In the presence of phospholipids, glycosaminoglycans potentiate factor Xa-mediated protein C activation by modulating factor Xa activity

Although the thrombin/thrombomodulin complex is considered the physiological activator of protein C, factor Xa (f.Xa) can also activate protein C in a reaction that is potentiated by glycosaminoglycans. To explore this phenomenon, we first examined the effect of glycosaminoglycans of varying degrees of sulfation on the kinetics of protein C activation by f.Xa in the presence of Ca2+ and phosphatidylcholine-phosphatidylserine vesicles (PCPS). Heparin increased the rate of protein C activation by f.Xa by 4-fold. In contrast, N-desulfated heparin had no effect on activation, whereas dextran sulfate, which is more sulfated than heparin, increased catalytic efficiency 21-fold. These data suggest that the capacity of glycosaminoglycans to catalyze protein C activation by f.Xa depends on their degree of sulfation. The affinities of individual glycosaminoglycans for protein C and f.Xa were measured in the absence or presence of PCPS by monitoring changes in extrinsic fluorescence when fluorescein-labeled f.Xa or protein C was titrated with the various glycosaminoglycans. Heparin binds protein C with low affinity in the absence or presence of PCPS. In contrast, the affinity of heparin for f.Xa is 86-fold higher in the presence of PCPS compared to that in the absence of PCPS. Similar results were obtained using surface plasmon resonance. These findings suggest that a high affinity glycosaminoglycan binding site is exposed when f.Xa binds to PCPS. The observation that heparin promotes f.Xa-mediated activation of prethrombin 1 only in the presence of phospholipid suggests that glycosaminoglycan binding modulates the active site of f.Xa. This study reveals that when f.Xa interacts with anionic phospholipids, glycosaminoglycans bind f.Xa more tightly, allosterically modulate its active site, and enhance its capacity to activate protein C.     

Antisense raf oligodeoxyribonucleotide is a radiosensitizer in vivo.

Raf-1, a cytosolic protein serine/threonine kinase, plays important roles in cell growth, proliferation, transformation, and cell survival. The aim of the present study was to evaluate the radiotherapeutic efficacy of a fully phosphorothioated and well-characterized antisense raf oligodeoxyribonucleotide (ODN) corresponding to the 3'-untranslated region of human c-raf-1 mRNA (ISIS 5132/5132). Using our recently developed liposome encapsulation of ODN approach, we first compared the pharmacokinetic parameters of a liposomal formulation of 5132 (LE-5132) and 5132. The peak plasma concentrations 5 minutes after ODN administrations (30 mg/kg i.v.) were 28.5 microg/ml and 13.5 microg/ml for LE-5132 and 5132, respectively. The decrease in plasma concentration of LE-5132 and 5132 followed a biexponential pattern, with initial distribution half-lives (t1/2alpha) of 34.8 minutes and 21.6 minutes, respectively. The terminal half-lives (t1/2beta) with LE-5132 and 5132 were 14.5 hours and 4.3 hours, respectively. The area under the plasma concentration-time curve (AUC) was 5.8 times higher with LE-5132 than with 5132. Significantly higher intact ODN levels could be measured in most organs within 48 hours of administration of LE-5132 compared with 5132 (liver 18.4-fold, spleen, 31-fold, heart 3-fold, lungs 1.5-fold). In kidneys, the level was lower with LE-5132 (0.77-fold). LE-5132 composition, unlike 5132, did not affect clotting time in vitro. Significant decline in the level of Raf-1 protein was observed in vitro in relatively radioresistant human laryngeal squamous cell carcinoma cells (SQ-20B) treated with LE-5132 compared with SQ-20B cells treated with equimolar concentration of 5132 or liposome-encapsulated mismatched 5132 (0.5 microM LE-5132, 71.3%+/-22.5%; 1.0 microM LE-5132, 79.6%+/-16.7%). In addition, LE-5132 appeared to be a more potent antitumor compound than 5132 (p < 0.001). These data established the suitability of LE-5132 for in vivo radiotherapeutic efficacy studies. Intravenous administration of LE-5132 into SQ-20B tumor-bearing athymic mice inhibited Raf-1 expression in tumor tissue compared with blank liposome-treated or untreated control groups. LE-5132 or ionizing radiation (IR) treatment alone caused significant but transient inhibition of SQ-20B tumor growth but not tumor regression. Remarkably, a combination of LE-5132 and IR treatments led to significant and sustained tumor regression for at least 27 days after the last treatment (< 0.001). Histopathologic examination of tumor samples revealed a significant proportion of cells containing fragmented chromatin in the LE-5132 + IR treatment group as compared with single agent and untreated control groups. These in vivo data support the notion that Raf-1 has proliferative and survival functions and advance the scientific and technologic bases for the use of antisense raf ODN in the management of radioresistant malignancies.     

Luteinising hormone,follicle stimulating hormone and gonadotropin releasing hormone immunoreactivity in two insects: Locusta migratoria migratoroides R&F and Sarcophaga bullata (Parker)

Summary

Materials immunologically related to luteinising hormone (LH), follicle stimulating hormone (FSH) and the gonadotropin releasing hormone (GnRH) were localised in cerebral tissue of Locusta migratoria and Sarcophaga bullata by means of the peroxidase-antiperoxidase method. Several polyclonal and a monoclonal antisera were used. From the third larval instar a positive reaction was observed in cells located in several parts of the brain. Each antiserum reacted with a constant number of well defined cells and nerve fibers. No differences between sexes were observed.     

Catecholamine-containing biodegradable microsphere implants as a novel approach in the treatment of CNS neurodegenerative disease

Biodegradable controlled-release microsphere systems made with the biocompatible biodegradable polyester excipient poly (dl-lactide-co-glycolide) constitute an exciting new technology for drug delivery to the central nervous system (CNS). Implantable controlled-release microspheres containing dopamine (DA) or norepinephrine (NE) provide a novel means to compare DA- or NE-induced restitution of function in unilateral 6-hydroxydopamine lesioned rats. A suspension of 3 μL of DA- or NE-containing microspheres or empty microspheres was implanted in 2 sites of the DA denervated striatum of rats previously unilaterally lesioned with 6-hydroxydopamine. Contralateral-rotational behavior induced by apomorphine was used as an index of lesion success and, following implantation of the microspheres, also as an index of functional recovery. Interestingly, both DA- and NE-microsphere-implanted rats displayed a 30–50% reduction in the number of apomorphine-induced rotations up to 8 wk postimplantation. Rats implanted with empty microspheres did not demonstrate significant changes in contralateral rotational behavior. Behavioral studies following implantation of a mixture of DA and NE microspheres revealed an 80% decrease in the number of apomorphine induced rotations up to 4 wk. On conclusion of the studies, immunocytochemical examination revealed growth of DA and tyrosine hydroxylase immunoreactive fibers in the striatum of DA and NE microsphere-implanted rats. Functional behavior appeared to correlate with the degree of fiber growth. Preliminary electron microscopic studies showed signs of axonal sprouting in the vicinity of the implanted microspheres. No growth was noted in rats implanted with empty microspheres. This report reviews the abilities of both microencapsulated NE and DA to assure functional recovery and to promote DA fiber (re)growth in parkinsonian rats. This novel means to deliver these substances to the central nervous system could be of therapeutic usefulness in Parkinson's disease.