Detection of antigenic proteins expressed by lymphocystis virus as vaccine candidates in olive flounder, Paralichthys olivaceus (Temminck & Schlegel).

Although the major capsid proteins (MCPs) of lymphocystis disease virus (LCDV) have been characterized, little is known about the host-derived immune response to MCPs and other LCDV antigenic proteins. To identify antigenic proteins of LCDV that could be used as vaccine candidates in olive flounder, Paralichthys olivaceus, we analysed the viral proteins responsible for its virulence by applying immuno-proteomics. LCDV proteins were separated by one-dimensional gel electrophoresis, transferred to polyvinylidene difluoride membrane, and probed with homogeneous P. olivaceus antisera elicited by LCDV natural infection and vaccination with formalin-killed LCDV. Four immune-reactive proteins were obtained at 68-, 51-, 41- and 21 kDa using antisera collected from natural infection while two proteins at 51- and 21 kDa exhibited response to antisera from vaccinated fish, indicating that the latter two proteins have vaccine potential. Using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and nanoelectrospray MS/MS, the 51 and 21 kDa proteins were identified as MCP and an unknown protein, respectively.

GRAB: a physiologic guanine nucleotide exchange factor for Rab3A, which interacts with inositol hexakisphosphate kinase.

Diphosphoinositol-pentakisphosphate (InsP7) and bis-diphosphoinositol tetrakisphosphate (InsP8) possess pyrophosphate bonds. InsP7 is formed from inositol hexakisphosphate (InsP6) by recently identified InsP6 kinases designated InsP6K1 and InsP6K2. We now report the identification, cloning, and characterization of a novel protein, GRAB (guanine nucleotide exchange factor for Rab3A), which interacts with both InsP6K1 and Rab3A, a Ras-like GTPase that regulates synaptic vesicle exocytosis. GRAB is a physiologic GEF (guanine nucleotide exchange factor) for Rab3A. Consistent with a role of Rab3A in synaptic vesicle exocytosis, GRAB regulates depolarization-induced release of dopamine from PC12 cells and nicotinic agonist-induced hGH release from bovine adrenal chromaffin cells. The association of InsP6K1 with GRAB fits with a role for InsP7 in vesicle exocytosis.

Immunoproteomic analysis of capsulate and non-capsulate strains of Lactococcus garvieae.

A comparative immunoproteomic study was carried out to investigate the immunogenicity of capsulate (KG9408) and non-capsulate (NSS9310) strains of Lactococcus garvieae. Immunoblot assays, following two-dimensional gel electrophoresis (2-DE) for L. garvieae strains, revealed a significant difference between anti-capsulate and anti-non-capsulate rabbit sera with respect to the number and antigenicity of antigenic spots. Anti-capsulate and anti-non-capsulate rabbit sera reacted with an average of 72 and 127 antigenic spots, respectively. The strong reaction of anti-non-capsulate sera with elongation factor (EF)-G and -Tu, and GMP synthase, of the L. garvieae strains identifies these as specific major antigens. This study clearly demonstrates the differences in 2-DE immunoblot profiles between the capsulate and non-capsulate strains of L. garvieae. These differences may be the reason for variations in immunogenicity between capsulate and non-capsulate strains. Glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, arginine deaminase and ornithine carbamoyltransferase were identified from the 2-DE immunoblot profiles of both strains. Therefore, these common antigens are potential markers for the development of vaccines against L. garvieae, irrespective of strain. Immunoproteomics, a powerful tool for studying antigens at the proteomic level, allowed a comparative investigation of the immunogenicity of capsulate and non-capsulate strains of L. garvieae for vaccine development.

Synergistic effects of combination sequential immunotherapies in a murine ovarian cancer model.

The antitumor effects of Corynebacterium parvum in a murine ovarian teratocarcinoma model depend upon a sequential activation of neutrophils and macrophages within the peritoneal cavity. We studied the sequential administration of biological response modifiers that independently activate each phase of the response. Tumor-challenged mice treated by i.p. injection of a pyridine-extracted fraction of cell-free Propionibacterium acnes (PA-PE, 1400 micrograms) demonstrated prolonged survival in less than 20% of the cases. An i.p. injection of a detoxified Salmonella endotoxin (DSE) preparation (150 micrograms) had no effect on tumor outgrowth. However, i.p. treatment with PA-PE (1400 micrograms), followed by 150 micrograms of DSE 1 day later, resulted in long-term survival (greater than 100 days) in 40 to 60% of mice. This antitumor effect was only evident when PA-PE was administered first (before DSE) and optimal when DSE was administered 24 h after PA-PE. The synergistic antitumor effect could be duplicated when tumor-challenged mice were first treated i.p. with peritoneal polymorphonuclear leukocytes, elicited by injection of PA-PE, and then treated with DSE 18 h later. These data indicate that appropriately timed injection of biological response modifiers with complementary effects can result in a synergistic prevention of tumor growth.

Intra-carotid thrombolytic therapy in acute ischemic stroke of carotid arterial territory.

Intra-carotid urokinase (UK) infusion in 20 patients with acute internal carotid artery (ICA) territorial ischemic stroke achieved immediate recanalization in 45% and the clinical outcome in patients with recanalization was superior to that of patients without recanalization. The procedure was most effective in patients with smaller arterial occlusions: 7 of 10 patients with MCA branch occlusions (M2 to M4) achieved recanalization compared to only 2 of 10 with distal ICA or M1 occlusions, which should be an important issue for the critical evaluation of the efficacy of thrombolytic therapy (TT). Hemorrhagic transformation was observed in 9 patients on CT scan; petechial hemorrhage in 5 and intraparenchymal hematoma formation in 4. Among 4 patients with hematoma formation, clinical deterioration was seen in 3 cases and the angiography at the immediate end of the UK infusion showed recanalization in only one patient. The average dose of UK in patients with parenchymal hematoma formation was higher than that of patients without hemorrhagic transformation (123.3 x 10(4) units vs 101 x 10(4) units). The administration of a large dose of UK, probably more than 100 x 10(4) units, and the absence of immediate recanalization seemed to increase the risk of parenchymal hematoma formation. Despite the effort of investigators, the in-hospital time delay for the TT was significant which was mainly related to the time consuming preparation for angiography especially during night. A more effective system for the earlier intervention of acute ischemic stroke needs to be developed.

Transient thyrotoxicosis from thyroiditis induced by sibutramine overdose: a case report.

Sibutramine is an antiobesity drug that inhibits the reuptake of serotonin and noradrenalin in the hypothalamus. A 37-year-old Korean man presented to the emergency room for the oral intake of 280 mg of sibutramine. The patient was in thyrotoxic state. The (99m)Technetium-pertechnetate thyroid scan showed irregular uptake of radioisotope and thyroid-stimulating hormone receptor antibody and thyroperoxidase antibody were negative. Thyroid function normalized after that. The patient had transient thyrotoxicosis with thyroiditis. We report a case of thyrotoxicosis accompanied by thyroiditis resulting from the intentional overdose of sibutramine.

Expression and serological application of a capsid protein of an iridovirus isolated from rock bream, Oplegnathus fasciatus (Temminck & Schlegel).

Iridoviruses infect a wide variety of wild and cultured fish. Those iridoviruses belonging to the genus Ranavirus, in the Iridoviridae family, cause systemic disease in infected animals with a high morbidity and mortality. This paper reports the cloning, sequencing, and expression of the rock bream iridovirus (RBIV) major capsid protein (MCP) in an Escherichia coli expression system for subsequent immunological studies. The completeness of the expressed protein was confirmed by peptide mass fingerprinting (PMF) analysis using MALDI-TOF MS. The recombinant MCP (rMCP)-specific mouse polyclonal antibody reacted with the viral 52 kDa protein, indicating that this rMCP induces an immunological response. Fish antibodies induced against iridovirus infection were also detected using ELISA when rMCP was used as an antigen. As a result, it was found that many cultured rock bream (92.5%) were naturally infected with iridovirus and that the rMCP might be useful for serological tests.

Fermented mushroom milk-supplemented dietary fibre prevents the onset of obesity and hypertriglyceridaemia in Otsuka Long-Evans Tokushima fatty rats.

AIM: Fermented milk product containing edible mushroom water extracts (mushroom yogurt; MY) has been reported to have glycaemic control and triglyceride-lowering effects in streptozotocin (STZ)-induced diabetic rats and Zucker diabetic fatty (ZDF) rats. Here, we investigated how MY-supplemented dietary fibre (10 and 20%, v/w) influences the onset of obesity and hypertriglyceridaemia in Otsuka Long-Evans Tokushima fatty (OLETF) rats. METHODS: The OLETF rats were fed a powdered chow diet supplemented with MY at the levels of 10 (v/w) and 20% for 6 weeks from 10 weeks of age, but the OLETF control rats were not supplemented. Their weight, fat distribution and lipid profile have been determined. RESULTS: The body weights in MY-fed rats were reduced compared with the control rats. The perirenal fat was decreased in both MY groups, but the visceral and epididymal fats reduced only in the MY 20% group. The concentrations of serum triglyceride and non-esterified fatty acid in MY-fed rats were decreased in a dose-dependent manner. However, the levels of other serum lipid profiles [total cholesterol, low-density lipoprotein cholesterol, and high-density lipoprotein cholesterol] were comparable among all rats. CONCLUSION: Anti-obesity and triglyceride lowering by MY-supplemented dietary fibre in OLETF rats might have resulted from the synergistic effect of components in the fermented mushroom-milk product.

Computed tomographic correlation with pituitary function in Sheehan's syndrome.

Twenty six patients with Sheehan's syndrome were studied with high-resolution computed tomography (CT) and the sequential pituitary stimulation test in order to correlate the CT findings of the sella turcica with the pituitary reserve functions. CT revealed 21 completely empty sella (CES), 4 partially empty sella (PES) and 1 normal sella. Panhypopituitarism occurred in 1 of 4 patients with PES and 20 of 21 with CES. One patient showing normal sella had a normal preservation of prolactin (PRL), thyroid stimulating hormone (TSH), follicle stimulating hormone (FSH) and lutenizing hormone (LH). In all patients with PES and CES, growth hormone (GH) responses to hypoglycemia and PRL responses to thyrotropin releasing hormone (TRH) were blunted. Three (75.0%) with PES had normal basal cortisol levels, which were more frequent than two (9.6%) with CES; however, most of the PES (3 of 4) and CES (20 of 21) demonstrated blunted cortisol responses to hypoglycemia. Three (75.0%) with PES and only one (4.8%) with CES had normal thyroxine levels and TSH responses to TRH. None with PES showed decreased basal and stimulated levels of FSH and LH, whereas 15 of 21 with CES did. The pituitary functions of the patients having considerable amounts of pituitary remnants visualized by CT were relatively preserved for TSH, cortisol, FSH and LH. Considering the above results, changes in the amounts of pituitary remnants detected by CT might correlate with hormonal secretory capacity.