Identification of epitopes associated with different biological activities on the glycoprotein of vesicular stomatitis virus by use of monoclonal antibodies

Summary Thirteen monoclonal antibodies (MAbs) to the glycoprotein (G) of vesicular stomatitis virus (VSV) serotype Indiana were prepared and examined for their effects on various biological activities of VSV, including in vitro infection, hemagglutination, adsorption to cells, and mediation of cell fusion. Competitive binding assays with these MAbs revealed the presence of at least seven distinct antigenic determinants (epitopes) on the G protein. In some cases, overlappings among epitopes to various degrees were observed as partial inhibition or binding enhancement. The MAbs to all the epitopes but one (epitopes 1–6) reacted with the denatured G protein in a Western immunoblot analysis. Four of the epitopes (epitopes 2, 4, 5, and 7) were involved in neutralization and two (epitopes 1 and 2) in hemagglutination inhibition. None of the MAbs inhibited the adsorption of radiolabeled VSV to BHK-21 cells; the MAbs to epitope 2 slightly enhanced the virus adsorption. All neutralization epitopes except epitope 2 (epitopes 4, 5, and 7) were associated with inhibition of VSV-mediated cell fusion. These results show a direct spatial relationship between the epitopes recognized by the MAbs and functional sites on G protein and further insights into the structure and function of G protein.     

Behçet Disease and the HLA System

Seventy-three unrelated patients with Behçet disease together with 33 members of seven families with at least two patients per family were tissue typed for 26 antigens of the HLA system. The patients with the complete type of Behçet disease were found to have HLA-B5 more significanth than healthy individuals. Family studies suggest that the genes closely linked to HLA locus influence the degree of severity of Behçet disease.     

Developmental changes in the fine structure and histochemical properties of mucous cells in the parotid gland of the infant Japanese macaque

Mucous cells have been known to occur in the terminal portions of the parotid gland in a few species of mammals during a limited period of their development. The aim of this study was to examine the occurrence and features of mucous cells in the parotid gland of the infant Japanese macaque. Light microscopy revealed that mucous cells in the macaque parotid gland were present in the terminal clusters and acini at postnatal day 15, were less prevalent at day 30, and continued to decrease in number over 3 months. Mucous cells were no longer recognized in the parotid gland in 6-month-old macaques. Electron microscopy showed that the mucous cells contained electron-lucent secretory granules and bipartite or tripartite secretory granules. By 3 months of age, there was a scarcity of mucous cells and a concomitant increase in transitional cells. These transitional cells were intermediate in structure between mucous and serous cells, and contained three types of granules: electron-lucent, bipartite or tripartite, and electron-dense. None of the cells showed apoptotic figures. Lectin histochemistry indicated that the mucous cells in the early postnatal period had sugar residues identical in nature to those seen in the granules from mature serous cells in the glands of 3-month-old macaques. Immunohistochemistry using an antibody against human alpha-amylase showed a weakly positive reactivity in the secretory granules of the mucous cells, starting from day 15. In the transitional cells, the electron-dense granules showed a stronger immunoreactivity than either the electron-lucent granules or the heterogeneously structured granules. These results suggest that the secretory granules of mucous cells have characteristics in common with those of serous cells, and that during the transitional period the mucous granules change from the initial electron-lucent to hetorogenous forms, finally becoming the electron-dense granules. The mucous cells in the parotid gland of the juvenile Japanese macaque are therefore suggested to be converted into serous cells.     

Ophthalmomyiasis caused by Sarcophaga crassipalpis (Diptera: Sarcophagidae) in a hospital patient

Nine sarcophagid larvae were found on the right eyelid, cornea, and bulbar conjunctiva of a debilitated patient in a hospital in Osaka, Japan. Inflammation of the right eyelid and conjunctival congestion, probably initiated or aggravated by the larvae, were found. The larvae were removed and reared for accurate identification, and, on the basis of the characteristics of the 3rd instar and adult flies, the species was identified as Sarcophaga crassipalpis Macquart. This is a report of ophthalmomyiasis caused by this facultative parasite in a human. Patients with diminished consciousness in hospitals need protection from flies.     

A promiscuous T cell hybridoma restricted to various I-A molecules

In a previous study, we identified T cell receptor and major histocompatibility complex (MHC) contact sites on the pigeon cytochrome c p43-58 peptide. Positions 46 and 54 of p43-58 were shown to be the MHC-binding sites. Specific amino acids were identified on the MHC-binding sites which bound to the relevant I-A molecule. In the present study, using NOD (I-A^g7) mice, we established a T cell hybridoma, NOE33-1-2, specific for a p43-58 analog 46R50E54A with arginine (R) and alanine (A) at positions 46 and 54, respectively. Interestingly, NOE 33-1-2 recognized 46R50E54A in the presence of not only I-A^g7, but also I-A^{d, s, u and v}. In contrast to previous reports that promiscuous T cells were able to recognize peptide antigens with various HLA-DR or I-E molecules consist of monomorphic α and polymorphic β chains, the promiscuous T cell clone NOE33-1-2 recognized peptides with various I-A molecules lacking the monomorphic chain.     

A middle meningeal artery which arises from the internal carotid artery in which the first branchial artery participates.

A middle meningeal artery arising from the internal carotid artery was found in the right half of the head of an 85-year-old male cadaver during student dissection practice. It arose from the lateral aspect of the internal carotid artery in the carotid canal, arrived at the foramen lacerum after running forward. It then ran backward under the trigeminal ganglion and took the usual course after passing its posterior margin. On one hand, the maxillary artery did not issue the middle meningeal artery, gave off only a small twig supplying the lateral pterygoid muscle at the corresponding position. It was corroborated by the fact that the foramen spinosum was absent in this example. During usual development, the middle meningeal artery primarily springs from the supraorbital branch of the stapedial artery that arises from the dorsal part of the second branchial artery. Later, by the formation of the external carotid artery connecting with the common trunk of the infraorbital and mandibular branches (maxillomandibular division) of the stapedial artery and by the atrophy of the proximal part of it, the middle meningeal artery is finally supplied by the external carotid artery. But in this example, it is supposed that the middle meningeal artery arose from a more distal position of the internal carotid artery owing to the persistence of the anastomosis between the dorsal part of the first branchial artery and the supraorbital branch and the interruption of the connection between the supraorbital branch and maxillomandibular division of the stapedial artery.     

Measurement of plasma von Willebrand factor cleaving protease in patients with varied thrombotic microangiopathy

Thrombotic thrombocytopenic purpura(TTP) is a multisystem disorders characterized by thrombocytopenia, microangiopathic hemolytic anemia associated with red cell fragmentation, and neurological and renal symptoms. Plasma of patients with TTP has been shown to contain unusually large von Willebrand factor(vWF) multimers that may cause platelet agglutination in vivo. Recently, a metalloprotease responsible for cleavage of vWF multimers has been isolated from normal human plasma and was found to be deficient in some patients with TTP. We examined the activity of the vWF-cleaving protease(vWF-CP), by modified Furlan's method, in plasma from patients with a familial TTP, 3 acquired TTP, 4 thrombotic microangiopathy(TMA) and 2 veno-occlusive disease(VOD) associated after allo-BMT. Diluted plasma samples of patients were incubated with protease-free vWF purified from normal human plasma, in the presence of urea and barium ions. The extent of vWF degradation was assayed by electrophoresis in SDS-agarose gels and immunoblotting. Activity of vWF-CP from 12 normal plasma have been shown as 77-180%(average 115%), whereas, no vWF-CP(below 5%) was observed in plasma from familial TTP, before and after plasma exchange, although FFP infusion therapy has been effective for this patient to recover thrombocytopenia. In 3 acquired TTP, 2 patients showed lack of vWF-CP activity in plasma, and inhibitors against vWF-CP have been elucidated by plasma cross-mixing test. After extensive plasma exchange and FFP infusion followed by corticosteroid therapy, normal vWF-CP was recovered in plasma from 2 acquired TTP patients. Among BMT patients, plasma from 4 BMT-TMA showed normal vWF-CP activities as 55-111%, whereas plasma from 2 BMT-VOD revealed low vWF-CP activity, as 24% and 37%, respectively. Thus, measurement of vWF-CP is crucial to predict differentiation of primary forms of TMA to establish the pathogenesis in varied endothelial dysfunction.     

Induction of hRAD9 is required for G2/M checkpoint signal transduction in gastric cancer cells.

DNA damage triggers the activation of checkpoints that delay cell cycle progression to allow for DNA repair. Loss of G2 checkpoints provides a growth advantage for tumor cells undergoing aberrant mitosis. However, the precise mechanisms of G2 checkpoints acting in gastric cancer are unknown. Here, we analyzed the G2 checkpoint function in two gastric cancer cells, MKN-28 cells containing a mutant p53 gene and MKN-45 cells which have wild-type p53. Two agents damaging DNA, camptothecin (CPT) or ultraviolet light (UV), were utilized to trigger a G2 phase cell cycle checkpoint response in these tumor cells. Both CPT and UV inhibited the growth of MKN-45 cells, whereas they did not affect the growth of MKN-28 cells. CPT induced cell cycle arrest at the G2/M phase and enhanced the expression of human RAD9 (hRAD9) in MKN-45 cells. In addition, hRAD9 showed perinuclear staining and similar localization with Bcl-2 in MKN-45 cells but not in MKN-28 cells after having applied CPT or UV light. These results suggest that besides p53 activity, the induction of hRAD9 is required for G2/M checkpoint signal transduction in gastric cancer cells.