The ability of non-invasive monitoring of deep-tissue developmental, metabolic, and pathogenic processes will advance modern biotechnology. Imaging of live mammals using fluorescent probes is more feasible within a “near-infrared optical window” (NIRW)1. Here we report a phytochrome-based near infra-red fluorescent protein (iRFP) with the excitation/emission maxima at 690/713 nm. Bright fluorescence in a living mouse proved iRFP to be a superior probe for non-invasive imaging of internal mammalian tissues. Its high intracellular stability, low cytotoxicity, and lack of the requirement to add external biliverdin-chromophore makes iRFP as easy to use as conventional GFP-like proteins. Compared to earlier phytochrome-derived fluorescent probes, the iRFP protein has better in vitro characteristics and performs well in cells and in vivo, having greater effective brightness and photostability. Compared to the far-red GFP-like proteins, iRFP has substantially higher signal to background ratio in a mouse model owing to its infra-red shifted spectra.

Plasmodium falciparum is unable to synthesize purine bases and relies upon purine salvage and purine recycling to meet its purine needs. We report that purines formed as products of polyamine synthesis are recycled in a novel pathway in which 5 -methylthioinosine is generated by adenosine deaminase. The action of P. falciparum purine nucleoside phosphorylase is a convergent step of purine salvage, converting both 5 -methylthioinosine and inosine to hypoxanthine. We used accelerator mass spectrometry to verify that 5 -methylthioinosine is an active nucleic acid precursor in P. falciparum. Prior studies have shown that inhibitors of purine salvage enzymes kill malaria, but potent malaria-specific inhibitors of these enzymes have not been described previously. 5 -Methylthio-immucillin-H, a transition state analogue inhibitor that is selective for malarial relative to human purine nucleoside phosphorylase, kills P. falciparum in culture. Immucillins are currently in clinical trials for other indications and may also have application as anti-malarials.

In humans and in mice, control of the intracellular pathogen, Mycobacterium tuberculosis (Mtb), requires IFN-γ. Although the adaptive immune response results in production of substantial amounts of IFN-γ in response to Mtb, the immune response is unable to eradicate the infection in most cases. We have previously reported evidence that Mtb inhibits macrophage responses to IFN-γ, suggesting that this may limit the ability of IFN-γ to stimulate macrophages to kill Mtb. We have also observed that uninfected macrophages, adjacent to infected macrophages in culture, exhibit decreased responses to IFN-γ. Here we report that IL-6 secreted by Mtb-infected macrophages inhibits the responses of uninfected macrophages to IFN-γ. IL-6 selectively inhibits a subset of IFN-γ-responsive genes at the level of transcriptional activation without inhibiting activation or function of STAT1. Inhibition of macrophage responses to IFN-γ by IL-6 requires new protein synthesis, but this effect is not attributable to suppressor of cytokine signaling 1 or 3. These results reveal a novel function for IL-6 and indicate that IL-6 secreted by Mtb-infected macrophages may contribute to the inability of the cellular immune response to eradicate infection.

We report a monomeric PSmOrange protein that is initially orange (excitation and emission at 548 and 565 nm) but becomes far-red (excitation and emission at 636 and 662 nm) after irradiation with blue-green light. Compared to its parental orange proteins, PSmOrange has greater brightness, faster maturation, higher photoconversion contrast, and better photostability. The red-shifted spectra of both forms of PSmOrange enable its simultaneous use with cyan-to-green photoswitchable proteins to study four intracellular populations. Photoconverted PSmOrange has, to date, the most far-red excitation peak, provides diffraction-limited and super-resolution imaging in far-red range, is optimally excited with common red lasers, and can be photoconverted subcutaneously in a mouse. PSmOrange photoswitching occurs via a two-step photo-oxidation process, which causes cleavage of the polypeptide backbone. The far-red fluorescence of photoconverted PSmOrange results from a novel chromophore containing N-acylimine with a coplanar carbon-oxygen double bond.

Plasmodium falciparum is a purine auxotroph, and starvation of purines is known to cause purine-less death in cultured cells (1-3). Parasites cultured in human erythrocytes can be killed rapidly by blocking PNP 1 with Immucillin-H and related Immucillins; however, these agents inhibit both P. falciparum and human PNPs, albeit with higher efficiency for human PNP (2, 4). The biochemical link between PNP inhibition and purine-less death is the formation of hypoxanthine, previously identified as a primary source of purines for P. falciparum (1, 3). The amino acid sequence of P. falciparum PNP as well as its substrate specificity differs from mammalian and bacterial enzymes (4, 5). We investigated this difference by structural analysis using Immucillin-H as a ligand to stabilize the catalytic site and as a starting point for synthesis of inhibitors more specific to PfPNP.Immucillin-H is effective in causing P. falciparum cell death by purine starvation even in the purine-rich environment of human erythrocytes. Hypoxanthine but not inosine can prevent the effect (2). The transition state of PfPNP has oxacarbenium ion character and ImmH binds to PfPNP with a dissociation constant of 860 pM, making it a powerful inhibitor for this target (Refs. 4 and 6; Fig. 1). However, ImmH was designed to be a transition state analogue of mammalian PNPs and binds with a constant of 56 pM to human PNP (7). Therefore, addition of this compound to human erythrocytes infected with P. falciparum has two effects, inhibition of human PNP at lower concentrations and inhibition of both human and PfPNPs at higher concentrations.Comparison of the PfPNP amino acid sequence against the data base of all PNPs indicated that this enzyme differs from mammalian and bacterial PNPs (see below). The structure of PfPNP in complex with ImmH and SO 4 revealed that the catalytic site has capacity for binding 5Ј-substituted nucleosides. 5Ј-Methylthioinosine and inosine are good substrates for PfPNP. We hypothesized that 5Ј-methylthio-substituted Immucillin would bind preferentially to PfPNP. This hypothesis was used to design, produce, and characterize 5Ј-methylthio-Immucillin-H (MT-ImmH), a novel transition state analogue inhibitor with high specificity for the P. falciparum PNP. EXPERIMENTAL PROCEDURESPfPNP-The coding sequence for PfPNP (4) was amplified by PCR from P. falciparum 3D7 genomic DNA, placed into the pTrcHis2-TOPO vector, expressed in Escherichia coli TOP 10, and purified to Ͼ95% homogeneity by nickel affinity chromatography. ImmH was synthesized by the convergent route (8). MT-ImmH was synthesized by the desilylation of the previously reported N-tert-butoxycarbonyl-7-O-tert-butyldimethylsilyl-2,3,6-trideoxy-3,6-imino-4,5-O-isopropylidene-D-allo-heptononitrile followed by methanesulfonylation of the 7-hydroxyl and displacement of the resulting mesylate with thiomethoxide. The resultant compound was converted into MT-Immucillin-H in an analogous fashion to that previously described (9, 10). Structure and stereochemistry of MT-ImmH was confirmed ...