Thrombolysis effect with FIIa from Agkistrodon acutus venom in different thrombosis model.

AIM: A fibrinolytic enzyme from Agkistrodon acutus venom, called FIIa, was tested for thrombolytic activity in animals. METHODS: Carotid thrombosis model in rats and rabbits and middle cerebral artery (MCA) thrombosis model in rats were used. RESULTS: Intravenous administration of FIIa, at a dosage of 0.625 mg/kg, resulted in thrombolysis of carotid thrombi. However, in middle cerebral artery thrombosis, the effective thrombolysis dose was 1.25 mg/kg. When the dosage of FIIa increased, the thrombolytic effect was stronger. Histological examination of kidney, liver, heart, and lung tissue showed no hemorrhage. CONCLUSION: It shows that FIIa from Agkistrodon acutus venom is able to solubilize thrombus in vivo without hemorrhage at an effective dose for thrombolysis.

[Relationship between heat stress suppression of neuroapoptosis and activation of nuclear kappa B in primary cultured rat cerebellar granule cells].

It has been well demonstrated that heat stress response (HSR) plays a crucial role in protecting cells from injury induced by various pathological stimuli. However, the protective mechanism of HSR is only poorly understood. The object of this article was to further investigate the relationship between the protective role of heat stress response and activation of NF-kappa B in primary cultured rat cerebellar granule cells. Heat stress was induced by hyperthermia (43+/-0.5 degrees centigrade), and DNA binding activity of NF-kappa B was determined with electrophoretic mobility shift assay (EMSA). Neuroapoptosis was measured by Hoechst 33258, agarose gel electrophoresis and flow cytometry (FCM) analysis. The results showed that the neurons treated with low potassium for l6 h could induce neuroapoptosis and promote the activity of nuclear kappa B. Heat stress treatment for 30, 60 and 90 min could suppress neuroapoptosis and the activity of nuclear kappa B induced by low potassium in a time-dependent manner. Activation of NF-kappa B using 100 nmol/L phorbol 12-myristate l3-acetate (PMA) could promote antiapoptotic action of heat stress response. In contrast, when NF-kappa B activation was inhibited by 10 micromol/L pyrrolidine dithiocarbamate derivatives (PDTC), heat stress did not provide protection against cell apoptosis induced by low potassium. The results suggest that the neuroprotection of heat stress has no relation to the suppression of NF-kappa B activity, and activation of NF-kappa B may promote antiapoptotic action of heat stress.

Antagonistic action of caffeine against LY294002-induced apoptosis in cerebellar granule neurons.

AIM: To study the effect of caffeine on apoptosis induced by inhibition of 1-phosphatidylinositol 3-kinase in cerebellar granule neurons. METHODS: Cerebellar granule neurons culture, agar gel electrophoresis, and stress-activated protein kinase (SAPK)/c-Jun N-terminal protein kinase (JNK) assay kit to measure SAPK/JNK activity. RESULTS: LY294002 evoked apoptosis concentration-dependently in cerebellar granule neurons. But death resulting from LY294002 was prevented by caffeine in a concentration-dependent manner. The survival effect of caffeine was not affected by inhibitors of ryanodine-sensitive Ca2+ release, nor was it inhibited by L-type channel blockers and N-methyl-D-aspartate (NMDA) receptor blocker. In addition, RP-cAMP, H89, and KN62 were not able to inhibit the protective effect of caffeine. Phosphorylation of c-Jun was necessary for the induction of apoptosis induced by LY294002 in cerebellar granule neurons. But caffeine directly inhibited the activation of JNK and decreased phospho-c-Jun in granule neurons. CONCLUSION: Caffeine inhibited the activation of JNK and decreased the phosphorylation of c-Jun to protect granule neurons from LY294002-induced apoptosis.

Preparation of monoclonal antibody against human m3 receptor.

AIM: To raise monoclonal antibody against human m3 receptor. METHODS: The m3 receptor selective peptide segments deduced from its gene were chemically synthesized, coupled to keyhole limpet hemocyanin carrier protein, and injected to Balb/c mice to raise monoclonal antibody. Antibody was purified by a combination of two-step precipitation methods and ion-exchange chromatography. The specificity of monoclonal antibody was tested by enzyme-linked immunosorbent assay, immunohistochemistry, and radioligand binding assay of receptors. RESULTS: The monoclonal antibody specifically bound to the protein of rat salivary gland and m3 peptide, but not m4 peptide. In radioligand binding assay of receptors, monoclonal antibody inhibited the binding of 3H-QNB to muscarinic receptor in rat salivary gland, but not in rat heart, and could not inhibit the binding of 3H-PZ to rat brain cerebral cortex membrane protein. Immunohistochemical study showed that the human salivary cell surface was strongly stained, whereas the human heart cell surface was not. CONCLUSION: Highly purified (96.3%) monoclonal antibody against the m3 receptor peptide recognized the m3 receptor.

Development of muscarinic m3 and m4 receptor antibodies with pharmacological activities.

AIM: To investigate the feasibility of developing subtype-selective anti-receptor antibodies with pharmacological activities for the study of subtypes of receptors. METHODS: New Zealand white rabbits were immunized with synthesized subtype-selective peptide segments of m3 and m4 receptors to develop antibodies. The effects of the antibodies on ligand-binding to muscarinic receptors were studied by competitive radioligand assay. The effects of the prepared antibodies on the contraction or relaxation activity of ACh in isolated rat ilea and aortic rings were studied. RESULTS: Antibodies against synthesized m3 and m4 receptor subtype-selective peptides were successfully prepared. Both antibodies inhibited [3H]QNB binding to muscarinic receptors with different maximal inhibitions which may be the proportions of m3 or m4 subtypes among the total muscarinic receptors in the tissues. The maximal inhibitory rates in rat cerebral cortex, myocardium, and salivary glands were 12.1% +/- 2.1%, 15.7% +/- 1.1%, and 63.6% +/- 2.8% for m3 antibodies, whereas 28% +/- 6%, 19.3% +/- 2.6%, and 1.6% +/- 1.4% for m4 antibodies respectively. The m3 antibodies inhibited the contraction activity of ACh in isolated rat ilea and the relaxation activity of ACh in isolated rat aortic rings. CONCLUSION: It is feasible to develop subtype-selective anti-receptor antibodies as new tools in the study of the functions of m3 and m4 subtypes of muscarinic receptors.

Changes in rat retinal ganglion neuronotrophic factor and its mRNA during postnatal development.

The monoclonal antibody specific to the retinal ganglion neuronotrophic factor (RGNTF) was used to localize and quantify the presence and amount of RGNTF during the postnatal development of the visual system in the rat. The results showed that in the 0-1-day age group, neurons at the superficial layers and deep part of superior colliculus, and as well as retinal ganglion cells were strongly stained with their RGNTF contents quantified to be 88%, 100% and 100%, respectively. In the 5-6-day age group, RGNTF contents were significantly reduced to merely 50%, 30% and 80%, respectively. The RGNTF contents reduced further to 0% as age increased to 2 years old. A 32P-DNA probe specific to the first 7 amino acid sequence of RGNTF at its N-terminal end was synthesized and used for in situ hybridization studies. The results revealed that strong hybridized signals (i.e. mRNA of RGNTF) were localized in the same neurons in the superficial layers and deep part of the superior colliculus only in the 0-1-day age group, and that no signals were found in retinae of all postnatal age groups. The significant reduction of RGNTF may be related to the RGC death during postnatal development. Neurons at the superficial layers and deep part of the superior colliculus are the sources of RGNTF for RGCs in the postnatal retina.

[Characteristics and inhibitory action of monoclonal and polyclonal antibodies against the retinal ganglion neuronotrophic factor (RGNTF)].

We have immunized Balb/c mice and rabbits with a minute quantity of a 30 kD neuronotrophic factor which was isolated from the extract of newborn rat tectum (Te) by Phast System gel electrophoresis. Splenic cells from the immunized mice were hybridized with NS-1 mouse myeloma cells. Three clones were selected from 576 wells of hybridomas and were capable of secreting monoclonal antibodies specific to the retinal ganglion neuronotrophic factor (RGNTF-MAbs), namely A1, D3 and E8. Subtyping of the three monoclonal antibodies revealed that A1 and D3 are IgG3 and E8 is IgM. They maintained secreting antibodies even after six months of culturing in vitro. In order to determine the specificities of these antibodies, we have used their ascites fluids containing antibodies at a different dilutions to study their effects on the survival of retinal ganglion cells in vitro. The results indicated that at the dilution ranges of 1:250 to 2000, all three monoclonal antibodies exhibited inhibition on the survival of retinal ganglion cells and the inhibition increased with increases in antibody concentrations; especially at a dilution of 1:250, the E8 monoclonal antibody reaching 70% inhibition and A1 and D3 reaching 66% and 62% inhibition, respectively. Polyclonal antibodies from rabbits exhibited similar but weaker results of inhibition. We can conclude that the monoclonal and polyclonal antibodies can specifically inhibit the activity of the 30 kD retinal ganglion neuronotrophic factor.