Kinetic characterization of tissue-type plasminogen activator (t-PA) and t-PA deletion mutants

The binding of t-PA to fibrin is mediated both by its "finger" (F) and its "kringle 2" (K2) domain. In addition, these domains are involved in the stimulation of t-PA activity by fibrin. We analyzed the kinetic characteristics of Glu-plasminogen activation by t-PA and a set of t-PA deletion mutants in the absence and the presence of desA-fibrin. In the absence of desA-fibrin, the activity of t-PA (variants) is determined by the presence of the protease domain, irrespective of the composition of the amino-terminal heavy chain. In the presence of the cofactor desA-fibrin, the activity of t-PA (variants) is dependent on the domain composition of the heavy chain. The activity of t-PA is stimulated 2,400 fold by desA-fibrin, whereas the activity of the mutant lacking the K1 domain (del. K1) increases 936 fold in the presence of this cofactor. Mutants lacking either the K2 domain (del. K2) or the F domain (del. F) exhibit an enhanced activity upon desA-fibrin addition of 200 and 210 fold, respectively. DesA-fibrin has no stimulatory effect on the activity of the mutant containing only the serine-protease domain (del.FE K1 K2) nor on the activity of the variant containing only the K1 domain and the serine-protease domain (del. FE K2). Furthermore, we determined the relative fibrin affinity of each t-PA variant, which is similarly dictated by the composition of the heavy chain.     

Ganglioside monoclonal antibody (A2B5) labels Alzheimer's neurofibrillary tangles

Ganglioside monoclonal antibody (A2B5) labels Alzheimer's neurofibrillary tangles both in isolated neurofibrillary tangle-bearing nerve cells and in partially purified preparations of tangle fibers. Antibody staining was preabsorbed by preincubation of antibody with neuronal ganglioside preparations. These results suggest that Alzheimer's neurofibrillary tangles have a ganglioside associated with them.     

Fetal antigen retained by mature neurons and ependyma studied with a monoclonal antibody (6B9)

A mouse monoclonal antibody (MAb 6B9, isotype IgM) was raised against autopsy tissue samples from the central nervous system (CNS) of multiple sclerosis (MS) patients. By immunofluorescence microscopy, MAb 6B9 intensely stains most or all cells in fetal rats. However, MAb 6B9 differentially stains various cell types in adult rats. Neurons, ependymal cells, and adrenal chromaffin cells are stained intensely, whereas astrocytes and oligodendrocytes are not stained. The 6B9-reactive antigen (6B9 antigen) is sensitive to periodic acid, but insensitive to treatment with protease, RNase, or hyaluronidase. Results from immunofluorescence microscopy on semithin sections and cultured neuroblastoma cells indicate that 6B9 antigen is intracellular. This is supported by immunoelectron microscopy, where labeling for 6B9 antigen appears in the cytoplasm distinct from any identifiable organelle. Further studies on 6B9 antigen should reveal its chemical nature as well as the significance of developmental changes in its distribution.     

Assay of human tissue-type plasminogen activator (t-PA) with an enzyme-linked immunosorbent assay (ELISA) based on three murine monoclonal antibodies to t-PA

An enzyme-linked immunosorbent assay (ELISA) for the measurement of human tissue-type plasminogen activator (t-PA) was developed. Microtiter plates were coated with a mixture of two monoclonal antibodies and bound t-PA was quantitated with a third monoclonal antibody linked to peroxidase. The lower limit of sensitivity of the assay was 0.2 ng of t-PA per ml. The concentration of antigen in citrated plasma of human subjects was found to be 3.4 +/- 0.8 ng/ml. The assay had a good reproducibility with values of 3.8, 6.5 and 4.9 percent respectively for the intra-, inter-assay and inter-dilution variation coefficients. The results of the ELISA assay on plasma samples from patients during thrombolytic therapy with t-PA correlated very well, over a wide concentration range, with those obtained with a previously described two-site immuno-radiometric assay (r = 0.96). This ELISA with monoclonal antibodies constitutes a stable and reproducible set of reagents for the measurement of t-PA antigen in biological fluids, avoiding the disadvantages of the use of radioisotopes and of polyclonal antibodies.     

Lithium enhancement of central 5-HT transmission induced by 5-HT precursors

The effects of acute and chronic lithium chloride administration on synaptic transmission between bulbospinal norepinephrine (NE) or 5-hydroxy-tryptamine (5-HT) pathways and sympathetic preganglionic neurons were tested in unanesthetized, spinal cats. Discharges recorded from sympathetic preganglionic white rami were evoked by stimulation of spinal reflex pathways or descending excitatory pathways in the cervical spinal cord. Acute lithium administration (2 meq/kg) produced insignificant depression of the reflex pathway but markedly depressed transmission through the intraspinal pathway, an effect that was prevented by depletion or blockage of 5-HT. These observations and the failure of lithium to alter the typical effects of L-dopa on both pathways indicate that lithium does not affect transmission through the excitatory NE pathway. L-Tryptophan (1,0 mg/kg) alone produced little or no depression of either pathway, but 3--4 hr after lithium, this dose of L-tryptophan gradually depressed transmission through both pathways by about 20%. After chronic lithium pretreatment (1 meq/kg twice a day for 3 days), L-tryptophan rapidly depressed transmission through spinal reflex and intraspinal pathways by 40% and 50% respectively. Chronic lithium pretreatment also more than doubled the depression of transmission through both pathways produced by 30 mg/kg of 5-HTP. The average of plasma lithium levels 8--10 hr after the last chronic dose was 1.5 meq/liter. These results support the proposal that lithium increases the uptake of L-tryptophan and 5-HTP by central 5-HT terminals and thereby enhances 5-HT synthesis which is reflected in increased transmission at central 5-HT synapses.     

Synaptic long-term enhancement (LTE) induced by a heterosynaptic neural input

The slow excitatory postsynaptic potential (s-EPSP) response of cat stellate ganglion can be enhanced for hours (long term enhancement, LTE) following a conditioning preganglionic train of low frequency pulses, even when test s-EPSPs are elicited by an unconditioned input line. This provides another model for associational alterations of synaptic efficacy.     

Kinetic analyses of the activation of Glu-plasminogen by urokinase in the presence of fibrin, fibrinogen or its degradation products

The kinetics of the activation of Glu-plasminogen (Glu-plg) and Lys-plasminogen (Lys-plg) by urokinase (UK) were studied in purified systems. The activation of plasminogen by UK in the purified systems followed Michaelis-Menten kinetics with a Michaelis constant (Km) of 1.45 microM and a catalytic rate constant (kcat) of 0.93/sec for Glu-plg as compared to 0.25 microM (Km) and 0.82/sec (kcat) for Lys-plg. In the presence of fibrin and fibrinogen or its plasmin degradation products (fragment D and fragment E), Km for Glu-plg hardly changed, whereas kcat for Glu-plg increased. Effect on increase in kcat was in the order of fibrin greater than fibrinogen greater than D greater than E. Fibrin, fibrinogen, D and E did not influence the activation of Lys-plg by UK. These results indicate that Glu-plg bound to fibrin, fibrinogen, D or E becomes easily activatable by UK. The activation of Lys-plg, however, is not influenced in the presence of fibrin, fibrinogen, D or E.     

Epitope mapping of the anti-urokinase monoclonal antibody 5B4 by isolated domains of urokinase

The amino terminal fragment (ATF) of urokinase-type plasminogen activator (uPA) is a degradation product comprising the entire growth factor-like and kringle domains. It has been previously shown that ATF is able to bind to the u-PA receptor through the growth factor-like domain and that the anti u-PA monoclonal antibody 5B4 (Mab 5B4) binds to ATF preventing u-PA receptor binding. To localize more precisely the epitope recognized by Mab 5B4, ATF was subfragmented by controlled enzymatic proteolysis with V8 protease. Three subfragments of 4,000 Mr (F-4k), 11,000 Mr (F-11k) and 12,000 Mr (F-12k) were purified from the reaction mixture and characterized. SDS-PAGE under reducing and non-reducing conditions, N-terminal aminoacid sequence analysis and C-terminal aminoacid analysis of each fragment indicate that F-4k and F-11k correspond to intact growth factor-like domain and kringle domain (residues 4-43 and 44-135 respectively) while F-12k corresponds to the kringle domain cleaved in the first loop at the glu52-gly53 bond. By Western blot and competitive binding experiments we show that Mab 5B4 recognizes an epitope located on the kringle domain of u-PA and that the binding is strongly reduced when the kringle contains an additional cleavage in its first loop. Since the receptor binding site of u-PA has been previously shown to be located on the growth factor-like domain, Mab 5B4 inhibits the binding of uPA to its cellular receptor likely by steric hindrance. Besides the proven utility in epitope localization of anti u-PA monoclonal antibodies, these u-PA fragments may represent powerful tools for studies of structure-function relationship of u-PA.     

Stimulation of oligodendrocyte differentiation in culture by growth in the presence of a monoclonal antibody to sulfated glycolipid

Perturbation of myelinogenesis by monoclonal antibodies against galactolipids is being used to study the role of these lipids in oligodendrocyte differentiation. We report here a marked stimulatory effect on oligodendrocyte differentiation when mixed primary cultures initiated from 19-21 day fetal rat telencephala are grown in the presence of a monoclonal antibody against sulfogalactolipids. When such cultures were grown in the presence of the IgM antibody 04 [Sommer and Schachner, Dev Biol 83:311-327 1981], the oligodendrocytes formed aggregates connected by fasciculated processes. Immunofluorescence microscopy and biochemical analyses of treated cultures demonstrated 2-3 fold increases in the fraction of 04-positive cells expressing myelin basic protein, and in the levels of myelin basic protein RNA, myelin basic protein, 2',3'-cyclic nucleotide 3'-phosphohydrolase activity, and 35SO4 incorporation into sulfatide. Greater than 90% of the cells positive for myelin basic protein in treated cultures were in aggregates. The specific activities of oligodendrocyte markers were unaffected in control cultures grown with nonspecific myeloma IgM. Since there was no increase in the total number of 04-positive cells in treated cultures, the increases in the specific activities of the myelin protein markers appears to be due to an increase in the fraction of cells expressing these markers. Time course studies demonstrated that both the rate and extent of oligodendrocyte differentiation were enhanced in treated cultures. These data are discussed with regard to possible mechanisms of the stimulation, considering not only potential direct effects of the antibody on the cell physiology, but also possible indirect effects due to antibody-induced aggregation.     

Immunohistochemical recognition of human neuroepithelial tumors by anti-Leu 7 (HNK-1) monoclonal antibody

Summary The immunoreactivity of the anti-Leu 7 (HNK-1) monoclonal antibody, a marker for natural killer cells, was evaluated with the peroxidase-antiperoxidase (PAP) method on sections of human paraffin-embedded tissues from 135 tumors of the central nervous system and five esthesioneuroblastomas. As shown independently by others, the antibody was found to react with most types of neoplastic neuroepithelial cells. Our findings indicate that the reaction is most often localized on the cytoplasmic membranes. The immunoreactive cell membranes were generally those of well-differentiated tumor cells and of neoplastic cells found in tumors that usually were not embryonal in nature. Parallel immunostaining either of the same or of successive sections with an anti-glial fibrillary acidic protein serum was of considerable assistance in discriminating between different immunoreactive cells, e.g., between astrocytes and cells presumed to be oligodendrocytes.Despite its cross-recognition of cells of various histogenesis, the anti-Leu 7 monoclonal antibody can, in well-defined circumstances, elucidate specific differential diagnostic problems involving neurogenic neoplasms that cannot be resolved with routine staining techniques.Supported by a grant from the Fondation Suisse de Bourses de Médecine et Biologie (EP) and by research grant CA 31271 from the National Cancer Institute, US Department of Health and Human Services (LJR)     

Immunohistochemical recognition of human nerve sheath tumors by anti-Leu 7 (HNK-1) monoclonal antibody

Summary Using relatively high dilutions of anti-Leu 7 monoclonal antibody and a four-step peroxidase-antiperoxidase (PAP) reaction in paraffin-embedded tissues, we tested the affinity of this antibody to the cells of 47 human nerve sheath tumors and 22 other tumors in which the differential diagnosis with nerve sheath neoplasms is known to arise. Of all the nerve sheath tumors studied 68%, including 80% of the schwannomas, contained anti-Leu 7-positive cells. All 22 non-schwannian neoplasms were entirely, negative. Specimens of eight experimental malignant rat schwannomas were also negative for anti-Leu 7 antibody. Our findings suggest that anti-Leu 7 monoclonal antibody is a promising marker that may facilitate the differential diagnosis between human Schwann cell and non-schwann cell neoplasms.Supported by a grant from the Fondation Suisse de Bourses de Médecine et Biologic (EP) and by research grant no. CA 31271 from the National Cancer Institute, US Dept. of Health and Human Services (LJR)     

Effects of 5-HT(2A) and 5-HT(2C) receptor antagonists on acute and chronic dyskinetic effects induced by haloperidol in rats

An important limitation of classical antipsychotic drugs such as haloperidol (HAL) is their liability to induce extrapyramidal motor symptoms acutely and tardive dyskinetic syndromes when given chronically. These effects are less likely to occur with newer antipsychotic drugs, an attribute that is often thought to result from their serotonin-2 (5-HT(2)) receptor antagonistic properties. In the present study, we used selected doses of the 5-HT(2A) antagonist M100,907, the 5-HT(2C) antagonist SB242,084 and the mixed 5-HT(2A/C) antagonist ketanserin to re-examine the respective roles of 2A vs. 2C 5-HT(2) receptor subtypes in both acute and chronic motor effects induced by HAL. Acutely, SB242,084 (0.5 mg/kg) reduced HAL-induced catalepsy, while M100,907 (0.5 mg/kg) and ketanserin (1 mg/kg) were without effect. None of the drugs reduced HAL-induced Fos expression in the striatum or frontal cortex, and M100,907 actually potentiated HAL-induced Fos expression in the n. accumbens. In rats chronically treated with HAL, both ketanserin and SB242,084 attenuated vacuous chewing movements, while M100,907 had no effect. In addition, 5-HT(2C) but not 5-HT(2A) mRNA levels were altered in several brain regions after chronic HAL. These results highlight the importance of 5-HT2(2C) receptors in both acute and chronic motoric side effects of HAL, and suggest that 5-HT(2C) antagonism could be targeted as a key property in the development of new antipsychotic medications.     

Acid phosphatase and 5'-nucleotidase activities in the brain of squirrels (Funambulus palmarum) with experimentally induced hyperhistidinaemia

In hyperhistidinaemic squirrels, changed activities of acid phosphatase and 5'-nucleotidase have been observed in the olfactory lobes and cerebral hemispheres. The possible significance of lowered activity of these lysosomal enzymes in the hyperhistidinaemic brain have been discussed.     

Immunohistochemical study on neuroglia identified by the monoclonal antibody against a macrophage differentiation antigen (Mac-1)

We have studied frozen sections of the developing and adult mouse central nervous system (CNS), with or without cold lesions, by immunohistochemical and histochemical methods. Using a monoclonal antibody against a macrophage differentiation antigen (Mac-1), we have shown that some neuroglia in the white matter of adult mice stained positively. In the developing CNS, with or without cold lesioning, Mac-1-positive glia were not detected. In the normal adult CNS, a small number of glia in the white matter stained faintly. After cold injury, the number of Mac-1-positive glia and their staining intensity increased for several months. Mac-1-positive glia were always negative for glial fibrillary acidic protein (GFA). Their morphology and distribution were similar to those of nucleoside diphosphatase-positive cells. Considering that the phagocytic activity of glia increases after injury to the CNS (Trachtenberg 1983) and that Mac-1 has been reported to be associated with the complement receptor (Beller et al. 1982), Mac-1-positive glia may play a role in phagocytosis in the damaged CNS.     

白藜芦醇对小鼠脑缺血再灌注损伤后MMP-9蛋白的影响

目的探讨白藜芦醇预治疗对小鼠暂时性脑缺血的脑保护作用及其对基质金属蛋白酶-9（MMP-9）表达及活性的影响。方法采用插线法制作小鼠暂时性大脑中动脉栓塞（MCAO）模型。白藜芦醇溶于20％环糊精／0．9％NaCL，50mg／kg强饲，1／d，于第7d行MCAO。MCAO2h再灌注22h取完整端脑，切成2mm冠状厚片，2％红四氮唑（TFC）染色，计算脑梗死灶的大小。分别于MCAO2h再灌注4h、10h及22h取患侧或对照侧端脑，提取总蛋白，采用Western blot和明胶酶谱分析方法研究MMP-9的表达及活性。结果白藜芦醇预治疗组MCAO2h再灌注22h时的梗死灶明显小于对照组（P〈0．01）。Western blot和明胶酶谱分析结果证明，白藜芦醇预治疗可降低脑缺血再灌注损伤所诱导的MMP-9蛋白的表达及其活性水平。结论白藜芦醇预治疗对脑缺血再灌注损伤有一定的保护作用，这可能得益于其抑制了MMP-9蛋白的高表达及活性增高。     

The novel monoclonal antibody 9F5 reveals expression of a fragment of GPNMB/osteoactivin processed by furin‐like protease(s) in a subpopulation of microglia in neonatal rat brain

To differentiate subtypes of microglia (MG), we developed a novel monoclonal antibody, 9F5, against one subtype (type 1) of rat primary MG. The 9F5 showed high selectivity for this cell type in Western blot and immunocytochemical analyses and no cross‐reaction with rat peritoneal macrophages (Mφ). We identified the antigen molecule for 9F5: the 50‐ to 70‐kDa fragments of rat glycoprotein nonmetastatic melanoma protein B (GPNMB)/osteoactivin, which started at Lys¹⁷⁰. In addition, 9F5 immunoreactivity with GPNMB depended on the activity of furin‐like protease(s). More important, rat type 1 MG expressed the GPNMB fragments, but type 2 MG and Mφ did not, although all these cells expressed mRNA and the full‐length protein for GPNMB. These results suggest that 9F5 reactivity with MG depends greatly on cleavage of GPNMB and that type 1 MG, in contrast to type 2 MG and Mφ, may have furin‐like protease(s) for GPNMB cleavage. In neonatal rat brain, amoeboid 9F5+ MG were observed in specific brain areas including forebrain subventricular zone, corpus callosum, and retina. Double‐immunοstaining with 9F5 antibody and anti‐Iba1 antibody, which reacts with MG throughout the CNS, revealed that 9F5+ MG were a portion of Iba1+ MG, suggesting that MG subtype(s) exist in vivo. We propose that 9F5 is a useful tool to discriminate between rat type 1 MG and other subtypes of MG/Mφ and to reveal the role of the GPNMB fragments during developing brain. GLIA 2016;64:1938–1961     

Memory impairment induced by chronic intracerebroventricular infusion of beta-amyloid (1-40) involves downregulation of protein kinase C

Signaling pathways underlying the cognitive deficit of the Alzheimer's disease (AD) are not completely understood. Protein kinase C (PKC), a major neuronal protein plays a critical role in cellular signal transduction and it is known to be subjected to modulation in AD. We showed previously that, chronic infusion of beta-amyloid (1-40) into rat cerebroventricle leads to deficit in spatial and non-spatial memory formation. As an attempt to identify the cellular correlates of the memory deficit, in the present study we investigated the PKC activation in different brain areas. Chronic infusion of beta-amyloid (1-40) for 14 days into the rat cerebroventricle decreased the activity of soluble protein kinase C (PKC) in the hippocampus. Subcellular translocation of PKC to membrane fraction in hippocampal slices of rats treated with beta-amyloid (1-40) was completely abolished under acute stimulation with 0.5 microM phorbol-dibutyrate (PDBu). We also reported a decreased affinity (k(D)) for PDBu binding in the hippocampus, cerebral cortex and striatum. The total number of binding sites for PDBu (B(max)) was increased, in the three brain areas analyzed on the day 14, but the changes were not statistically significant. Our data indicate that chronic accumulation of beta-amyloid (1-40) into the rat brain reduced activation of PKC, effect that would substantially contribute to the memory deficit found in these animals.     

Identification and Immunolocalization by monoclonal antibody of NSP-5, a surface polypeptide of neural cells

A monoclonal antibody, termed anti-NSP-5 (anti-Neural cell Surface Protein-5) was obtained from an hybridoma generated by fusing rat myeloma cells with splenocytes of a rat immunized with membranes from the cerebella of weaver mutant mice. This antibody reacted with the surface membrane of a subset of neurones in cultures from cerebella and dorsal root ganglia. In both culture systems, only tetanus toxin-positive cells were stained by the antibody. In sections of adult cerebellum a punctate pattern of staining was seen in the molecular layer, the Purkinje cell layer and the upper part of the granule cell layer. The white matter was strongly positive whereas granule cell and Purkinje cell bodies were clearly negative. In sections from adult dorsal root ganglia anti-NSP-5 labeled most sensory neurones including their axones in the dorsal roots.The expression of the antigen was developmentally regulated. It could not be detected in cerebellar cultures prepared from animals younger than 7 days, in good agreement with the data obtained on tissue sections. Similarly, the antigen could not be detected by immunoblotting in neonatal spinal cord, but a NSP-5-reactive band was present at postnatal day 7. The antibody bound a polypeptide of around MW 180 000 in extracts prepared from adult mouse spinal cord or cerebellum. When purified by immunoaffinity chromatography the antigen co-eluted with numerous