ABSTRACT
The complete sequence of herpes simplex virus type 2 (HSV-2) glycoproteins B and C (gB & gC) were cloned into plasmid expression vectors and evaluated in murine and guinea pig genital HSV-2 models. Balb/c mice were immunized with either pgB-2 or pgC-2 plasmids intramuscularly (IM) or intradermally (ID). The vaccines induced HSV-2-specific neutralizing and ELISA IgG antibody, but little or no enhancement of viral clearance from the vagina was detected following intravaginal challenge. Immunization of guinea pigs with pgB-2 or pgC-2 induced ELISA IgG antibody; however, antibody titers were approximately one log(10) unit lower than that seen in HSV-2 convalescent sera. IM immunization of guinea pigs with either plasmid also did not decrease vaginal viral shedding following vaginal challenge, but the severity of the acute disease and the subsequent number of recurrent lesion days were reduced in animals immunized with pgB-2. Lastly, IM immunization of latently infected guinea pigs with a combined gB-2 and gC-2 plasmid vaccine significantly reduced the number of subsequent HSV-2 recurrences. DNA vectors expressing gB-2 or gC-2 were both immunogenic, although the gB-2 plasmid induced higher titers of antibody and significantly reduced primary and recurrent herpetic disease in the guinea pig model. These results also suggest that immunotherapy with plasmid expression vectors may be effective against recurrent genital HSV-2 disease.
Subject(s)
Herpes Genitalis/prevention & control , Simplexvirus/immunology , Vaccines, DNA , Viral Envelope Proteins/genetics , Animals , Chlorocebus aethiops , Disease Models, Animal , Enzyme-Linked Immunosorbent Assay , Female , Guinea Pigs , Mice , Plasmids , Rabbits , Simplexvirus/genetics , Vagina/virology , Vero Cells , Viral Envelope Proteins/immunologyABSTRACT
Lysyl oxidase (EC 1.4.3.13) is a copper-dependent enzyme acting principally on collagen and elastin catalyzing the formation of aldehyde cross-links. It is also believed to possess a tumor suppressor activity as the anti-oncogene of ras. While rat, human, and mouse lysyl oxidase cDNAs have been cloned, little is known about the structure of the gene, its organization, or regulation. This paper describes the cloning of an intronic segment of the human lysyl oxidase gene. Sequence analysis defined the location of an intron that separates the prepro-coding segments from the segment encoding the catalytic domain. Genomic restriction mapping and gene copy number data established that multiple lysyl oxidase mRNA transcripts originate from a single gene and thus are products of alternative splicing. Northern analysis of adult and fetal fibroblast RNA showed a dominant approximately 4.3-kilobase lysyl oxidase mRNA transcript that varied in abundance as a function of cell line. These data are consistent with a complex mechanism regulating the expression of the lysyl oxidase gene.
Subject(s)
Chromosome Mapping , Genes , Protein-Lysine 6-Oxidase/genetics , Adult , Animals , Base Sequence , Blotting, Northern , Cells, Cultured , DNA/genetics , DNA/isolation & purification , Fetus , Fibroblasts/enzymology , Genes, Tumor Suppressor , Humans , Mice , Molecular Sequence Data , Oligodeoxyribonucleotides , RNA, Messenger/genetics , Rats , Restriction Mapping , Sequence Homology, Nucleic Acid , Skin/enzymology , Software , Transcription, GeneticABSTRACT
Nine different nylon and nitrocellulose membranes were compared utilizing four different methods of attaching the nucleic acid target. Nylon membranes repeatedly demonstrated increased sensitivity as compared to nitrocellulose membranes. Sensitivity could also be enhanced by mildly denaturing the target prior to attachment onto the membrane. This was achieved by either UV cross-linking or baking.
