Rhodnius prolixus, a blood-sucking bug, is a unique insect that is known to produce nitric oxide (NO) in the salivary glands to use as a vasodilator for blood sucking. We report here the cloning of the NO synthase (NOS) cDNA from these salivary glands and its expression in a baculovirus system. This cDNA encodes a protein of 1174 amino acids with a calculated molecular mass of 132331 Da. The primary structures of mammalian NOS, including the putative cofactor-recognition sites for heme, tetrahydrobiopterin (BH,), calmodulin, FMN, FAD and NADPH are all conserved in salivary-gland NOS. Recombinant salivary-gland NOS differed from nerve NOS and endothelial NOS in that it lacked a large N-terminal domain and an N-terminal myristylation sequence, respectively. Salivary-gland NOS produced in a baculovirus system showed NOS activity and demonstrated that salivary-gland NOS was soluble and was Ca2+ and calmodulin dependent, similarly to mammalian constitutive NOS isoforms. Recombinant salivary-gland NOS was purified to near homogeneity and migrated at 130 kDa on SDS/PAGE.Keywords : insect; salivary gland ; nitric-oxide synthase; CaZ+ and calmodulin dependence; baculovirus.NO is produced in various mammalian tissues where it plays physiologically important roles [I]. NO acts as neurotransmitter in the peripheral and the central nervous systems and is involved in brain development, memory formation and learning. In the circulatory system, NO is produced in endothelial cells, and acts as a regulator of vascular tone and platelet aggregation. NO is induced in macrophages, neutrophils and liver cells, and acts as an important effector of the cell-mediated immune system. The enzyme critical for NO production, NO synthase (NOS), is found in these different tissues, and various isofoms have been purified and characterized, and some have been cloned [2, 31. Mammalian NOS have been grouped into two general categories: constitutive (Ca*' kalmodulin dependent, e.g. endothelial NOS and neuronal NOS) and inducible (Ca"/calmodulin independent, e.g. macrophage) types.By contrast, in invertebrates, the production and utilization of NO is not as well understood, although similar physiological functions are probably present. In some insects and molluscs, the existence of NOS in brain has been demonstrated, and the function of NO as a neurotransmitter was suggested [4, 51. In the silkworm, Bombyx mori, NOS activity in the fat body was induced by lipopolysaccharide, an agent that mimics bacterial assault, which suggested the involvement of NO in the insect immune defense system [ 3 ] . This activity required Ca2+, calmodulin, NADPH, FAD, FMN and tetrahydrobiopterin (BH,), similarly to mammalian NOS. Correspondence to M . Yuda, Department of Medical Zoology,Fux: f 8 l 592 31 5215. Abhreviufions. NO, nitric oxide; NOS, nitric-oxide synthase; RACE, rapid amplification of cDNA ends ; BH,, tetrahydrobiopterin ; MOI, multiplicity of infection.Note. The nucleotide sequence data published here have been submitted to the GenBank database and ar...

Anti-FRP mAbs induced polykaryocyte formation of U2ME-7 cells (CD4+U937 cells transfected with the HIV gpl60 gene). Anti-FRP-1 mAb immunoprecipitated gp8O-85, gpl20 and homodimers of these peptides, and anti-FRP-2 mAb reacted with gp135 identically to the A3 subunit of integrin. Both anti-FRP-1 and anti-FRP-2 mAb-induced cell fusion was blocked by anti-(l integrin antibody, fibronectin or inhibiting anti-FRP-1 antibody. Therefore, anti-FRP mAbs were thought to induce the fusion via an integrin system(s). FRP-mediated fusion was temperature, cytoskeleton, energy and Ca2+ dependent. These experiments showed a possible regulatory function of cell fusion by an integrin system(s).

CD8+ CTLs and virus-neutralizing antibodies have been associated with spontaneous and vaccine-induced immune control of retroviral infections. We previously showed that a single immunization with an env gene-encoded CD4+ T cell epitope protected mice against fatal Friend retrovirus infection. Here, we analyzed immune cell components required for the peptide-induced anti-retroviral protection. Mice lacking CD8+ T cells were nevertheless protected against Friend virus infection, while mice lacking B cells were not. Virus-producing cells both in the spleen and bone marrow decreased rapidly in their number and became undetectable by 4 weeks after infection in the majority of the peptide-immunized animals even in the absence of CD8+ T cells. In the vaccinated animals the production and class switching of virus-neutralizing and anti-leukemia cell antibodies were facilitated; however, virus-induced erythroid cell expansion was suppressed before neutralizing antibodies became detectable in the serum. Further, the numbers of virus-producing cells in the spleen and bone marrow in the early stage of the infection were smaller in the peptide-immunized than in unimmunized control mice in the absence of B cells. Thus, peptide immunization facilitates both early cellular and late humoral immune responses that lead to the effective control of the retrovirus-induced disease, but CD8+ T cells are not crucial for the elimination of virus-infected cells in the peptide-primed animals.

Previously, we obtained a neutralizing monoclonal antibody directed against the hemagglutinin-neuraminidase (HN) protein of human parainfluenza type 2 virus (PIV2), which was able to prevent cell fusion without affecting the hemagglutinating and neuraminidase activities. In this study, four escape mutants of PIV2 have been obtained under pressure of the monoclonal antibody. Intriguingly, the HN protein of each mutant proved to have two amino acid substitutions, one of which is at 83Asn or 91Lys, and another one is at 150Leu, 160Ala, or 186Met. One mutant designated F13, which has substitutions at 83Asn and 186Met in the HN protein, could not cause cell fusion in HeLa cells despite its multiple replication, while the other mutants formed typical syncytial cells. The deduced amino acid sequence of F13 fusion (F) protein proved to be identical to that of wild-type F protein, and furthermore, protein expression analyses have revealed that the low-fusion phenotype of F13 was due to its mutated HN protein, whose antigenicity to the monoclonal antibody was abolished by the single mutation at 83Asn. These observations have suggested that the principal epitope for the monoclonal antibody resides in the presumed stalk domain of the HN protein, which may play an important role in promoting cell fusion.