Allen B. Clarkson

Kupffer cells are obligatory for Plasmodium yoelii sporozoite infection of the liver

Previous studies suggested Plasmodium sporozoites infect hepatocytes after passing through Kupffer cells, but proof has been elusive. Here we present new information strengthening that hypothesis. We used homozygous op/op mice known to have few Kupffer cells because they lack macrophage colony stimulating factor 1 required for macrophage maturation due to a deactivating point mutation in the osteopetrosis gene. We found these mice to have 77% fewer Kupffer cells and to exhibit reduced clearance of colloidal carbon particles compared with heterozygous phenotypically normal littermates. Using a novel quantitative reverse transcription polymerase chain reaction assay for P. yoelii 18S rRNA, we found liver infection of op/op mice to be decreased by 84% compared with controls. However, using another way of limiting Kupffer cells, treatment with liposome‐encapsulated clodronate, infection of normal mice was enhanced seven‐ to 15‐fold. This was explained by electron microscopy showing temporary gaps in the sinusoidal cell layer caused by this treatment. Thus, Kupffer cell deficiency in op/op mice decreases sporozoite infection by reducing the number of portals to the liver parenchyma, whereas clodronate increases sporozoite infection by opening portals and providing direct access to hepatocytes. Together these data provide strong support for the hypothesis that Kupffer cells are the portal for sporozoites to hepatocytes and critical for the onset of a malaria infection.

Mitochondrial development in Trypanosoma brucei brucei transitional bloodstream forms

Intermediate and short stumpy bloodstream forms of Trypanosoma brucei brucei are transitional stages in the differentiation of mammal-infective long slender bloodstream forms into the procyclic forms found in the midgut of the tsetse vector. Although the mitochondria of the proliferative long slender forms do not accumulate rhodamine 123, the mitochondria of the transitional forms attain this ability thus revealing the development of an electromotive force (EMF) across the inner mitochondrial membrane. The EMF is inhibited by 2,4-dinitrophenol, rotenone and salicylhydroxamic acid but not by antimycin A or cyanide. Consequently, NADH dehydrogenase, site I of oxidative phosphorylation, is the source of the EMF and the plant-like trypanosome alternative oxidase (TAO) supports the electron flow serving as the terminal oxidase of the chain. Although the TAO is present in the long slender forms as well, it serves only as the terminal oxidase for electrons from glycerol-3-phosphate dehydrogenase. The data presented here, combined with older data, lead to the conclusion that the mitochondria of transitional intermediate and short stumpy forms likely produce ATP. This putative production is either by F1F0 ATPase driven by the complex I proton pump or by mitochondrial substrate level phosphorylation, or most likely by both. These conclusions contrast with the previously held dogma that all bloodstream form mitochondria are incapable of ATP production.

Deferoxamine and eflornithine (DL-alpha-difluoromethylornithine) in a rat model of Pneumocystis carinii pneumonia.

The iron chelator deferoxamine and the polyamine biosynthesis inhibitor eflornithine (DL-alpha-difluoromethylornithine) were examined for anti-Pneumocystis carinii activity in the rat model of P. carinii pneumonia. The activity of deferoxamine at 250, 500, and 1,000 mg/kg given intraperitoneally provides evidence that iron chelation is a promising novel approach to P. carinii chemotherapy. Results with eflornithine at 2, 3, and 4% in drinking water confirm and extend previously reported activity in the rat model.

Effect of Nicotine on Lung S-Adenosylmethionine and Development of Pneumocystis Pneumonia

Because S-adenosylmethionine (AdoMet) is required by Pneumocystis carinii in vitro, Pneumocystis infection depletes plasma AdoMet of rats and humans, nicotine reduces AdoMet of guinea pig lungs, and smoking correlates with reduced episodes of Pneumocystis pneumonia (PCP) in AIDS patients, we tested the effect of nicotine treatment on PCP using a rat model. Intraperitoneal infusion of 400 μg of R-(+) nicotine kg-1 h-1 intraperitoneal for 21 days caused a 15-fold reduction in lung AdoMet although neither plasma nor liver were changed. Infusion of 4 and 400 μg kg-1 h-1 into immunosuppressed rats, beginning when rats were inoculated with P. carinii, caused 85 and 99.88% reductions, respectively, in P. carinii cysts at sacrifice 21 days later; P. carinii nuclei were reduced by 91.2 and >99.99%, respectively. This effect was reversed by concomitant administration of AdoMet with nicotine. Treatment with AdoMet alone increased infection intensity. We conclude that AdoMet is a critical and limiting nutrient for Pneumocystis thus can serve as a therapeutic target for PCP. Regarding the mechanism, nicotine treatment caused no change in rat lung activity of AdoMet synthesizing methionine ATP transferase activity nor was there any evidence of increased AdoMet utilization for methylation reactions. Except of a doubling of putrescine, nicotine treatment also did not change lung polyamine content. However, key polyamine anabolic and catabolic enzymes were upregulated, and there were corresponding changes in polyamine metabolic intermediates. We conclude that chronic nicotine treatment increases lung polyamine catabolic/anabolic cycling and/or excretion leading to increased AdoMet-consuming polyamine biosynthesis and depletion of lung AdoMet.

Response of rat model of Pneumocystis carinii pneumonia to continuous infusion of deferoxamine.

The iron-chelating drug deferoxamine mesylate (DFO) is active against Pneumocystis carinii in vitro and in rat and mouse models of P. carinii pneumonia. Because DFO has a short half-life, daily divided or continuous dosage was expected to improve the dose response, as is the case with DFO treatment of malaria. Therefore, results of single daily intraperitoneal injections were compared with results of an evenly divided four-times-daily dosage and the efficacy of delivery with implanted infusion pumps. The highest bolus dosage (1,000 mg kg-1 of body weight day-1) was as effective as the standard combination of trimethoprim with sulfamethoxazole. Unexpectedly, very little improvement was observed with the divided or continuous dosage, and several mechanisms that could account for this are discussed.

Polyamine-Regulated Translation of Spermidine/Spermine-N1-Acetyltransferase

ABSTRACT Rapid synthesis of the polyamine catabolic enzyme spermidine/spermine-N1-acetyltransferase (SSAT) in response to increased polyamines is an important polyamine homeostatic mechanism. Indirect evidence has suggested that there is an important control mechanism involving the release of a translational repressor protein that allows the immediate initiation of SSAT protein synthesis without RNA transcription, maturation, or translocation. To identify a repressor protein, we used a mass spectroscopy-based RNA-protein interaction system and found six proteins that bind to the coding region of SSAT mRNA. Individual small interfering RNA (siRNA) experiments showed that nucleolin knockdown enhances SSAT translation. Nucleolin exists in several isoforms, and we report that the isoform that binds to SSAT mRNA undergoes autocatalysis in the presence of polyamines, a result suggesting that there is a negative feedback system that helps control the cellular content of polyamines. Preliminary molecular interaction data show that a nucleolin isoform binds to a 5′ stem-loop of the coding region of SSAT mRNA. The glycine/arginine-rich C terminus of nucleolin is required for binding, and the four RNA recognition motif domains are included in the isoform that blocks SSAT translation. Understanding SSAT translational control mechanisms has the potential for the development of therapeutic strategies against cancer and obesity.

SILAC analysis of oxidative stress‐mediated proteins in human pneumocytes: New role for treacle

To better understand lung oxidant stress responses, we examined A549 lung cells exposed to H2O2 using “stable isotope labeling by amino acids.” We identified 466 cytosolic and 387 nuclear proteins; H2O2 exposure produced ≥twofold differences in 31, all were downregulations. None were previously reported as oxidant stress response proteins, although they share common functions. One of the responders, treacle, was linked to p53, an important oxidative stress response. The Treacher Collins–Franceschetti syndrome can result from treacle mutation and insufficiency was suggested to cause increased p53 leading to the syndrome. However, results here indicate p53 and treacle responses to H2O2 are independent: treacle remains suppressed after p53 recovery; the threshold for treacle reduction is well above that for p53 induction; and treacle suppression by short interfering RNA does not modify the p53 response. Evidence of treacle antioxidant activity include reduction being driven by proteasome degradation independently of mRNA, typical for oxidant‐absorbing proteins, and increased sensitivity to H2O2 consequent to short interfering RNA suppression. Data here show a link between oxidative stress and treacle reduction, demonstrate that treacle does not control p53, provide evidence of a treacle oxidant defense role, support the hypothesis that oxidant stress plays a role in the Treacher Collins–Franceschetti syndrome, and raise the possibility that treacle plays an anti‐oxidant role in lungs.

Mechanism and Tissue Specificity of Nicotine-mediated Lung S-Adenosylmethionine Reduction

We previously reported that chronic nicotine infusion blocks development of Pneumocystis pneumonia. This discovery developed from our work demonstrating the inability of this fungal pathogen to synthesize the critical metabolic intermediate S-adenosylmethionine and work by others showing nicotine to cause lung-specific reduction of S-adenosylmethionine in guinea pigs. We had found nicotine infusion to cause increased lung ornithine decarboxylase activity (rate-controlling enzyme of polyamine synthesis) and hypothesized that S-adenosylmethionine reduction is driven by up-regulated polyamine biosynthesis. Here we report a critical test of our hypothesis; inhibition of ornithine decarboxylase blocks the effect of nicotine on lung S-adenosylmethionine. Further support is provided by metabolite analyses showing nicotine to cause a strong diversion of S-adenosylmethionine toward polyamine synthesis and away from methylation reactions; these shifts are reversed by inhibition of ornithine decarboxylase. Because the nicotine effect on Pneumocystis is so striking, we considered the possibility of tissue specificity. Using laser capture microdissection, we collected samples of lung alveolar regions (site of infection) and respiratory epithelium for controls. We found nicotine to cause increased ornithine decarboxylase protein in alveolar regions but not airway epithelium; we conclude that tissue specificity likely contributes to the effect of nicotine on Pneumocystis pneumonia. Earlier we reported that the full effect of nicotine requires 3 weeks of treatment, and here we show recovery is symmetrical, also requiring 3 weeks after treatment cessation. Because this time frame is similar to pneumocyte turnover time, the shift in polyamine metabolism may occur as new pneumocytes are produced.

Pneumocystis S-adenosylmethionine transport: a potential drug target.

Pneumocystis pneumonia (PCP) is a life-threatening condition in immunosuppressed patients. Current treatments are inadequate, and new drug leads are needed. This fungus depends on its host for S-adenosylmethionine (AdoMet), a critical metabolic intermediate ordinarily synthesized by individual cells as needed. Pneumocystis contains a gene coding for the AdoMet-synthesizing enzyme methionine ATP transferase (MAT), and the protein is expressed. However, the fungus lacks MAT activity, and infection causes the depletion of host plasma AdoMet. The uptake of Pneumocystis AdoMet was shown to be exquisitely specific, which suggests the transport of AdoMet as a potential drug target. Here we report on the discovery of PcPET8, a Pneumocystis gene with homology to mitochondrial AdoMet transporters. When expressed by Saccharomyces cerevisiae, it locates properly to the mitochondrion and complements a strain of S. cerevisiae lacking its native mitochondrial AdoMet transporter. The importance of AdoMet transport is demonstrated by the ability of the AdoMet analogue sinefungin to block the uptake of Pneumocystis AdoMet and inhibit growth in culture. Because PcPET8 is likely critical for Pneumocystis, the yeast construct has potential as a surrogate for testing compounds against Pneumocystis.

Effect of a Bis-Benzyl Polyamine Analogue on Pneumocystis carinii

ABSTRACT Pneumocystis carinii is the causative agent of P. carinii pneumonia (PCP), an opportunistic infection associated with AIDS and other immunosuppressed conditions. Although polyamine metabolism of this fungus has been shown to be a chemotherapeutic target, this metabolism has not been thoroughly investigated. Reported here is the effect of one polyamine analogue,N,N′-bis{3-[(phenylmethyl)amino]propyl}-1,7-diaminoheptane (BBS), on P. carinii. BBS inhibits the growth of P. carinii in culture, but at concentrations higher than those required to inhibit the growth of other pathogens. However, BBS is at least as active in an animal model of PCP as in other models of diseases studied. BBS causes some reduction in P. cariniipolyamine content and polyamine biosynthetic enzyme activities, but the effect is less than that observed with other pathogens and very much less than the effect of the polyamine biosynthesis inhibitordl-α-difluoromethylornithine. BBS enters P. carinii cells via a polyamine transporter, unlike all other cells that have been studied. P. carinii cells do not remove the benzyl groups of BBS, as is reported for mammalian cells. The most likely mode of action is displacement of natural polyamines. Overall, the activity of BBS provides further evidence that polyamines and polyamine metabolism are rational targets for the development of drugs to treat PCP. Because the details of BBS-P. cariniiinteraction differ from those of other cells studied, polyamine analogues may provide a highly specific treatment for PCP.