Purification of a membrane-derived proteinase capable of activating a galactosyltransferase involved in volume regulation

UDPgalactose:sn-glycerol-3-phosphate α-D-galactosyltransferase (IFP-synthase, EC 2.4.1.96) shows low activity in extracts prepared from standard volume cells of Poterioochromonals malhamensis under certain conditions. This inactive enzyme has been partially purified by chromatography on DEAE-cellulose, Sephadex G-150 and α-lactalbumin-agarose. It can be activated by an auxiliary enzyme which can be eluted from membranes and which has been purified to homogeneity by chromatography on DEAE-Sephacel and immobilized hemoglobin and fetuin. The activating enzyme is inhibited by chymostatin, antipain and diisopropylfluorophosphate and does not require divalent ions. It consists of a single peptide chain of molecular weight 46 000, can split certain proteins and appears to be a serine proteinase operating around a pH of 6.0. The activating proteinase is irreversibly generated in the crude homogenates on addition of Ca²⁺ and also shows increased activity shortly after cell shrinkage. This might indicate that it represents one of the possibilities to render the galactosyltransferase active as a result of the physiological stimulus.     

In vitro antigen ELISA for quality control of tetanus vaccines

Consistency of production is recognised as an important aspect of vaccine manufacture and suitably validated in vitro assays are required for quality control testing of these products. For the manufacture and batch release of tetanus vaccines, antigen content and integrity, and degree of adsorption of antigen to the adjuvant are critical parameters that should be monitored for consistency. Here we describe the development and use of an Enzyme Linked Immunosorbent Assay (ELISA) to quantify tetanus antigen in combined vaccine products and to measure the degree of adsorption of antigen to adjuvant. Whilst the antigen assay cannot be assumed to predict potency for different products, it can be used as part of a panel of in vitro methods to provide a more informative product profile and to monitor trends in production. The antigen assay is particularly valuable for providing quantitative information on every final lot when modifications of in vivo potency tests, such as single dilution assays, are used.     

Sesame Utilization in China: New Archaeobotanical Evidence from Xinjiang

Sesame Utilization in China: New Archaeobotanical Evidence from Xinjiang. A cache of sesame (Sesamum indicum L.) seeds, discovered in the Thousand Buddha Grottoes at Boziklik, Turpan, China, dating to ca. 700 years before present (BP), is hard evidence of their use in China since that time. Morphological and anatomical features suggest a white sesame cultivar. The sizeable quantity unearthed implies that sesame was a valued commodity that could provision the monks and enrich the diet of ancient inhabitants as an oil source.     

THE CYST-THECA RELATIONSHIP IN CALCIODINELLUM OPEROSUM EMEND. (PERIDINIALES, DINOPHYCEAE) AND A NEW APPROACH FOR THE STUDY OF CALCAREOUS CYSTS

The paratabulate calcareous cyst of Calciodinellum operosum Deflandre was recorded in a sediment trap sample collected in the Bay of Naples (Tyrrhenian Sea, Italy). The germination of this resting stage produced a phototrophic vegetative cell that had the typical plate pattern of a Scrippsiella species. The cyst morphotypes, observed in a clonal culture of this species, ranged from cysts with well-developed paratabulation to cysts in which the paratabulation was barely visible, to cysts covered by irregularly shaped crystals. The analysis of thin sections of the calcareous cysts using the polarized light microscope equipped with crossed nicols and a gypsum plate showed that the optical orientation of the calcite crystals was tangential in all the morphotypes examined. We suggest that the crystallographic method we describe might provide insights for calcareous cyst taxonomy and phylogeny .     

Human lymphocyte tubulin: Purification and characterization in normal and leukemic cells

Tubulin has been purified from human blood and tonsil lymphocytes. Using gel filtration, the molecular weight of human lymphocyte tubulin was estimated to be 119 000. The proteins was shown to consist of two subunits, with molecular weights of 61 000 and 58 000 comparable to the α and β polypeptides of human brain tubulin. A partial identity reaction was observed between lymphocyte tubulin and human tubulin when tested by double immunodiffusion against a rabbit anti-human brain tubulin antibody. In the presence of GTP, the purified protein polymerized to form microtubules. Tubulin was localized to the cell's juxtacentriolar region by immunofluorescence and electron microscopy. When assayed by a colchicine-binding assay corrected for time decay, the binding affinity was 1.50 ± 0.86 · 10⁶M^?1 and a level in normal lymphocytes of 1.21 · 10^?2 ± 0.79 g/g of soluble protein was determined. Since chronic lymphocytic leukemia lymphocytes have an anomalous capping behavior as well as an unusual susceptibility to colchicine toxicity, the properties and levels of tubulin were determined in these cells. Similar values were obtained for the level, decay rate, molecular weight, and K_a for colchicine as for normal lymphocytes. Chronic lymphocytic leukemia lymphocyte tubulin polymerized in a normal fashion. It thus appears that a decrease in the quantity or function of tubulin does not account for these anomalies in the chronic lymphocytic leukemia lymphocyte.     

Isolation of Osteoprogenitors from Human Jaw Periosteal Cells: A Comparison of Two Magnetic Separation Methods

Human jaw periosteum tissue contains osteoprogenitors that have potential for tissue engineering applications in oral and maxillofacial surgeries. To isolate osteoprogenitor cells from heterogeneous cell populations, we used the specific mesenchymal stem cell antigen-1 (MSCA-1) antibody and compared two magnetic separation methods. We analyzed the obtained MSCA-1⁺ and MSCA-1⁻ fractions in terms of purity, yield of positive/negative cells and proliferative and mineralization potentials. The analysis of cell viability after separation revealed that the EasySep method yielded higher viability rates, whereas the flow cytometry results showed a higher purity for the MACS-separated cell fractions. The mineralization capacity of the osteogenic induced MSCA-1⁺ cells compared with the MSCA-1⁻ controls using MACS was 5-fold higher, whereas the same comparison after EasySep showed no significant differences between both fractions. By analyzing cell proliferation, we detected a significant difference between the proliferative potential of the osteogenic cells versus untreated cells after the MACS and EasySep separations. The differentiated cells after MACS separation adjusted their proliferative capacity, whereas the EasySep-separated cells failed to do so. The protein expression analysis showed small differences between the two separation methods. Our findings suggest that MACS is a more suitable separation method to isolate osteoprogenitors from the entire jaw periosteal cell population.