A study of three IACUCs and their views of scientific merit and alternatives

Two ethical issues facing Institutional Animal Care and Use Committees [IACUCs] today are assessing scientific merit and the use of alternatives in research proposals. This study evaluated 3 IACUCs using a 19-question survey, with a 77.8% response rate. Although 76% of members answered that scientific merit should be more dili-gently assessed if more than slight pain is caused, 14% believed that assessing scien-tific merit is not the role of the IACUC. Nearly 86% agreed that the search for alterna-tives should be more diligent for protocols that incur more than slight pain to the animals involved. Some members believed that alternatives were not actively enough sought after, while others believed no viable alternatives existed. Additional guide-lines are needed to clarify these issues.     

Aspergillus nidulans polarity mutant swoA is complemented by protein O-mannosyltransferase pmtA

Previously swoA was identified in Aspergillus nidulans as a single locus, temperature-sensitive (ts) mutant aberrant in polarity maintenance. swoA was complemented by transformation with a plasmid genomic library. The sequence of the complementing gene was identical to a previously submitted GenBank sequence for pmtA, a protein O-mannosyltransferase. The pmtA/swoA-2 gene hybridized to three cosmids that are located on chromosome V and the swoA mutation was mitotically mapped to chromosome V. PMTs are endoplasmic reticulum-resident proteins responsible for the first step of O-glycosylation. Structural predictions suggest that PmtA contains seven membrane spans similar to PMTs from Saccharomyces cerevisiae and other organisms. Phylogenetic analysis indicates that PmtA is most closely related to the S. cerevisiae sub-family of PMTs containing Pmt2, Pmt3 and Pmt6. The mutant pmtA/swoA-2 locus contained a lesion that changed Y662 to a stop codon, eliminating the final membrane spanning domain and interrupting a domain essential for function in other PMTs.     

Interactions between aromatase (estrogen synthase) and dopamine in the control of male sexual behavior in quail

In male quail, like in other vertebrates including rodents, testosterone acting especially through its estrogenic metabolites is necessary for the activation of male sexual behavior. Also, the administration of dopamine agonists and antagonists profoundly influences male sexual behavior. How the steroid-sensitive neural network and dopamine interact physiologically, remains largely unknown. It is often implicitly assumed that testosterone or its metabolite estradiol, stimulates male sexual behavior via the modification of dopaminergic transmission. We have now identified in quail two possible ways in which dopamine could potentially affect sexual behavior by modulating the aromatization of testosterone into an estrogen. One is a long-acting mechanism that presumably involves the modification of dopaminergic transmission followed by the alteration of the genomic expression of aromatase. The other is a more rapid mechanism that does not appear to be dopamine receptor-mediated and may involve a direct interaction of dopamine with aromatase (possibly via substrate competition). We review here the experimental data supporting the existence of these controls of aromatase activity by dopamine and discuss the possible contribution of these controls to the activation of male sexual behavior.     

Aminoglycoside-derived cationic lipids as efficient vectors for gene transfection in vitro and in vivo

Background

Cationic lipids are at present very actively investigated for gene transfer studies and gene therapy applications. Basically, they rely on the formation of DNA/lipid aggregates via electrostatic interactions between their cationic headgroup and the negatively charged DNA. Although their structure/activity relationships are not well understood, it is generally agreed that the nature of the positive headgroup impacts on their transfection activity. Thus, we have directed our efforts toward the development of cationic lipids with novel cationic moieties. In the present work, we have explored the transfection potential of the lipophilic derivatives of the aminoglycoside kanamycin A. Indeed, aminoglycosides, which are natural polyamines known to bind to nucleic acids, provide a favorable scaffold for the synthesis of a variety of cationic lipids because of their structural features and multifunctional nature.

Methods and results

We report here the synthesis of a cationic cholesterol derivative characterized by a kanamycin A headgroup and of its polyguanidinylated derivative. The amino‐sugar‐based cationic lipid is highly efficient for gene transfection into a variety of mammalian cell lines when used either alone or as a liposomal formulation with the neutral phospholipid dioleoylphosphatidylethanolamine (DOPE). Its polyguanidinylated derivative was also found to mediate in vitro gene transfection. In addition, colloidally stable kanamycin‐cholesterol/DOPE lipoplexes were found to be efficient for gene transfection into the mouse airways in vivo.

Conclusions

These results reveal the usefulness of cationic lipids characterized by headgroups composed of an aminoglycoside or its guanidinylated derivative for gene transfection in vitro and in vivo. Copyright © 2002 John Wiley & Sons, Ltd.

     

Yersinia: an update

The 8th International Symposium on Yersinia was held in Turku, Finland, 4–8 September 2002.     

Design of a targeted peptide nucleic acid prodrug to inhibit hepatic human microsomal triglyceride transfer protein expression in hepatocytes

In this study, we present the design and synthesis of an antisense peptide nucleic acid (asPNA) prodrug, which displays an improved biodistribution profile and an equally improved capacity to reduce the levels of target mRNA. The prodrug, K(GalNAc)(2)-asPNA, comprised of a 14-mer sequence complementary to the human microsomal triglyceride transfer protein (huMTP) gene, conjugated to a high-affinity tag for the hepatic asialoglycoprotein receptor (K(GalNAc)(2)). The prodrug was avidly bound and rapidly internalized by HepG2s. After iv injection into mice, K(GalNAc)(2)-asPNA accumulated in the parenchymal liver cells to a much greater extent than nonconjugated PNA (46% +/- 1% vs 3.1% +/- 0.5% of the injected dose, respectively). The prodrug was able to reduce MTP mRNA levels in HepG2 cells by 35-40% (P < 0.02) at 100 nM in an asialoglycoprotein receptor- and sequence-dependent fashion. In conclusion, hepatocyte-targeted PNA prodrugs combine a greatly improved tropism with an enhanced local intracellular availability and activity, making them attractive therapeutics to lower the expression level of hepatic target genes such as MTP.     

Chromosomal sequences from Klebsiella pneumoniae flank the SHV-5 extended-spectrum beta-lactamase gene in pACM1

The nucleotide sequence was determined for Anon 13, a 1250-bp SmaI fragment located approximately 2.8 kb downstream from bla(SHV-5) in pACM1. Anon 13 is 99% identical to a segment of the unpublished sequence of the Klebsiella pneumoniae chromosome. Genes of the K. pneumoniae sequence are undefined, but conceptual amino acid translations of two ORFs in Anon 13 are homologous to L-fuculose-1-phosphate aldolase (FucA) and a conserved hypothetical protein present in the chromosomes of several species of bacteria. In addition, restriction mapping indicates that the region of homology between the K. pneumoniae chromosome and pACM1 is as least 7.9 kb and includes both Anon 13 and bla(SHV). These observations demonstrate the chromosomal origin of the bla(SHV-5) on pACM1.     

Enumeration of viable anaerobic bacteria by solid phase cytometry under aerobic conditions

Classical enumeration methods for anaerobes are time-consuming and require special conditions. Solid phase cytometry (SPC) is a recent laser scanning technique for the quantitative detection of fluorescently labelled bacteria on a membrane filter that eliminates the need for a growth phase. Fluorescent labelling of cells results from the cleavage by intracellular esterases of a fluorescein type ester to yield a free fluorescein derivative, which is retained only in cells with an intact cytoplasmic membrane. However, as the standard labelling procedure is carried out under the conditions of aerobiosis, labelling of anaerobic bacteria does not appear to be obvious. We have labelled eight strains of vegetative anaerobic bacteria (i.e. Bacteroides thetaiotaomicron, Clostridium bifermentans, C. butyricum, C. perfringens, Fusobacterium nucleatum, Porphyromonas canoris, P. gingivalis, Propionibacterium acnes) and two strains of spores (C. butyricum, C. perfringens,) within 4 h under aerobic conditions. However, anaerobiosis remained necessary for spores of C. sordellii, C. sporogenes, C. tyrobutyricum. For vegetative cells of all strains, plots of SPC versus plate counts were linear with slopes exceeding 1.0, indicating that SPC consistently yielded higher numbers of bacteria.