Solubilization and characterization of the acceptor for Clostridium botulinum type B neurotoxin from rat brain synaptic membranes.

The acceptor for Clostridium botulinum type B neurotoxin was solubilized from rat brain synaptic membrane with nonionic detergent, nonanoyl-N-methylglucamide (MEGA-9). The solubilized acceptor was assayed for the binding activity by precipitating the acceptor with acetone in the presence of phosphatidylcholine. 125Ilabeled neurotoxin specifically bound to the lipid vesicles having incorporated the acceptor together with gangliosides. The lipid vesicles having incorporated either the acceptor or gangliosides alone showed extremely low binding activity. The treatment of the solubilized acceptor with lysyl endopeptidase and glycopeptidase F but not with sialidase resulted in decreased toxin binding, indicating that the putative acceptor is a glycoprotein accompanying an N-linked carbohydrate moiety. The observations suggest also that a protein acceptor/ganglioside complex may be required to form the functional toxin receptor.

A new model of transient cerebral ischemia in neonatal rats.

A new model of transient cerebral ischemia in 10-day- old rats is described. Under microscopic guidance, the right external and internal carotid arteries were electrically coagulated. A solid 0.47 mm diameter nylon thread was inserted into the right common carotid artery toward the ascending aorta up to 10-12 mm from the upper edge of the sternomastoid muscle (preischemic rats). A 60-min cerebral ischemia was induced by clamping the left external and internal carotid arteries (ischemic rats), followed by 3-h recirculation. 31P magnetic resonance (MRS) spectroscopic studies revealed that severe intracellular acidosis occurred and ATP disappeared completely for a least the last 20 min of ischemia. Cerebral blood flow (CBF), measured by the hydrogen clearance technique, decreased to approximately 11% of the preischemic level in the frontal cortex soon after the induction of ischemia. On resuscitation, ATP recovered completely and the preischemic intracellular pH level was restored within 180 min. CBF has recovered to approximately 30% of the preischemic level at 5 min after resuscitation. The CBF recovery was not compete even at 180 min after resuscitation. With this model, the effects of pure ischemia without hypoxia on the neonatal brain and the process of recovery from transient ischemia can be studied.

The high-affinity binding of Clostridium botulinum type B neurotoxin to synaptotagmin II associated with gangliosides GT1b/GD1a.

125I-labeled botulinum type B neurotoxin was shown to bind specifically to recombinant rat synaptotagmins I and II. Binding required reconstitution of the recombinant proteins with gangliosides GT1b/GD1a. Scatchard plot analyses revealed a single class of binding site with dissociation constants of 0.23 and 2.3 nM for synaptotagmin II and synaptotagmin I, respectively, values very similar to those of the high- (0.4 nM) and low-affinity (4.1 nM) binding sites in synaptosomes. The high-affinity binding of neurotoxin to synaptosomes was specifically inhibited by a monoclonal antibody recognizing with the amino-terminal region of synaptotagmin II. These results suggest that this region of synaptotagmin II participates in the formation of the high-affinity toxin binding site by associating with specific gangliosides.

Distribution of soluble N-ethylmaleimide fusion protein attachment proteins (SNAPs) in the rat nervous system.

Soluble N-ethylmaleimide-sensitive fusion protein (NSF) attachment protein (SNAP) plays an essential role in vesicular transport and the release of neurotransmitters and hormones through associations with NSF and SNAP receptors (SNAREs). Three isoforms (alpha, beta and gamma) of SNAP are expressed in mammals. We have generated isoform-specific antibodies and studied the expression and distribution of these SNAP isoforms in the rat nervous system. Each antibody specifically recognized alpha-, beta- or gamma-SNAP in an isoform-specific manner in immunoblots of brain homogenate. Alpha- and gamma-SNAP were ubiquitously expressed in various tissues, whereas beta-SNAP was expressed only in brain. After subcellular fractionation of brain homogenates, all three isoforms were recovered in both soluble and particulate fractions. Immunohistochemistry revealed that alpha- and beta-SNAP were generally differentially distributed both in synaptic and non-synaptic regions, including brain white matter. The presynaptic location of both alpha- and beta-SNAP was confirmed by immunoelectron microscopy. At the neuromuscular junction, immunoreactive alpha-SNAP was identified in synaptic vesicles, while in the cerebellum, beta-SNAP was present in the presynaptic membranes of basket neuron and mossy fiber terminals. From these results we suggest that both alpha- and beta-SNAP may play an important role in neurotransmitter release as well as in constitutive vesicular transport.

Immunochemical identification of the ADP-ribosyltransferase in botulinum C1 neurotoxin as C3 exoenzyme-like molecule.

Botulinum C1 neurotoxin and C3 exoenzyme were purified to apparent homogeneity from the culture filtrate of Clostridium botulinum type C strain 003-9. Both preparations catalyzed ADP-ribosylation of the same substrate, the Mr 22,000 rho gene product (Gb). When the light and heavy chains of C1 toxin were separated, ADP-ribosyltransferase activity in the toxin was quantitatively recovered in the light chain fraction. Anti-C1 toxin antiserum precipitated the ADP-ribosyltransferase activity and the neurotoxicity of C1 toxin in parallel, whereas it had no effect on C3 exoenzyme. On the other hand, anti-C3 exoenzyme antiserum precipitated the ADP-ribosyltransferase activities of both C3 exoenzyme and C1 toxin. This antibody, however, did not precipitate the neurotoxicity of C1 toxin. The ADP-ribosyltransferase in C1 toxin was quantitatively adsorbed onto the anti-C3 antibody column and separated from the majority of C1 toxin protein. The enzyme was then eluted with acidic urea and Western blotting analysis of this eluate revealed the appearance of a protein band positively stained with anti-C3 antibody at a position similar to that of C3 exoenzyme. Quantitative determination by enzyme-linked immunosorbent assay showed that the C3-like immunoreactivity is present in the C1 toxin molecules at the molecular ratio of 1 to 1,000. These results suggest that the ADP-ribosyltransferase activity in C1 toxin is expressed by a C3-like molecule which is present in a small amount in the toxin preparation and appears to bind to the toxin component(s). The above results also indicate that the ADP-ribosyltransferase in C1 toxin is not related to its neurotoxin action.

Dissociation of SNAP-25 and VAMP-2 by MgATP in permeabilized adrenal chromaffin cells.

In digitonin-permeabilized adrenal chromaffin cells, Ca(2+)-induced catecholamine release can be resolved into at least two sequential steps: a MgATP-dependent priming step and a MgATP-independent Ca(2+)-triggered step. Botulinum neurotoxins types A and E cleaved SNAP-25, and blocked MgATP-independent Ca(2+)-induced catecholamine release from the permeabilized chromaffin cells. When the permeabilized cells were primed by pretreatment with MgATP, the amount of SNAP-25 associated with VAMP-2 decreased, and the fraction of SNAP-25 proteolyzed by the neurotoxins increased. These results suggest that dissociation of SNAP-25 and VAMP-2 occurs during the MgATP-dependent priming step, and SNAP-25 plays some important roles in the subsequent MgATP-independent step.

Binding of botulinum type B neurotoxin to Chinese hamster ovary cells transfected with rat synaptotagmin II cDNA.

We have previously identified synaptotagmin, a synaptic vesicle membrane protein from rat brain, as a binding protein for Clostridium botulinum type B neurotoxin. In this report, rat synaptotagmin II was expressed by transfection in Chinese hamster ovary cells and interaction with the neurotoxin was studied. In stable transfectants, the NH(2)-terminal region of synaptotagmin was exposed to the extracellular medium. Synaptotagmin-expressing cells were shown to possess an extremely low binding activity for the radiodinated toxin. However, toxin-binding was markedly increased to cells which had been treated with gangliosides G T1b or G D1a. In synapses, the intravesicular NH(2)-terminus of synaptotagmin becomes exposed at the cell surface after following exocytosis. These findings suggest that the NH(2)-terminal domain of synaptotagmin II forms the binding site for type B neurotoxin by associating with specific gangliosides in presynaptic plasma membranes.

Efficacy of thyrotropin-releasing hormone in the treatment of spinal muscular atrophy.

Children with spinal muscular atrophy were treated by the administration of thyrotropin-releasing hormone. In three infants with spinal muscular atrophy type I, thyrotropin-releasing hormone showed little efficacy, but in children with types II and III, there was improvement in motor function and electromyographic findings after the thyrotropin-releasing hormone therapy. Thyrotropin-releasing hormone has a neurotrophic effect on the spinal anterior motor neurons of spinal muscular atrophy patients and thus may be warranted for the management of spinal muscular atrophy.

Intracellular alkalosis during hypoxia in newborn mouse brain in the presence of systemic acidosis: a phosphorus magnetic resonance spectroscopic study.

We investigated the in vivo changes in cerebral energy metabolism and pHi in newborn mice noninvasively during 8 h of hypoxia with FiO2 = 5%, using phosphorus magnetic resonance spectroscopy continuously. The intracellular brain pH (pHi) increased from 7.20 +/- 0.03 to 7.36 +/- 0.03 (P < 0.05) at 1 h of hypoxia and then decreased gradually. On the other hand, the mixed arterial and venous blood pH decreased gradually during hypoxia, reaching a minimum value of 7.16 +/- 0.01 at the end of the hypoxia. There was no significant difference in PCO2 between control (47.4 +/- 0.8 mm Hg) and 1-h hypoxic (49.0 +/- 1.1 mm Hg) mice. The blood glucose concentration was significantly increased at 1 h of hypoxia. These results indicate that the alkaline shift in pHi during hypoxia was caused neither by systemic alkalosis due to hypocapnia nor hypoglycemia.

Patient with unusual alternating hemiplegia.

A patient with unusual alternating hemiplegia in childhood is reported. The frequency of hemiplegic episodes was lower than that in other reported patients. Interictal positron emission tomography revealed decreased cerebral blood flow in the left hemisphere. After cerebral angiography, postanesthetic irritability occurred which evolved into coma with hemiplegic episodes. We believe that cerebral angiography in a child with alternating hemiplegia may cause hemiplegic episodes and coma. The episodes disappeared almost completely for 6 years after treatment with flunarizine, thus indicating the benefit of the medication for the treatment of alternating hemiplegia.

Anaerobic oxidation of dissolved hydrogen sulfide in continuous culture of the phototrophic bacterium Prosthecochloris aestuarii.

The anaerobic oxidation of dissolved H2S to elemental sulfur was studied at 23 degrees C and pH 6.5+/-0.3 in continuous culture of the phototrophic green sulfur bacterium Prosthecochloris aestuarii. The number of cells formed in the cultures was proportional to the amount of H2S oxidized, and the growth yield was independent of light intensity. The specific growth rate was significantly dependent on the dissolved H2S concentration and light intensity. The kinetic data were analyzed with a rate expression as a function of each rate-limiting factor. Under illumination by white fluorescent lamps, the specific oxidation rate of P. aestuarii reached a maximum of 2.02 x 10(-14) mol-H2S.h(-1).cell(-1) when the dissolved H2S concentration was 2.1 mM at 5000 lx. Simultaneous use of near infrared LED (light-emitting diode) and white fluorescent lamps provided a 35% increase in the maximum specific H2S oxidation rate.

Role of tenascin in human immunoglobulin A nephropathy.

Tenascin is a component of the extracellular matrix that responds rapidly to inflammation or injury. The activity index and chronicity index of immunoglobulin A nephropathy are mainly used to decide whether or not steroid therapy is indicated, but are sometimes difficult to evaluate histologically. We investigated whether tenascin staining of the glomeruli was an indicator of the activity or chronicity indices in patients with immunoglobulin A nephropathy. Tenascin staining was evaluated immunohistochemically in 38 renal specimens, including 32 from patients with immunoglobulin A nephropathy and six from control kidneys, and the extent of staining was scored. Tenascin staining was correlated with the chronicity index (r = 0.643, P < 0.0003) but not with the activity index.

A case of nephrotic syndrome with rapid spontaneous remission in an elderly patient.

In July 1994, a 70-year-old woman was diagnosed as having nephrotic syndrome with proteinuria of 8 to 10 g/day and a serum albumin level of 1.8 g/dl. She was hospitalized in August 1994 for investigation. The urinary findings then normalized, with urinary protein and occult blood both negative and total urinary protein excretion at 0 g/day. A renal biopsy was performed, and spontaneous remission of minimal change nephrotic syndrome was diagnosed. This is an interesting case involving rapid remission of minimal change nephrotic syndrome in an elderly patient.

[Zonisamide monotherapy against absence attacks: report of two cases].

We report two cases of absence seizures, in which zonisamide monotherapy was effective. In one case the attacks recurred 6 months after the beginning of zonisamide therapy but ceased again with an increase of the dosage. The EEG prior to treatment showed 3 Hz generalized spike-wave patterns and 3 Hz high voltage slow wave burst in the right occipital area in one case, and 3.5-4 Hz generalized spike-wave patterns and focal spike-wave complexes in the left frontal to central areas in the other. One of the generalized spike-wave patterns were preceded by right occipital delta wave burst in one case, and bilateral occipital sharp-wave complex in the other. It is suggested that zonisamide monotherapy may be effective against absence seizures with focal paroxysmal discharges.

Effect of cell permeable peptide of c-Jun NH2-terminal kinase inhibitor on the attenuation of renal ischemia-reperfusion injury in pigs.

The outcomes of organ transplantation have improved due to better immunosuppressive drugs, surgical techniques, and management of complications. However, ischemia-reperfusion injury remains a challenge affecting graft survival. In this study, we employed injection of a protein transduction domain (PTD) to inhibit the c-Jun NH2-terminal kinase (JNK) pathway thereby attenuating ischemia-reperfusion injury in a porcine model. The PTD-JNK inhibitor (JNKI) was administered into the renal artery, allowing it to be taken into various elements including vascular endothelial cells by endocytosis via the PTD. Serum creatinine and blood urea nitrogen concentrations were lower among PTD-JNKI than controls. In addition, renal tissue blood flow was maintained in the PTD-JNKI group, resulting in less tissue injury and fewer apoptotic cells. These results suggested that the PTD technique improved renal transplantation outcomes.

Antidepressant-like effect of sildenafil through oxytocin-dependent cyclic AMP response element-binding protein phosphorylation.

Oxytocin (OT) levels in plasma increase during sexual response and are significantly lower in patients with depression. A drug for the treatment of sexual dysfunction, sildenafil, enhances the electrically evoked release of OT from the posterior pituitary. In this study, we showed that sildenafil had an antidepressant-like effect through activation of an OT signaling pathway. Application of sildenafil reduced depression-related behavior in male mice. The antidepressant-like effect was blocked by an OT receptor (OTR) antagonist and was absent in OTR knockout (KO) mice. Sildenafil increased the phosphorylation of cAMP response element-binding protein (CREB) in the hippocampus. The OTR antagonist inhibited sildenafil-induced CREB phosphorylation and sildenafil had no effect on CREB phosphorylation in OTR KO mice. These results suggest sildenafil to have an antidepressant-like effect through the activation of OT signaling and to be a promising drug for the treatment of depression.

Effect of milrinone on ischemia-reperfusion injury in the rat kidney.

BACKGROUND: Milrinone (MIL), a phosphodiesterase (PDE) 3 inhibitor, exhibits cardiotonic and angioectatic effects. Various PDE inhibitors have been shown to suppress inflammatory cytokines. In this study, we evaluated the angioectatic and anti-inflammatory cytokine effects of MIL on renal function after warm ischemia in a rat ischemia-reperfusion (I-R) injury model. MATERIALS AND METHODS: MIL or control solution was perfused from the left renal artery to the right kidney, and the left kidney was excised. The right renal artery, vein, and ureter were clamped and then released after 50 minutes to produce warm ischemia. We evaluated control (n = 7), MIL (n = 7), and sham operation (n = 7) groups for serum creatinine, blood urea nitrogen (BUN), blood flow, expression of tumor necrosis factor (TNF)-α mRNA, apoptosis index, and histological evidence of acute tubular necrosis. RESULTS: Serum creatinine and BUN concentrations peaked at 24 hours after reperfusion. MIL treatment significantly reduced serum creatinine (control group 1.27 ± 0.45 mg/dL vs MIL group 0.77 ± 0.19 mg/dL, P < .05; sham 0.35 ± 0.2 mg/dL) and BUN (control 67.6 ± 13.6 mg/dL vs MIL 51.0 ± 8.8 mg/dL, P < .05; sham 23.0 ± 4.2 mg/dL) levels at 24 hours. Thereafter, serum creatinine and BUN concentrations in the MIL group remained significantly lower compared with the control group for 120 hours (P < .05). MIL group exhibited significantly higher tissue blood flow, less acute tubular necrosis, lower expression of TNF-α mRNA in renal tissue, and lower apoptotic index (P < .05). CONCLUSIONS: MIL maintained renal tissue blood flow by its vasodilatory effect, suppressed expression of TNF-α mRNA by increasing intracellular cyclic adenosine monophosphate, and ultimately decreased tubular cell apoptosis, thus protecting renal function after warm I-R injury.

Living donor renal transplantation using grafts with multiple arteries procured by laparoscopic nephrectomy.

OBJECTIVE: Kidney grafts with multiple renal arteries (MRAs) are not uncommon, but they do make transplantation more difficult. Laparoscopic graft nephrectomy has become the standard; however, the safety and reliability must be maintained for both a donor and a recipient even in case of MRAs. This study evaluated the short-term outcomes of living donor renal transplant using grafts with MRAs procured by laparoscopic nephrectomy. PATIENTS AND METHODS: This study reviewed all living donor kidney transplantations performed from January 2008 to June 2009, which were divided into 3 groups according to the number of renal graft arteries. The serum creatinine level, warm ischemic time (WIT), rewarming time, total ischemic time (TIT), operative time, acute rejection episodes, and complications in each group were evaluated. RESULTS: The serum creatinine level showed no difference among the groups. Longer TIT was observed in the MRAs group, but WIT and rewarming time did not differ. The acute rejection rate was not different. There were no vessel complications in any donors and recipients. CONCLUSION: Harvesting kidney grafts with MRAs by laparoscopic nephrectomy requires a longer TIT; however, transplantation can be performed safely and reliably for both donors and recipients.