Tissue distribution of a plasmid DNA encoding Hsp65 gene is dependent on the dose administered through intramuscular delivery

In order to assess a new strategy of DNA vaccine for a more complete understanding of its action in immune response, it is important to determine the in vivo biodistribution fate and antigen expression. In previous studies, our group focused on the prophylactic and therapeutic use of a plasmid DNA encoding the Mycobacterium leprae 65-kDa heat shock protein (Hsp65) and achieved an efficient immune response induction as well as protection against virulent M. tuberculosis challenge. In the present study, we examined in vivo tissue distribution of naked DNA-Hsp65 vaccine, the Hsp65 message, genome integration and methylation status of plasmid DNA. The DNA-Hsp65 was detectable in several tissue types, indicating that DNA-Hsp65 disseminates widely throughout the body. The biodistribution was dose-dependent. In contrast, RT-PCR detected the Hsp65 message for at least 15 days in muscle or liver tissue from immunized mice. We also analyzed the methylation status and integration of the injected plasmid DNA into the host cellular genome. The bacterial methylation pattern persisted for at least 6 months, indicating that the plasmid DNA-Hsp65 does not replicate in mammalian tissue, and Southern blot analysis showed that plasmid DNA was not integrated. These results have important implications for the use of DNA-Hsp65 vaccine in a clinical setting and open new perspectives for DNA vaccines and new considerations about the inoculation site and delivery system.     

A flavoenzyme model: Facile oxidation of thiols by a flavin immobilized in cationic polyelectrolytes

The reactions of a polymer-bound flavin with thiols (2-mercaptoethanol, glutathione, thiophenol, and 1,4-butanedithiol) are remarkably accelerated, when compared with that of a monomeric flavin. The rate enhancements observed were 30- to 6000-fold. In particular, thiophenol which had been believed not to be oxidized by flavin in nonenzymatic systems was oxidized most rapidly among the monothiols examined. The reaction rates were improved by incorporation of a dodecyl group into the flavin-containing polymer. Therefore, the hydrophobic nature of the cationic polymer matrix was concluded to be responsible for the large rate enhancement among other factors.     

Suppressed recombination on mouse chromosome 15 defined regions of chromosomal inversions associated with koala (koa) and hairy ears (eh) mutations.

Koala (Koa) and hairy ears (Eh) mutations of mice are associated with chromosomal inversions in the distal half of chromosome 15. Since these two mutant mice show some common phenotypic features including extra hair on pinna and craniofacial dysmorphogenesis and have similar inverted regions, we determined the inverted regions of these two chromosomal inversions to examine whether a common gene is responsible for the phenotypes of these two mutants. The inverted regions were identified as the recombination-suppressed regions by linkage analysis. The length of the recombination-suppressed regions of Koa and Eh were approximately 52 and 47 Mb, respectively, and these inverted regions were not the same. These results indicate that the phenotypes of Koa and Eh mutant mice are likely to be caused by different genes.     

A complex of collagenase with low molecular weight inhibitors in the culture medium of embryonic chick skin explants.

Collagenase inhibitors with molecular weights of about 6,000 and 12,000 were isolated from latent chick skin collagenase treated with 3 M NaI and from the culture medium of embryonic skin explants. It is suggested that these inhibitors, which are possibly derived from connective tissue macromolecule metabolites, are candidates for regulating factors of collagenase activity in vivo.     

Effects of aging on alpha 1-adrenoceptor mechanisms and the inhibitory effect of diltiazem on noradrenaline maximum response in isolated rat aortic preparation

The effects of aging on alpha 1-adrenoceptor mechanisms in aortic preparations isolated from 3-, 6-, 10-, 18-, and 40-week-old rats were studied and compared with serotonin receptor mechanisms in the same preparations. The potency (pD2 value) of noradrenaline increased with age from 3 to 10 weeks, but decreased thereafter with age from 10 to 40 weeks. The affinity (pKA value) of noradrenaline and of prazosin (pA2 value) did not alter with aging. The change in potency or the pD2 value of noradrenaline was proportional to receptor reserve (pD2-pKA value) for noradrenaline, suggesting that the change of potency of noradrenaline with age was due to a change of receptor reserve, but not to change of drug affinity to alpha 1-adrenoceptors. The potency (pD2 value) and affinity (pKA value) of serotonin, and the affinity (pA2 value) of ketanserin, did not alter with aging, suggesting that serotonin receptor mechanisms in rat aorta did not change with age. The inhibitory effect of diltiazem on noradrenaline maximum response decreased with age from 3 to 10 weeks, but increased with age from 10 to 40 weeks. An inverse relationship between changes of diltiazem inhibition and receptor reserve of noradrenaline was found. Diltiazem's inhibitory effect on serotonin maximum response did not alter with aging.     

The simultaneous determination of the products of estrogen 2- and 4-hydroxylase action; the use of high-performance liquid chromatography with electrochemical detection

Both trace-labeled and high-specific activity ¹²⁵I-labeled derivatives of hexadecapeptide gastrin (G) were prepared by reaction with the iodinated form of the imidoester, methyl p-hydroxybenzimidate. Reaction conditions for preparation of trace-labeled iodinated imidoester gastrin (IIE-G) were: excess imidoester to G (IIE:G, 20:1), pH 9.2, and a reaction time of 24 h. Following purification by gel filtration and silica gel chromatography, an IIE-G component was isolated which appeared homogeneous on thin-layer chromatography and retained the same biological and immunological properties as unmodified G. Somewhat different conditions were necessary to prepare high specific activity iodinated imidoester gastrin (IIE¹-G). These included reducing the volume (20 μl) and pH (7.5) at which the imidoester was iodinated and adjusting the concentrations of reactants to the same molar amounts as 5 mCi of carrier-free ¹²⁵I. Sufficient amounts of IIE¹-G were obtained by reversing the ratio of G and IIE¹ and reacting with a G excess (GIIE¹, 10:1). The purified IIE¹-G had a specific activity exceeding 1500 μCi/nmol and was used to establish a specific and sensitive radioimmunoassay for gastrin.     

Neutralizing Activities of Early and Late IgG Fragments from Rabbits Immunized with Herpes Simplex Virus

Rabbits immunized with herpes virus were bled periodically and bivalent and univalent fragments of IgG from each serum sample were prepared by enzymatic digestion. The 2-week F(ab′)₂ showed a low neutralizing activity only after addition of anti-IgG. F(ab′)₂ of the 4-week serum retained almost all of the neutralizing activity of IgG, while its univalent fragments demonstrated none even when tested with anti-IgG. In contrast to these early IgG fragments, univalent fragments of the 9-week and 20-week IgG neutralized the virus to considerable extents in the absence of anti-IgG; after addition of anti-IgG the activity equaled that of intact IgG in the cases of Fab′ and Fab-II, though the activity of Fab-I was relatively low. The three univalent fragments were all sensitive to heating at 70 C and to ultraviolet irradiation, whereas intact IgG resisted these treatments. F(ab′)₂ was resistant to the heating and less sensitive to ultraviolet irradiation than univalent fragments. Neutralization kinetic curve experiments to test blocking effects of IgG fragments against the neutralization by intact IgG suggested that the early Fab′ did combine with the virus and that the late Fab′ exerted a higher blocking effect than the early Fab′.     

Mitochondrial DNA variation and genetic structure in populations of Drosophila melanogaster

The understanding of the genetic structure of a species can be improved by considering together data from different types of genetic markers. In the past, a number of worldwide populations of Drosophila melanogaster have been extensively studied for several such markers, including allozymes, chromosomal inversions, and quantitative characters. Here we present results from a study of restriction- fragment-length polymorphisms of mitochondrial DNA (mtDNA) in 92 isofemale lines from many of the same geographic populations of D. melanogaster. Eleven restriction enzymes were used, of which four revealed restriction-site polymorphism. A total of 24 different haplotypes were observed, of which 18 were unique to single populations. In many populations, the unique haplotypes have reached high frequency without being observed in neighboring populations. A Wagner parsimony tree reveals that mutationally close variants show geographical clumping, suggesting local differentiation of mtDNA in populations. The Old-World and the New-World populations are differentiated, with the predominant Old-World haplotype being virtually absent from the New World. These results contrast with those for the nuclear genes, in which many loci show parallel clines in different continents, and suggest a common origin of D. melanogaster populations in North America.