Genetic heterogeneity of betanodaviruses in juvenile production trials of Pacific bluefin tuna, Thunnus orientalis (Temminck & Schlegel).

Pacific bluefin tuna, Thunnus orientalis (Temminck & Schlegel), is one of the most important commercially exploited fish species in the world, and juvenile production techniques have been developed for its culture and stock enhancement in Japan. However, recent juvenile production has often failed because of the occurrence of viral nervous necrosis caused by betanodaviruses. In this study, we examined the genetic variability of betanodaviruses detected in the diseased juveniles to understand the transmission of the disease in a tuna hatchery. A total of 94 nucleotide sequences of betanodavirus (partial sequence of the coat protein gene, RNA2) were obtained from fish samples by reverse-transcriptase polymerase chain reaction amplification and 13 haplotypes were recognized among the sequences. The haplotype distributions in the viral populations from the diseased juveniles were related to the broodstocks from which the juveniles originated, suggesting that vertical transmission had occurred in the hatchery. The statistical parsimony network of viral haplotypes suggests that the nucleotide substitutions among the samples were accumulated in a recent population growth.

PCR-based detection of betanodaviruses from cultured and wild marine fish with no clinical signs.

Betanodaviruses are the causative agents of viral nervous necrosis (VNN) or viral encephalopathy and retinopathy (VER) in cultured marine fish. A total of 131 apparently healthy fish from 30 species were collected in two geographically remote aquaculture areas, Yashima Bay (Kagawa Prefecture) and Tamanoura Bay (Nagasaki Prefecture), in Japan. The brains of fish were examined by reverse transcriptase-polymerase chain reaction (RT-PCR) and nested PCR to detect the coat protein gene of betanodavirus. In Yashima Bay, two and 13 of 20 cultured fish were positive for nodavirus in RT-PCR and nested PCR, respectively, and four of five wild fish were positive only in nested PCR. In Tamanoura Bay, 28 and 99 of 106 wild fish were positive for the virus in RT-PCR and nested PCR, respectively. All the sequences of the nested PCR products (177 nt) from 27 fish species (10 cultured and 17 wild) were highly homologous to each other (99-100%) and were closely related to that of the known betanodavirus, redspotted grouper nervous necrosis virus (RGNNV). These results illustrate that large populations of cultured and wild marine fish in aquaculture areas are subclinically infected with genetically closely related betanodaviruses, suggesting an importance of such infected fish as a carrier or reservoir of betanodaviruses.

The C terminus of brome mosaic virus coat protein controls viral cell-to-cell and long-distance movement.

To investigate the functional domains of the coat protein (CP; 189 amino acids) of Brome mosaic virus, a plant RNA virus, 19 alanine-scanning mutants were constructed and tested for their infectivity in barley and Nicotiana benthamiana. Despite its apparent normal replicative competence and CP production, the C-terminal mutant F184A produced no virions. Furthermore, virion-forming C-terminal mutants P178A and D182A failed to move from cell to cell in both plant species, and mutants D181A and V187A showed host-specific movement. These results indicate that the C-terminal region of CP plays some important roles in virus movement and encapsidation. The specificity of certain mutations for viral movement in two different plant species is evidence for the involvement of host-specific factors.

Orbital blowout fracture with persistent mobility deficit due to fibrosis of the inferior rectus muscle and perimuscular tissue.

A case of orbital blowout fracture accompanied by fibrosis of the inferior rectus muscle resulting in an irreversible orbital mobility deficit is reported. An 8-year-old girl with an orbital blowout fracture was treated with steroids for 10 days, as with other cases in our department. She exhibited a disturbance of vertical eye movement and a positive forced duction test result. Although surgery was performed on day 13, and on day 27 due to poor recovery after the first operation, almost no improvement of the ocular movement was noted. The results of a traction test, performed during the second operation, suggested that the inferior rectus muscle had adhered to the periosteum. Magnetic resonance imaging performed 3 days after the second operation revealed fibrosis of the inferior rectus muscle and perimuscular tissue, resulting in an irreversible disturbance of the vertical ocular movement. The present findings suggest that the need for and timing of surgery in patients with blowout fractures should be determined on an individual basis.

Expression of chimeric P450 genes encoding flavonoid-3', 5'-hydroxylase in transgenic tobacco and petunia plants(1).

Flavonoid-3',5'-hydroxylase (F3'5'H), a member of the cytochrome P450 family, is the key enzyme in the synthesis of 3', 5'-hydroxylated anthocyanins, which are generally required for blue or purple flowers. A full-length cDNA, TG1, was isolated from prairie gentian by heterologous hybridization with a petunia cDNA, AK14, which encodes F3'5'H. To investigate the in vivo function of TG1 and AK14, they were subcloned into a plant expression vector and expressed under the control of the CaMV35S promoter in transgenic tobacco or petunia, both of which originally lack the enzyme. Transgenic petunia plants had a dramatic change in flower color from pink to magenta with a high content of 3',5'-hydroxylated anthocyanins. In contrast, transgenic tobacco plants had minimal color change with at most 35% 3',5'-hydroxylated anthocyanin content. These results indicate that the products of TG1 and AK14 have F3'5'H activity in planta and that interspecific gene transfer alters anthocyanin pigment synthesis. The difference in apparent F3'5'H activity between tobacco and petunia is discussed.

Detection of activated eosinophils in nasal polyps of an aspirin-induced asthma patient.

Aspirin-induced asthma (AIA) is frequently accompanied by nasal polyps. Eosinophil infiltration is a characteristic feature of nasal polyps associated with AIA. Even though steroids are well known to be effective on managing AIA and its nasal polyps, histochemical examinations after steroid therapy and at recurrence, involving eosinophil infiltration of nasal polyps, have been less studied. To know the histochemical effects of steroid treatment on eosinophil accumulation in nasal polyps of AIA and the histochemical feature of a recurring polyp and to detect distributional differences between storage and secreted forms of eosinophil cationic proteins, we carried out immunocytochemical labelling with antibodies against EGI (recognizing resting and activated eosinophils) and EG2 (recognizing only activated eosinophils), and determined eosinophil infiltration in nasal polyps that were obtained before and after steroid treatment, and at recurrence of polyps. A large number of eosinophils in AIA polyps were found before steroid treatment and at recurrence, and they were predominantly composed of activated eosinophils (EG2-positive). In contrast, eosinophil infiltration was rare in polyps obtained immediately after steroid treatment. This finding suggests that eosinophil infiltration may be associated with nasal polyp formation in AIA, and that activation of eosinophils plays an important role in accumulation of eosinophils and polyp formation beginning with the initial stage.

Isolated mucocele in an Onodi cell.

A rare case of retrobulbar optic neuropathy caused by an isolated mucocele in an Onodi cell is presented. A 63-year-old man suffering from left recurrent optic neuropathy had shown improved visual acuity with steroid treatment at the age of 48 and 56 years. Impaired visual acuity and blurred vision in the left eye appeared again, and a marked inferior decrease in the left visual field was noted. Although axial computed tomography (CT) showed no abnormal findings, magnetic resonance imaging (MRI) revealed a small squamous-shaped lesion (17 x 17 x 7.3 mm) in the left posterior ethmoidal air cell (Onodi cell) hanging over the left optic nerve. Endoscopic sinus surgery was performed. The optic canal was extruding into the Onodi cell cavity, and the superior bony wall of the canal was eroded by the mucocele. The isolated mucocele in the Onodi cell seemed to be compressing the optic nerve downward an causing retrobulbar optic neuropathy. An Onodi cell lesion, even if it was isolated and small, would cause optic neuropathy. MRI and coronal CT scanning should be performed for the diagnosis and preoperative planning in a case of retrobulbar optic neuropathy.

Characterization of a 34-kDa soybean binding protein for the syringolide elicitors.

Syringolides are water-soluble, low-molecular-weight elicitors that trigger defense responses in soybean cultivars carrying the Rpg4 disease-resistance gene but not in rpg4 cultivars. 125I-syringolide 1 previously was shown to bind to a soluble protein(s) in extracts from soybean leaves. A 34-kDa protein that accounted for 125I-syringolide 1 binding activity was isolated with a syringolide affinity-gel column. Partial sequences of internal peptides of the 34-kDa protein were identical to P34, a previously described soybean seed allergen. In soybean seeds, P34 is processed from a 46-kDa precursor protein and was shown to have homology with thiol proteases. P34 is a moderately abundant protein in soybean seeds and cotyledons but its level in leaves is low. cDNAs encoding 46-, 34-, and 32-kDa forms of the soybean protein were cloned into the baculovirus vector, pVL1392, and expressed in insect cells. The resulting 32- and 34-kDa proteins, but not the 46-kDa protein, exhibited ligand-specific 125I-syringolide binding activity. These results suggest that P34 may be the receptor that mediates syringolide signaling.

Specific Binding of the Syringolide Elicitors to a Soluble Protein Fraction from Soybean Leaves.

Syringolides are glycolipid elicitors produced by Gram-negative bacteria expressing Pseudomonas syringae avirulence gene D. The syringolides mediate gene-for-gene complementarity, inducing the hypersensitive response only in soybean plants carrying the Rpg4 disease resistance gene. A site(s) for 125I-syringolide 1 was detected in the soluble protein fraction from soybean leaves, but no evidence for ligand-specific binding to the microsomal fraction was obtained. The Kd value for syringolide 1 binding with the soluble fraction was 8.7 nM, and binding was greatly reduced by prior protease treatment or heating. A native gel assay was also used to demonstrate ligand-specific binding of labeled syringolide 1 with a soluble protein(s). Competition studies with 125I-syringolide 1 and several structural derivatives demonstrated a direct correlation between binding affinity to the soluble fraction and elicitor activity. However, differential competition binding studies disclosed no differences in syringolide binding to soluble fractions from Rpg4/Rpg4 or rpg4/rpg4 soybean leaves. Thus, the observed binding site fulfills several criteria expected of an intracellular receptor for the syringolides, but it is most likely not encoded by the Rpg4 gene. Instead, the Rpg4 gene product may function subsequent to elicitor binding, possibly in intracellular signal transduction.

A structural model for the mechanisms of elicitor release from fungal cell walls by plant beta-1,3-endoglucanase.

The release of elicitor-active carbohydrates from fungal cell walls by beta-1,3-endoglucanase contained in host tissues has been implicated as one of the earliest processes in the interaction between soybean (Glycine max) and the fungal pathogen Phytophthora megasperma f. sp. glycinea leading to host defense responses such as phytoalexin production. The present study was conducted to evaluate the primary structure of the glucanase-released elicitor (RE). Gel-filtration chromatography of carbohydrates released from mycelial walls by purified soybean beta-1,3-endoglucanase resolved them into the four fractions (elicitor-active RE-I, -II, and -III and elicitor-inactive RE-IV). Sugar composition analysis indicated that all of the fractions were composed almost entirely of glucose. 1H- and 13C-nuclear magnetic resonance analysis indicated the presence of both beta-1,3- and beta-1,6-linkages for the elicitor-active RE-I, -II, and -III fractions and only beta-1,3 linkage for the elicitor-inactive RE-IV fraction. Methylation analysis and degradation studies employing beta-1,3-endo- and beta-1,3-exoglucanase further suggested that the basic structure of elicitor-active RE consists of beta-1,6-linked glucan backbone chains of various lengths with frequent side branches composed of beta-1,3-linked one or two glucose moieties. From these structural analyses of RE, a structural model of how RE is originally present in fungal cell walls and released by host beta-1,3-endoglucanase is also proposed.

A case of cerebellar infarction occurred with the 8th cranial nerve symptoms.

A rare case, 32-year-old man, of cerebellar infarction with the occurrence of the 8th cranial nerve symptoms was reported. On the neuro-otological examination, hearing test and caloric test showed a severe hearing loss and no response on the right side, respectively, and the spontaneous horizontal nystagmus fixed to the left direction was observed. Magnetic resonance (MR) imaging showed the infarctions in the areas of anterior inferior cerebellar artery (AICA) and posterior inferior cerebellar artery (PICA) on the right side. On the 14th illness day, the 8th cranial nerve symptoms disappeared, and on the 12th illness day, right hearing level and caloric response were significantly improved. We suggested that such an early recovery of the subjective symptoms and neuro-otological findings may be attributable to the recanalized circulation disturbance or the development of collateral circulation.

Progress of caloric response of vestibular neuronitis.

Progress of caloric response and subjective symptoms of 60 patients with vestibular neuronitis was evaluated by a long term follow-up study. Normalization of caloric responses was confirmed in 25 (41.7%) out of 60 patients, 20 of whom had recovered within 2 years of the onset of vertigo. The rate of the patients with canal paresis was about 90% after 1 month of the onset, and 80% after 6 months, while 50% of them still showed canal paresis after 5 or 10 years had passed. Complete relief from subjective symptoms was recognized in 34 (56.7%) cases during the follow-up period. We conclude that the prognosis of vestibular neuronitis is not always good, because vestibular function did not recover within normal levels in about half of the patients in spite of complete relief from subjective symptoms in many of them.

Cell culture study of the vestibular ganglion cells. Morphology and immunohistochemical activity.

Incubation of vestibular ganglion cells from the rat fetus and chick embryos was successfully done demonstrating bipolar cells and two types of multipolar cells, small round cells and large cells, in the cell cultures produced. Vestibular ganglion cells were found to be highly irregular in size. Furthermore, the presence of neurotransmitters (choline acetyltransferase and substance P) was confirmed immunohistochemically. Substance P positive cells had many bipolar cells and some multipolar cells. However, choline acetyl transferase positive cells had some small multipolar cells but few bipolar cells. These findings suggest that all vestibular ganglion cells do not have the same function.

Serovirological study of vestibular neuronitis.

A serovirological study to clarify the pathogenesis of vestibular neuronitis was made on 44 patients. The diagnosis of vestibular neuronitis was made under the diagnostic criteria. Sera from all 44 cases were collected twice or more at defined intervals. Of these, 36 cases were treated as paired sera. Seventeen out of 36 paired cases showed significant change in serum viral antibody titer (HSV, 2 cases; CMV, 1 case; EBV, 7 cases; rubella, 2 cases; adeno., 2 cases; influ. A, 1 case; influ. B, 2 cases). It was assumed that infection caused by these detected viruses played an important role in the onset of vertigo in each case.

Mucoepidermoid carcinoma of the vocal cord. Report of a case.

A case of mucoepidermoid carcinoma of the vocal cord is described in a 51-year-old man who presented with a thickened right vocal cord at the midportion without any limitation of movement. Histological examination revealed a mucoepidermoid carcinoma of low-grade malignancy. Partial laryngectomy was performed, but 10 months after the operation a squamous cell carcinoma developed on the other vocal cord. Primary vocal cord involvement of this tumor is rare. The literature is reviewed.