Akira Yoshida

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.

Neurotransmitter Release from Synaptotagmin-Deficient Clonal Variants of PC 12 Cells

Synaptotagmin (p65) is an abundant synaptic vesicle protein of neurons and contains regions similar to the regulatory domain of protein kinase C. These domains are thought to be involved in calcium-dependent interaction with membrane phospholipids during exocytosis. To assess the functional role of synaptotagmin, synaptotagmin-deficient clonal variants of PC12 cells were isolated. All of the variant cells released catecholamine and adenosine triphosphate in response to elevated intracellular concentrations of calcium, which suggests that synaptotagmin is not essential for secretion of catecholamine and adenosine triphosphate from PC12 cells.

Estimation of infarct size by myocardial emission computed tomography with thallium-201 and its relation to creatine kinase-MB release after myocardial infarction in man.

We evaluated emission computed tomography (ECT) for thallium-201 (201TI) myocardial imaging in estimating infarct size (IS). In 18 patients in whom IS was estimated enzymatically at the time the acute episode, planar 21T1 perfusion scintigraphy and ECT with a rotating gamma camera were performed 4 weeks after the first myocardial infarction. From the size of 201T1 perfusion defects, the infarct area in planar images and the infarct volume in reconstructed ECT images, were measured by computerized planimetry. When scintigraphic IS was compared with the accumulated creatine kinase-MB isoenzyme release (CK-MBr), infarct volume determined from ECT correlated closely with CK-MBr -(r 0.89), whereas infarct area measured from planar images correlated less satisfactorily with the enzymatic IS ( an average infarct area from three views, r = 0.69; for the largest infarct area, r = 0.73). Although conventional scintigraphic evaluation is useful for detecting and localizing infarction, quantification ischemic injury with this two-dimensional technique has a significant inherent limitation. The ECT approach can provide a more accurate three-dimensional quantitative estimate of infarction, and can corroborate the enzymatic estimate of IS.

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.

Human recombinant erythropoietin directly stimulates B cell immunoglobulin production and proliferation in serum‐free medium

The effect of human recombinant erythropoietin (Epo) on B cell responses was studied in a serum‐free medium. Epo enhanced IgM production and thymidine uptake by a human IgM‐producing lymphoblasloid cell line, CBL. This effect was specific to Epo since enhancement was blocked by anti‐Epo antibody but not by control antibody. Among the various cytokines, interleukin‐4 (IL‐4) enhanced IgM production and thymidine uptake while IL‐6 enhanced IgM production without affecting thymidine uptake. In contrast, other cytokines including IL‐1β, IL‐2, IL‐5, interferon‐alpha (IFN‐α), interferon‐gamma (IFN‐γ), or granulocyte/macrophage colony‐stimulating factor (GM‐CSF) were without effect. However, the enhancing effect of Epo is different from that of IL‐4 or IL‐6, since Epo effect was not blocked by anti‐IL‐4 antibody or anti‐IL‐6 antibody. Moreover, specific binding of Epo was detected on CBL cells. Epo also enhanced immunoglobulin (IgG, IgM and IgA) production and thymidine uptake by purified tonsil small resting B cells stimulated by Staphylococcus aureus Cowan strain I (SAC) or by large activated B cells. In contrast, Epo had no effect on unstimulated smalt resting B cells. These results indicate that Epo could directly stimulate activated and differentiated B cells and could enhance B cell immunoglobulin production and proliferation.

Vasoactive intestinal peptide stimulates immunoglobulin production and growth of human B cells

The effect of vasoactive intestinal peptide (VIP) on human lymphoblastoid B cell lines and tonsil B cells was studied. VIP increased immunoglobulin production and proliferation by lymphoblastoid B cell line. GM‐1056, in a dose‐dependent manner. As little as 10‐12 M of VIP was effective, and higher concentrations of VIP induced an approximately five‐fold increase in IgA production. Moreover, this enhancement was blocked by VIP antagonist. Similarly, VIP enhanced IgM and IgG production by other lymphoblastoid B cell lines, CBL and IM‐9, respectively. In contrast to VIP, another neuropeptide substance P (SP) or somatostatin failed to enhance immunoglobulin production and thymidine uptake. VIP also enhanced IgA production and thymidine uptake by purified tonsil B cells. However, in contrast to B cell lines, VIP failed to enhance IgM and IgG production by tonsil B cells. SP or somatostatin failed to enhance immunoglobulin production or thymidine uptake by tonsil B cells. These results indicate that VIP acts as B cell stimulatory factor and that VIP may also have preferential effect on IgA production on tonsil B cells.

Differential effect of vasoactive intestinal peptide, somatostatin, and substance P on human IgE and IgG subclass production

We studied the effect of vasoactive intestinal peptide (VIP), somatostatin (SOM), and substance P (SP) on IL-4-stimulated human IgE and IgG subclass production. VIP and SOM, but not SP, inhibited IgE production without affecting IgM or IgA production by mononuclear cells (MNC) from nonatopic donors from 10 pM to 10 nM. These neuropeptides also differentially modulated IgG subclass production. While IgG1 production was not affected by VIP, SOM, or SP, all of the neuropeptides enhanced IgG2 production. By contrast, SOM and SP, but not VIP, inhibited IgG3 production, whereas VIP and SP, but not SOM, enhanced IgG4 production. The effect by neuropeptides was specific since each peptide effect was specifically blocked by each antagonist. To achieve this effect, neuropeptides must be added at the start of the culture and be present throughout the entire culture period. The inhibition of IgE production was not mediated by known inhibitors of IgE production, IFN-gamma or PGE2, because the addition of anti-IFN-gamma mAb (10 micrograms/ml) or indomethacin (0.1 microM) did not overcome the inhibition of IgE production. In contrast to MNC, neuropeptides did not affect IgG subclass production in purified B cells. IgE production was not induced by IL-4 in purified B cells. Neuropeptides also failed to modulate IgG subclass production in cultures of B cells with either T cells or monocytes. However, they modulated IgE production and IgG subclass production in B cells in the presence of T cells and monocytes. In purified B cells, IL-4 plus anti-CD40 mAb induced IgE production which was not inhibited by VIP or SOM. However, VIP or SOM, but not SP, inhibited IgE production in B cells cultured with both T cells and monocytes. Finally, the mechanism of modulation of IgE and IgG4 production was dependent on IL-4-induced switching, since neuropeptides modulated IgG4 and IgE production in surface IgG4-negative (sIgG4-) and sIgE- B cells, respectively. In contrast, modulation of IgG2 and IgG3 production was not due to switching, since neuropeptides did not affect either IgG2 or IgG3 production in sIgG2- or sIgG3- B cells, respectively.

Antigens Associated with N‐ and L‐Type Calcium Channels in Lambert‐Eaton Myasthenic Syndrome

Abstract: In Lambert‐Eaton myasthenic syndrome neurotransmitter release is reduced by an autoimmune response directed against the calcium channel complex of the nerve terminal. Autoantibodies were detected by immunoprecipitation assays using solubilized receptors labeled with ligands selective for N‐type (125I‐ω conotoxin GVIA) and L‐type ([3H]PN200‐110) calcium channels. Sera with a high antibody titer (>3 nM) against rat brain N‐type channels contained autoantibodies that immunoprecipitated neuronal and muscle L‐type channels. These IgG fractions stained a 55‐kDa protein in immunoblots of purified skeletal muscle dihydropyridine receptor, suggesting that they contain autoantibodies against the β subunit of the calcium channel. A distinct antibody population in the same fractions reacted with a nerve terminal 65‐kDa protein that is unrelated to the β subunit and displays properties similar to those of synaptotagmin.

Enhancement of in vitro spontaneous IgE production by topical steroids in patients with atopic dermatitis

BACKGROUNDnAtopic dermatitis (AD) is an inflammatory skin disease. Although topical steroids are widely used for AD, management of severe AD is not satisfactory because of relapse or occasional aggravation of symptoms. Moreover, glucocorticoids induce in vitro IgE production. On the other hand, topical sodium cromoglycate (SCG) solution is a safe and effective treatment for AD.nnnMETHODSnWe treated 43 patients with AD with SCG solution (n = 21) or with topical steroids, beclomethasone dipropionate (BD) ointment (n = 22). After 2 weeks, clinical evaluation and spontaneous immunoglobulin production by peripheral blood B cells or surface IgE+ B cells from patients in the SCG and BD groups were assessed.nnnRESULTSnBoth SCG and BD treatment remarkably improved eczema. However, although SCG treatment decreased spontaneous IgE production by B cells without affecting production of IgG, IgM, or IgA, BD treatment selectively increased spontaneous IgE production. SCG treatment also decreased IgE production by surface IgE+ B cells, whereas BD treatment increased it.nnnCONCLUSIONnTopical steroid treatment increases in vitro spontaneous IgE production by B cells. This indicated that topical steroids may decrease inflammation; however, a large-scale study on the effect of topical steroids on IgE production in vitro and in vivo may be necessary.

Recognition of regional hypertrophy in hypertrophic cardiomyopathy using thallium-201 emission-computed tomography: Comparison with two-dimensional echocardiography☆

The configuration of the hypertrophied myocardium was evaluated by thallium-201 emission-computed tomography and 2-dimensional (2-D) sector scan in 10 patients with obstructive hypertrophic cardiomyopathy (HC), 10 with nonobstructive HC with giant negative T waves and 10 with concentric left ventricular (LV) hypertrophy. Thallium-201 myocardial imaging was reconstructed into multiple 12-mm-thick slices in 3 planes. The thickness ratio of the ventricular septum and the LV posterior wall in the short-axis plane and the ratio of the ventricular septum and the apical wall in the long-axis plane were analyzed. In the patients with obstructive HC the ventricular septal wall thickness index was increased, and the ratio of septal to posterior wall thickness index (1.45 +/- 0.23) was greater than that in the patients with nonobstructive HC with giant negative T waves or in those with concentric LV hypertrophy (1.03 +/- 0.20 and 0.98 +/- 0.11, respectively; p less than 0.01 for each). In the patients with nonobstructive HC with giant negative T waves, increased apical wall thickness with apical cavity obliteration was characteristic, and the ratio of ventricular septal to apical wall thickness index (0.66 +/- 0.14) was less than that in the patients with obstructive HC or in those with concentric LV hypertrophy (1.46 +/- 0.38 and 1.04 +/- 0.09, respectively; p less than 0.001 for each). In contrast, technically satisfactory 2-D sector scanning (83%) demonstrated various configurations of the hypertrophied ventricularseptum, but could not detect apical hypertrophy in 4 of the 10 patients with nonobstructive HC with giant negative T waves whose LV cineangiograms demonstrated apical hypertrophy. Thus, thallium-201 emission-computed tomography is useful in evaluating the characteristics of LV hypertrophy and assists 2-D sector scan, especially in patients with apical hypertrophy in HC.