SummaryExposure of BALB/c mice to mosquitoes infected with irradiated Plasmodium berghei confers protective immunity against subsequent sporozoite challenge. Immunized mice challenged with viable sporozoites develop parasitemia when treated orally with substrate inhibitors of nitric oxide synthase (NOS). This suggests that the production of nitric oxide (NO) prevents the development of exoerythrocytic stages of malaria in liver. Liver tissue from immunized mice expressed maximal levels of mKNA for inducible NOS (iNOS) between 12 and 24 h after challenge with sporozoites. Intraperitoneal injection of neutralizing monoclonal antibody against interferon 3/(IFN-3') or in vivo depletion of CD8 + T cells, but not CD4 + T cells, at the time of challenge blocked expression of iNOS mlLNA and ablated protection in immunized mice. These results show that both CD8 § T cells and IFN-~/are important components in the regulation of iNOS in liver which contributes to the protective response of mice immunized with irradiated malaria sporozoites. IFN-3,, likely provided by malaria-specific CD8 + T cells, induces liver cells, hepatocytes and/or Kupffer cells, to produce NO for the destruction of infected hepatocytes or the parasite within these cells. W ithin minutes after an infected Anopheles mosquito bites the vertebrate host, malaria sporozoites migrate to the liver and invade hepatocytes. There, the parasite matures, and after several days the infected hepatocytes lyse, releasing thousands of merozoites. Once in circulation, the parasite infects erythrocytes causing parasitemia. Prior exposure to irradiated sporozoites confers protective immunity (1, 2). This immunity is directed against liver stage malaria, and does not protect against the blood stage malaria.CD8 + T cells and IFN-3' are required for protective immunity to sporozoite challenge. In vivo depletion of CD8 § T cells or neutralization of IFN-3~ blocks induction of effector activity at the hepatic stage, resulting in parasitaemia (3-5). In vitro studies show that IFN-qr kills parasites by stimulating malaria-infected hepatocytes to produce nitric oxide (NO), and the addition of monomethyl-r-arginine (NGMMLA), a substrate inhibitor of nitric oxide synthase (NOS), to primary cultures of mouse hepatocytes reversed the antiparasitic effects of IFN-'y (6, 7). Human hepatocytes also respond to IFN-3' for NO production (8). As to whether human hepatocytes exhibit antimalaria activity when stimulated to produce NO, remains to be examined.At present, the antimalaria effector mechanism triggered by sporozoites in immunized animals is not fully understood. Presumably malaria-specific CD8 + T cells act directly against infected hepatocytes by recognizing malaria antigen on the cell surface (i.e., induction of CTLs) or malaria-specific lymphocytes release cytokines, such as IFN-3,, upon parasite stimulation, which induces an antimalarial response (3)(4)(5)(9)(10)(11)(12). The relationship between CD8 + T cells, IFN-% and NO-mediated protection in sporozoite-immunized mice was ...

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The Bacillus spoOH gene codes for c H, which, as part of the RNA polymerase holoenzyme EorH, is responsible for the transcription of several genes which are expressed at the beginning of the sporulation process. In this communication, we examined the regulation of the spoOH gene of Bacillus subtilis by using lacZ reporter gene assays, quantitative RNA determinations, and Western immunoassay. The expression of the spoOH gene increases as the culture enters the mid-logarithmic stage of growth. This increased expression requires the genes spoOA, spoOB, spoOE, and spoOF, and the requirement for at least spoOA and spoOB can be bypassed when the abrB gene is mutated. The expression of the spoOH gene is constitutive in the presence of the abrB mutation, being expressed at higher levels during vegetative growth. In addition, the sof-l mutation, in the spoOA structural gene, can bypass the need for spoOF in spoOH expression. (25,44). aG is produced only in the forespore (41), and the gene for rK is transcribed after a chromosomal rearrangement which occurs only in the mother cell (39). It has also been demonstrated that a 14-kDa protein changes the transcriptional specificity of EurK (24). Thus, the functioning of minor species of RNA polymerase containing these u factors can be regulated by processing, by localization within the bacterial cell, and by transcriptional activators.Ea.H, the RNA polymerase containing cr, which is encoded by spoOH (9, 51), is required for the in vivo and in vitro transcription of promoters spoVGpl (3, 51), rpoDp3 (4), and citGp2 (10, 42). These promoters are turned on just when the culture reaches the stationary phase of growth (To) in rich medium. Therefore, the activity of this minor species of * Corresponding author.

Results for clade 1 viruses were more consistent among laboratories when a standard antibody was used.

(8,9) or DNA recombinants (10, 11). However, the task of assigning these polypeptides to specific functions has just begun. Indeed, until now, no poxvirus proteins with known functions have been mapped by molecular or classical genetic methods.Early mRNAs of vaccinia virus that have been examined in detail do not appear to be spliced or shortened at their 5' ends (12-15). For one early mRNA, the nucleotides within the 60-bp sequence immediately upstream ofthe initiation site are 89% adenylate and thymidylate, and no homologies to prokaryotic or eukaryotic sequences were found further upstream (16). This finding led to the proposal that the vaccinia virus RNA polymerase (17, 18) has its own novel DNA recognition sequences.Although additional early vaccinia virus genes are being examined to develop a consensus sequence, such information alone will be insufficient to establish functional significance.In vitro mutagenesis provides a direct way ofcorrelating the two parameters: nucleotide sequence and function. Such studies are facilitated by the use of selectable genetic markers. For vaccinia virus, the most suitable marker is thymidine kinase (TK). Dubbs and Kit (19) demonstrated that TK activity increases after infection of TKV or TK-mouse L cells with wildtype vaccinia virus but not with a TK-mutant, providing evidence that the enzyme is virus-coded. However, no information existed regarding the genetic locus of the vaccinia TK. For herpesvirus, which also encodes TK (20)

Foscarnet (phosphonoformic acid) is a pyrophosphate analog that inhibits the replication of human immunodeficiency virus type 1 (HIV-1) in vitro and in patients with AIDS. HIV-1 resistance to foscarnet has not been reported despite long-term foscarnet therapy of AIDS patients with cytomegalovirus disease. We therefore attempted to select foscarnet-resistant HIV-1 in vitro by serial endpoint passage of virus in 400 M foscarnet. After 13 cycles of passage in MT-2 cells, virus exhibiting Ն8.5-fold foscarnet resistance was isolated. The reverse transcriptase (RT) from resistant virions exhibited a similar level of foscarnet resistance in enzyme inhibition assays (ϳ10-fold resistance). Foscarnet-resistant virus showed increased susceptibility to 3-azido-3-deoxythymidine (90-fold) and to the HIV-1-specific RT inhibitors TIBO R82150 (30-fold) and nevirapine (20-fold). DNA sequence analysis of RT clones from resistant virus revealed the coexistence of two mutations in all clones: Gln-161 to Leu (CAA to CTA) and His-208 to Tyr (CAT to TAT). Sequence analysis of six clinical HIV-1 isolates showing reduced susceptibility to foscarnet revealed the Tyr-208 mutation in two, the Leu-161 mutation in one, and a Trp-88-to-Ser or -Gly mutation in four isolates. Site-specific mutagenesis and production of mutant recombinant viruses demonstrated that the Leu-161, Ser-88, and Tyr-208 mutations reduced HIV-1 susceptibility to foscarnet 10.5-, 4.3-, and 2.4-fold, respectively, in MT-2 cells. In the crystal structure of HIV-1 RT, the Gln-161 residue lies in the ␣E helix beneath the putative deoxynucleoside triphosphate (dNTP) binding site. The Gln-161-to-Leu mutation may affect the structure of the dNTP binding site and its affinity for foscarnet. The location of the Trp-88 residue in the ␤5a strand of HIV-1 RT suggests that the Ser-88 mutation affects template-primer binding, as do several mutations that affect RT susceptibility to nucleoside analogs.Foscarnet (trisodium phosphonoformic acid) is a pyrophosphate analog that inhibits the polymerases of diverse DNA and RNA viruses, including herpes simplex viruses, varicella-zoster virus, cytomegalovirus (CMV), hepatitis B virus, influenza virus, human immunodeficiency virus type 1 (HIV-1), and other retroviruses (for a review see reference 26). Foscarnet is licensed and widely prescribed for the treatment of CMV retinitis in patients with AIDS. It is also the current drug of choice for acyclovir-or ganciclovir-resistant herpesvirus infections (6). Several clinical trials have demonstrated that foscarnet has antiretroviral activity in vivo (5,7,12,29). In an early trial of foscarnet for the treatment of CMV retinitis, Reddy et al. (29) observed sustained reductions in serum HIV-1 p24 antigen levels for a median of 16 weeks after initiation of foscarnet therapy. In a more recent study of foscarnet as primary therapy of HIV-1, reductions in serum p24 antigen were observed in all patients who received at least 1 week of foscarnet therapy (7). This direct antiretroviral effect of foscarnet ha...

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The transcriptional regulatory gene spoOH encodes an RNA polymerase sigma factor called sigma H that directs gene expression at an early stage of sporulation in the Gram-positive bacterium Bacillus subtilis. We now report that conditions that induce sporulation cause a rapid increase in the cellular concentration of sigma H. This increase could account for the stimulated transcription of certain sigma H-controlled genes at the onset of sporulation. Experiments in which the expression of spoOH was monitored by use of a spoOH-lacZ fusion and in which expression of spoOH was artificially manipulated by use of an isopropyl-beta-D-thiogalacto-side-inducible promoter indicate that sporulation-induced increases in the amount of sigma H are not controlled at the level of the transcription of its structural gene. Rather, we infer the existence of post-transcriptional control mechanisms that govern sigma H levels, and we present evidence suggesting that increases in the amount of sigma H at the start of sporulation are due to increased translation or stability of the spoOH mRNA and, to a lesser extent, decreased turnover of spoOH protein.