubiquitylation of the E3 ubiquitin ligases HectH9 and E6-AP. The UBE1L2 mRNA is most abundantly expressed in the testis, suggesting an organ-specific regulation of ubiquitin activation.     

Effect of breed and sperm concentration on the changes in structural, functional and motility parameters of ram-lamb spermatozoa during storage at 4 degrees C

The objectives of this study were (1) to determine the changes in structural, functional and motility parameters of ram-lamb semen stored at two different concentrations at 4 degrees C for 8 days in egg-yolk based extender and (2) to determine the effect of breed of ram-lambs on the changes in structural, functional and motility parameters of ram-lamb semen from different breeds stored at two different concentrations at 4 degrees C for 8 days in egg-yolk based extender. Two different concentrations suitable for laparoscopic and cervical insemination were employed in this experiment. A total of 14 ram-lambs (Polled Dorset-5, Suffolk-5, Katahdin-4) with satisfactory breeding potential were selected. Semen samples were collected by electro-ejaculation. Semen samples were extended to 50 and 200 million sperm per ml with a commercial egg yolk based extender (Triladyl, Minitube of America, Verona, WI, USA) at room temperature and were stored at 4 degrees C. The sperm DNA fragmentation index (DFI), percentages of high mitochondrial membrane potential (hMMP) and plasma membrane integrity (PMI) were assessed using flow cytometry as part of structural and functional parameters on Days 0, 1, 4, 6, and 8. A computer assisted sperm analyser (HTM-IVOS, Version 10.8, Hamilton Thorne Research, Beverly, MA, USA) was used to assess the sperm motility parameters on Days 0, 1, 4, 6, and 8. PROC MIXED procedure was used to determine the effect of days of storage, concentration and breed. The concentration and days of storage significantly affected the sperm structural, functional and motility parameters (P<0.0001). Significant concentration x days of storage interaction was found for all structural and functional parameters. There was a significant concentration x days of storage interaction for average path velocity, curvilinear velocity, straightness and linearity. Overall changes in the sperm structural, functional and sperm motility parameters over the storage period were less dramatic in the 200 x 10(6) ml(-1) concentration when compared to 50 x 10(6) ml(-1) concentration. The hMMP and total progressive motility were influenced by breed. In conclusion, the quality of structural, functional and motility parameters declined as days of storage were increased and the magnitude of changes in the parameters was less dramatic at the higher concentration.     

Estrogen receptor alpha interacts with 17β-hydroxysteroid dehydrogenase type 10 in mitochondria

Estrogen receptor alpha (ERα) is present in the nucleus, the cytosol and in mitochondria. The rat ERα ligand binding domain was employed as bait in a bacterial two-hybrid screening of a human heart cDNA library to detect novel protein-protein interaction partners of ERα in the heart. 17β-Hydroxysteroid dehydrogenase type 10 (17β-HSD10), which converts potent (17β-estradiol) to less potent estrogens (estrone), co-localized with 17β-HSD10 in the mitochondria of rat cardiac myocytes. GST pull-down experiments confirmed the interaction of ERα and 17β-HSD10. These findings suggest that the ERα estrogen receptor might be involved in regulating intracellular estrogen levels by modulating 17β-HSD10 activity.     

Maßgeschneiderte Mikroorganismen

Tailor‐made microorganisms Microbial diversity provides unlimited resources for the development of novel industrial processes and products. Since the beginning of the 20th century microorganisms have been successfully applied for the large scale production of bio‐based products. In recent years, modern methods of strain development and Synthetic Biology have enabled biotech engineers to design even more sophisticated and tailor‐made microorganisms. These microbes serve industrial processes for the production of bulk chemicals, enzymes, polymers, biofuels as well as plant‐derived ingredients such as Artemisinin in an ecologically and economically sustainable and attractive fashion. In the future, production of advanced biofuels, microbial fuel cells, CO₂ as feedstock and microbial cellulose are research topics as well as challenges of global importance. Continuous efforts in microbiology and biotechnology research will be pivotal for white biotechnology to gain more momentum in transforming the chemical industry towards a knowledge based bio‐economy.