Isolation, purification and partial characterization of early pregnancy factor (EPF) from sera of pregnant women.

Early pregnancy factor (EPF) is a pregnancy protein, which is secreted into the maternal serum 12-16 hours after fertilization. It is thought to be an immunosuppressive molecule. EPF is detected in pregnant woman's serum by the rosette inhibition assay (RIA). In this study, EPF was purified from the pregnant woman's sera by using ion exchange chromatography and analyzed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The proteins which showed a positive result with the RIA, were found to be 35 kDa and 17 kDa molecular weights. The biological activities of these proteins were stable upon heat treatment at 56 degrees C for 30 min. Proteins isolated and purified in this study might be of great significance to the field of human reproduction with particular reference to pregnancy and recurrent abortion.

Process stability assessed by selecting Shewhart's psi statistical analysis technique of the influence of matrix modifier and furnace program in the optimization and precision of zinc determinations by graphite furnace atomic absorption spectroscopy.

The method previously used in the Toxicology Laboratories of King Faisal Specialist Hospital and Research Center for determining the zinc concentration in serum by Zeeman atomic absorption spectrometer was improved by modifying the matrix modifier and by changing the heated graphite furnace atomization (HGA) program. After trying several methods we failed to achieve the required precision and the accuracy of methods for serum zinc determination. We changed the matrix modifier to a fifty percent mixture (v/v) of 3.90 grams per liter of ammonium phosphate in Type 1 water with 0.2% nitric acid and 1.0 gram per liter of magnesium nitrate in acidic water (0.2% HNO3) with 0.1% triton X-100 was used as matrix modifier. A twenty-five fold dilution of the sample in matrix modifier was injected on the L'vov's platform of the furnace. In order to reduce the high sensitivity of Zn the furnace program was modified. The method is found very robust. The average reproducibility between inter-runs and intra-run is less than 1.59% with a high degree of accuracy. We used two levels of controls i.e. normal or low level and abnormal or high level. The linearity and the detection limit of the assay were 0.9992 and 0.010 micromol/L respectively. Average recovery of the analyte was 98.65%. The X-Bar and R charts were constructed by using Shewhart's statistical analysis technique to assess the test methodology. It was found that the assay is capable and stable for routine clinical and research analysis. The capability index (C(P)) of the assay, an indicator of the precision, was calculated.

Immunomodulatory effect of Nigella sativa proteins fractionated by ion exchange chromatography.

Whole Nigella sativa (N. sativa) proteins were purified on a DEAE Sephadex A50 ion exchange column. Complete fractionation was achieved in four peaks. Analysis of the purified peaks was carried out by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Whole N. sativa showed a number of protein bands ranging from 94-10 kDa molecular mass. In mixed lymphocyte cultures (MLC), whole N. sativa and its purified proteins were found stimulatory as well as suppressive and this effect varied from one donor to another. Maximum stimulation (mean + S.E. of % relative index was 63.73 + 20.78) was observed with fractionated N. sativa proteins (P1) (10 microg/ml) in MLC. In MLC, also N. sativa peaks (P1 and P2) were stimulatory at all concentrations (10 microg/ml, 1 microg/ml or 0.1 microg/ml) used. However, a uniformly suppressive effect of N. sativa and its all four peaks at a concentration of 10 microg/ml was noticed when lymphocytes were activated with pokeweed mitogen (PWM). The effect of N. sativa proteins was further evaluated on the production of cytokines which were measured by using specific enzyme-linked immunosorbent assay. Large quantities of IL-1beta were secreted by whole N. sativa in culture medium with non-activated peripheral blood mononuclear cells (PBMC) (450 pg/ml) and with allogeneic cells (410 pg/ml). Fractionated N. sativa was less effective when compared with whole N. sativa proteins. No effect on IL-4 secretion was seen either by using non-activated, PWM-activated or allogeneic-cells. Whole N. sativa suppressed as well as stimulated the production of IL-8 in non-activated and PWM-activated PBMC respectively. All N. sativa peaks with protein concentration of 2 microg/ml were stimulatory for the induction of IL-8 by PWM-activated cells. However, no effect on IL-8 was seen either with whole N. sativa or its peaks when allogeneic PBMC were used. Stimulatory effect of whole N. sativa and fractionated proteins was also noticed on the production of TNF-alpha either using non-activated or mitogen activated cells.

Purification and some properties of a hydrophilic prokallikrein and a hydrophobic prokallikrein from sheep kidney cortex.

1. A hydrophilic and a hydrophobic prokallikrein were purified from sheep kidney cortex and some of their biochemical properties were compared. 2. The effects of several inhibitors on the activity of the hydrophilic and hydrophobic kallikreins were investigated. 3. The thermal stability and response of the hydrophobic kallikrein towards various detergents were also studied.

Purification of a second kallikrein from bovine pancreas.

Two kallikreins were identified in a homogenate of bovine pancreas in terms of their differential elution from an anion-exchange chromatography column. The kallikreins were quantified by their ability to release kinin from a partially purified preparation of bovine kininogen. The second kallikrein, designated kallikrein B, was purified by a three-step procedure following anion-exchange chromatography consisting of affinity chromatography on a benzamidine-agarose resin, gel filtration and hydrophobic interaction chromatography. An overall purification factor of 556-fold was achieved with a 58% recovery of enzymatic activity. The final material was shown to be homogeneous by a number of electrophoretic analyses. The relative molecular mass of pro-kallikrein B was found to be 26,700 by gel filtration and that of kallikrein B to be 26,000 by SDS gel electrophoresis. Gel isoelectric focusing revealed the presence of several isoenzymic forms ranging in isoelectric point from pH 4.05 to 4.35.

Immunosuppression by human seminal plasma fractionated by DEAE Sephadex A-50 ion exchange chromatography.

We fractionated the whole human seminal plasma on DEAE Sephadex A-50 ion exchange columns. Complete separation was achieved in seven peaks using different salt concentrations in phosphate buffer pH 6. The seminal plasma proteins were separated by sodium dodecyl-sulphate polyacrylamide gel electrophoresis. Human seminal plasma (SP) and its fractions were used in mixed lymphocyte reaction in vitro. Fractions 3, 4, and 7 were found to suppress the proliferation of human peripheral blood mononuclear cells to phytohemagglutinin and pokweed mitogen at a concentration of 10 micrograms ml-1 while stimulatory effect was observed at lower concentrations (1 microgram and 2.5 micrograms ml-1). Whole human SP and other fractions failed to suppress the proliferation of lymphocytes in vitro. Furthermore, the effect of human SP and its fractions was also investigated on phagocytic function of polymorphonuclear leukocytes (PMNs) using luminol dependent chemiluminescence assay stimulated with phorbol myristate acetate and opsonized yeast. Fractionated SP was found to have a suppressive effect on the luminol-dependent chemiluminescence of PMNs in the whole blood.

Bovine pancreatic kallikrein: purification and radioimmunoassay.

Kallikrein was isolated from bovine pancreatic tissue by a sequential procedure of anion-exchange, hydrophobic interaction and gel filtration chromatographies. A monospecific polyclonal antiserum to the pure enzyme was raised in rabbits and was used to set up a specific radioimmunoassay for bovine tissue kallikrein.