bFGF and heparin but not laminin are necessary factors in the mediums that affect NSCs differentiation into cholinergic neurons.

OBJECTIVES: Neural stem cells (NSCs) are self-renewed, pluripotent cells that can differentiate into neurons, astrocytes and oligodendrocytes. Cholinergic neurons are an important kind of neurons that play an essential role in the treatment of Parkinsonism and epilepsy. We are interested in how different mediums affect NSCs differentiation into cholinergic neurons. METHODS: NSCs were isolated from the striatum corpora of embryonic brain in a 14-day pregnant rat. Cells were cultured in basic mediums [F12/DMEM (1:1) including 1% B27 (v/v) and 20 ng/ml EGF] but with different combinations of three supplements: bFGF (20 ng/ml), heparin (5 mug/ml) and laminin (1 mug/ml). After 7 days culturing, cells were immunized with choline acetyltransferase (ChAT), a marker enzyme of cholinergic neuron. RESULTS: We found ChAT could not be detected in the basic mediums with only one supplement. Then, we tested the combination of two out of three. We found that ChAT positive cells could only be detected in the medium with bFGF and heparin (FH). However, when we added the laminin into the FH, more ChAT positive cells appeared. DISCUSSION: This finding suggests that bFGF and heparin are essential in the mediums that affect NSCs differentiation into cholinergic neurons, and laminin is an important positive factor in this process.

Heparin binds to lipopolysaccharide (LPS)-binding protein, facilitates the transfer of LPS to CD14, and enhances LPS-induced activation of peripheral blood monocytes.

Heparin is one of the most effective drugs for preventing and treating thromboembolic complications in surgical patients. Recent evidence suggests that heparin enhances the proinflammatory responses of human peripheral blood monocytes to Gram-negative endotoxin (LPS). We have identified LPS-binding protein (LBP) as a novel heparin-binding plasma protein. The affinity of LPB to heparin was KD = 55 +/- 8 nM, as measured by surface plasmon resonance. Using a fluorescence-based assay, we showed that clinically used heparin preparations significantly enhance the ability of LBP to catalytically disaggregate and transfer LPS to CD14, the LPS receptor. The presence of clinically relevant heparin concentrations in human whole blood increased LPS-induced production of the proinflammatory cytokine IL-8. Fondaparinux, which is identical with the antithrombin III-binding pentasaccharide in heparin, did not bind to LBP or alter LBP function. Thus, this novel anticoagulant drug is a potential candidate for safe administration to patients who have endotoxemia and require anticoagulation.

Effect of sperm capacitation and fertilization media on IVF and early embryo development of prepubertal goat oocytes.

Experiments were carried out to develop an improved IVF system for prepubertal goat oocytes matured in vitro. Cumulus oocyte complexes (COC) were obtained by slicing ovaries from slaughtered prepubertal goats. Oocytes were matured in TCM199 supplemented with 20% estrous goat serum (EGS) + 10 micrograms/mL FSH + 10 micrograms/mL LH + 1 microgram/mL estradiol 17 beta for 27 h at 38.5 degrees C in 5% CO2 in air. In Experiments 1 and 2, freshly ejaculated spermatozoa were capacitated in 1 of 3 media: TALP/H, modified Defined Medium (mDM) and mH-M199 with 50 micrograms/mL heparin for 45 min. Matured oocytes were fertilized in TALP, mDM or mH-M199 in Experiment 1 and in TALP in Experiment 2. In Experiment 3, three media were used for sperm capacitation and fertilization: Treatment A (control group): spermatozoa were capacitated in mDM with 50 micrograms/mL heparin for 45 min and fertilized in TALP medium with 1 microgram/mL hypotaurine; Treatment B: spermatozoa were capacitated in mDM with 50 micrograms/mL heparin + 388 micrograms/mL caffeine for 30 min and fertilized in TALP medium without hypotaurine; Treatment C: spermatozoa were capacitated in mDM with 50 micrograms/mL heparin for 45 min and fertilized in TALP medium with PHE (20 microM penicillamine, 10 microM hypotaurine and 2 microM epinephrine). At 24 h post insemination, the ova were transferred to a granulosa cell monolayer, and early embryo development was evaluated until Day 8. In experiment 2, the results show, that mDM plus heparin for sperm capacitation and TALP medium with hypotaurine for oocyte fertilization provided the highest proportion of penetrated oocytes, both total number (79.6%) and normal fertilization (55.1%), whereas the use of caffeine (44.6 and 31.2%, total and normal fertilization rate, respectively) and PHE (31.8 and 20.6%, total and normal fertilization rate, respectively) as motility enhancers did not improve the results obtained in the control group (48.7% and 37.2%, total and normal fertilization rate, respectively). These were no differences for the results of morulae and blastocysts.

Modification of lymphocyte migration by sulfated polysaccharides.

The role of sulfated polysaccharides in lymphocyte migration has been analyzed in vivo using lymphocytes labeled with an intracellular DNA-binding fluorochrome Hoechst 33342. The influence of a panel of sulfated polysaccharides on entry (by injecting the sulfated polysaccharide prior to the labeled cells) and displacement from lymphoid organs (by injecting the sulfated polysaccharide after the labeled cells have localized) indicated that different sulfated polysaccharides have selective effects on entry and displacement, and furthermore positioning of subpopulations within organs. Additional experiments suggested that receptors for sulfated polysaccharides on high endothelial venules may interact with complementary structures on lymphocytes. The data supporting this conclusion were: (a) the normal localization behavior of lymphocytes preincubated with sulfated polysaccharides; (b) an inverse relationship between the expression of lymphocyte surface receptors for sulfated polysaccharides and the ability of the lymphocytes to enter lymphoid organs and (c) the selective binding of sulfated polysaccharide-coupled fluoresceinated beads to high endothelial venules. In this case only the beads coupled with the sulfated polysaccharides that inhibited entry bound to the high endothelial venules. These findings are discussed in terms of a fundamental cellular recognition system utilizing sulfated polysaccharides.