Gene expression profiling in postmortem Rett Syndrome brain: differential gene expression and patient classification.

The identification of mutations in the transcriptional repressor methyl-CpG-binding protein 2 (MECP2) gene in Rett Syndrome (RTT) suggests that an inappropriate release of transcriptional silencing may give rise to RTT neuropathology. Despite this progress, the molecular basis of RTT neuropathogenesis remains unclear. Using multiple cDNA microarray technologies, subtractive hybridization, and conventional biochemistry, we generated comprehensive gene expression profiles of postmortem brain tissue from RTT patients and matched controls. Many glial transcripts involved in known neuropathological mechanisms were found to have increased expression in RTT brain, while decreases were observed in the expression of multiple neuron-specific mRNAs. Dramatic and consistent decreases in transcripts encoding presynaptic markers indicated a specific deficit in presynaptic development. Employing multiple clustering algorithms, it was possible to accurately segregate RTT from control brain tissue samples based solely on gene expression profile. Although previously achieved in cancers, our results constitute the first report of human disease classification using gene expression profiling in a complex tissue source such as brain.

Viremia and AIDS in rhesus macaques after intramuscular inoculation of plasmid DNA encoding full-length SIVmac239.

We have succeeded in stably maintaining the entire genome of SIVmac239 as a plasmid clone. Supercoiled proviral plasmid DNA was inoculated intramuscularly into two adult rhesus macaques and into a neonate. All three animals became viremic and seroconverted. Viral kinetics were followed prospectively by quantitative competitive reverse transcriptase polymerase chain reaction (QC-RT-PCR), measurement of proviral DNA load in peripheral blood mononuclear cells (PBMCs) by PCR, and virus isolation by cocultivation. The infant developed high virus loads and succumbed to AIDS and SIV-associated nephropathy at 10 weeks postinoculation. Both adults are still living but have progressed to AIDS; one adult has also developed severe thrombocytopenia. We conclude that infection through intramuscular inoculation of cloned plasmid DNA encoding the entire proviral genome is reproducible and will provide a useful tool for studying viral pathogenesis.

Live attenuated, multiply deleted simian immunodeficiency virus causes AIDS in infant and adult macaques.

A substantial risk in using live attenuated, multiply deleted viruses as vaccines against AIDS is their potential to induce AIDS. A mutant of the simian immunodeficiency virus (SIV) with large deletions in nef and vpr and in the negative regulatory element induced AIDS in six of eight infant macaques vaccinated orally or intravenously. Early signs of immune dysfunction were seen in the remaining two offspring. Prolonged follow-up of sixteen vaccinated adult macaques also showed resurgence of chronic viremia in four animals: two of these developed early signs of disease and one died of AIDS. We conclude that this multiply deleted SIV is pathogenic and that human AIDS vaccines built on similar prototypes may cause AIDS.

Phylogenetic relationship of the complete Rauscher murine leukemia virus genome with other murine leukemia virus genomes.

We report the complete nucleotide sequence of the genome of Rauscher murine leukemia virus (R-MuLV), the replication-competent helper virus present in the Rauscher virus complex, and its phylogenetic relationship with other murine leukemia virus genomes. An overall sequence identity of 97.6% was found between R-MuLV and the Friend helper virus (F-MuLV), and the two viruses were closely related on the phylogenetic trees constructed from either gag, pol, or env sequences. Moloney murine leukemia virus (Mo-MuLV) was the next closest relative to R-MuLV and F-MuLV on all trees, followed by Akv and radiation leukemia virus (RadLV). The most distantly related helper virus was Hortulanus murine leukemia virus (Ho-MuLV). Interestingly, Cas-Br-E branched with Mo-MuLV on the gag and pol trees, whereas on the env tree, it revealed the highest degree of relatedness to Ho-MuLV, possibly due to an ancient recombination with an Ho-MuLV ancestor. In summary, a phylogenetic analysis involving various MuLVs has been performed, in which the postulated close relationship between R-MuLV and F-MuLV has been confirmed, consistent with the pathobiology of the two viruses.

Structure and function of a virally encoded fungal toxin from Ustilago maydis: a fungal and mammalian Ca2+ channel inhibitor.

BACKGROUND: The P4 strain of the corn smut fungus, Ustilago maydis, secretes a fungal toxin, KP4, encoded by a fungal virus (UMV4) that persistently infects its cells. UMV4, unlike most other (non-fungal) viruses, does not spread to uninfected cells by release into the extracellular milieu during its normal life cycle and is thus dependent upon host survival for replication. In symbiosis with the host fungus, UMV4 encodes KP4 to kill other competitive strains of U. maydis, thereby promoting both host and virus survival. KP4 belongs to a family of fungal toxins and determining its structure should lead to a better understanding of the function and evolutionary origins of these toxins. Elucidation of the mechanism of toxin action could lead to new anti-fungal agents against human pathogens. RESULTS: We have determined the atomic structure of KP4 to 1.9 A resolution. KP4 belongs to the alpha/beta-sandwich family, and has a unique topology comprising a five-stranded antiparallel beta-sheet with two antiparallel alpha-helices lying at approximately 45 degrees to these strands. The structure has two left-handed beta alpha beta cross-overs and a basic protuberance extending from the beta-sheet. In vivo experiments demonstrated abrogation of toxin killing by Ca2+ and, to a lesser extent, Mg2+. These results led to experiments demonstrating that the toxin specifically inhibits voltage-gated Ca2+ channels in mammalian cells. CONCLUSIONS: Similarities, although somewhat limited, between KP4 and scorpion toxins led us to investigate the possibility that the toxic effects of KP4 may be mediated by inhibition of cation channels. Our results suggest that certain properties of fungal Ca2+ channels are homologous to those in mammalian cells. KP4 may, therefore, be a new tool for studying mammalian Ca2+ channels and current mammalian Ca2+ channel inhibitors may be useful lead compounds for new anti-fungal agents.