Reduction of Light-Induced Anthocyanin Accumulation in Inoculated Sorghum Mesocotyls1 : Implications for a Compensatory Role in the Defense Response

Sorghum (Sorghum bicolor L. Moench) accumulates the anthocyanin cyanidin 3-dimalonyl glucoside in etiolated mesocotyls in response to light. Inoculation with the nonpathogenic fungus Cochliobolus heterostrophus drastically reduced the light-induced accumulation of anthocyanin by repressing the transcription of the anthocyanin biosynthesis genes encoding flavanone 3-hydroxylase, dihydroflavonol 4-reductase, and anthocyanidin synthase. In contrast to these repression effects, fungal inoculation resulted in the synthesis of the four known 3-deoxyanthocyanidin phytoalexins and a corresponding activation of genes encoding the key branch-point enzymes in the phenylpropanoid pathway, phenylalanine ammonia-lyase and chalcone synthase. In addition, a gene encoding the pathogenesis-related protein PR-10 was strongly induced in response to inoculation. The accumulation of phytoalexins leveled off by 48 h after inoculation and was accompanied by a more rapid increase in the rate of anthocyanin accumulation. The results suggest that the plant represses less essential metabolic activities such as anthocyanin synthesis as a means of compensating for the immediate biochemical and physiological needs for the defense response.

Reovirus reverse genetics: Incorporation of the CAT gene into the reovirus genome

We have modified the infectious reovirus RNA system so as to generate a reovirus reverse genetics system. The system consists of (i) the plus strands of nine wild-type reovirus genome segments; (ii) transcripts of the genetically modified cDNA form of the tenth genome segment; and (iii) a cell line transformed so as to express the protein normally encoded by the tenth genome segment. In the work described here, we have generated a serotype 3 reovirus into the S2 double-stranded RNA genome segment of which the CAT gene has been cloned. The virus is stable, replicates in cells that have been transformed (so as to express the S2 gene product, protein σ2), and expresses high levels of CAT activity. This technology can be extended to members of the orbivirus and rotavirus genera. This technology provides a powerful system for basic studies of double-stranded RNA virus replication; a nonpathogenic viral vector that replicates to high titers and could be used for clinical applications; and a system for providing nonselectable viral variants (the result of mutations, insertions, and deletions) that could be valuable for the construction of viral vaccine strains against human and animal pathogens.

Comparative interleukin (IL-2)/interferon IFN-gamma and IL-4/IL-10 responses during acute infection of macaques inoculated with attenuated nef-truncated or pathogenic SICmac251 virus.

Comparison of immune responses to infection by a pathogenic or a nonpathogenic immunodeficiency virus in macaques may provide insights into pathogenetic events leading to simian AIDS. This work is aimed at exploring cytokine expression during infection by simian immunodeficiency virus (SIV). We used semiquantitative reverse transcription-PCR to monitor interleukin (IL)-2/interferon (IFN)-gamma (Th1-like), and IL-4/IL-10 (Th2-like) expression in unmanipulated peripheral blood mononuclear cells (PBMCs), during the acute phase of infection of eight cynomolgus macaques (Macaca fascicularis) with a pathogenic primary isolate of SIVmac251 (full-length nef), and of four other cynomolgus macaques by an attenuated molecular clone of SIVmac251 (nef-truncated). All the monkeys became infected, as clearly shown by the presence of infected PBMCs and by seroconversion. Nevertheless, PBMC-associated virus loads and p27 antigenemia in monkeys infected by the attenuated virus clone remained lower than those observed in animals infected with the pathogenic SIVmac251 isolate. A rise of IL-10 mRNA expression occurred in both groups of monkeys coincident with the peak of viral replication. In monkeys infected with the pathogenic SIVmac251, IL-2, IL-4, and IFN-gamma mRNAs were either weakly detectable or undetectable. On the contrary, animals infected by the attenuated virus exhibited an overexpression of these cytokine mRNAs during the first weeks after inoculation. The lack of expression of these cytokines in monkeys infected with the pathogenic primary isolate may reflect early immunodeficiency.

Antimycobacterial activities of antisense oligodeoxynucleotide phosphorothioates in drug-resistant strains.

Strains of Mycobacterium smegmatis, a mycobacterium which shares genetic sequences, grows more rapidly, and is nonpathogenic in man as compared with Mycobacterium tuberculosis, were utilized for the initial development of new antimycobacterial therapy. Drug-resistant strains of M. smegmatis which are known to arise in a manner identical to the emergence of drug-resistant strains of M. tuberculosis were isolated and utilized as models for the antimycobacterial activities of modified and unmodified oligodeoxynucleotide phosphorothioates in broth cultures. Under normal conditions, oligodeoxynucleotide phosphorothioates do not enter mycobacteria, and several strategies were successfully utilized to afford entry of oligonucleotides into the mycobacterial cells. One involved the presence of very low levels of ethambutol, which enables the entry of oligonucleotides into mycobacteria because of its induced alterations in the cell wall, and another involved the utilization of oligonucleotides covalently attached to a D-cycloserine molecule, whereby entry into the mycobacterial cell is achieved by a receptor-mediated process. Another low molecular weight, covalently attached ligand that enabled the entry and subsequent antimycobacterial activities of oligodeoxynucleotide phosphorothioates in the absence of a cell wall modifying reagent was biotin. Significant sequence-specific growth inhibition of wild-type, as well as of drug-resistant, M. smegmatis was obtained by modified oligonucleotides complementary in sequence to a specific region of the mycobacterium aspartokinase (ask) gene when utilized in combinations with ethambutol (as compared to ethambutol alone) or as D-cycloserine or biotin covalent adducts without the presence of any other cytotoxic or cytostatic agent.