Plasmodium falciparum is responsible for the majority of life-threatening cases of malaria. Plasmodia species cannot synthesize purines de novo, whereas mammalian cells obtain purines from de novo synthesis or by purine salvage. Hypoxanthine is proposed to be the major source of purines for P. falciparum growth. It is produced from inosine phosphorolysis by purine nucleoside phosphorylase (PNP) Malaria is caused by the protozoan parasites Plasmodium falciparum, ovale, vivax, and malariae and is responsible for an estimated 1-2 million deaths per year (1, 2). Controlling this disease with current anti-malarials has become more difficult because of emerging drug-resistant strains (1, 2). Therefore, the validation of alternative anti-malarial targets is crucial to development of new chemotherapeutic approaches. The purine salvage pathway has been identified as a potential anti-malarial target (3, 4). Unlike its host, Plasmodium cannot synthesize purines de novo and depends exclusively on purine salvage for RNA and DNA synthesis (3, 5-8). Erythrocytes also lack enzymes for de novo purine synthesis. The parasite requires a continuous supply of purines for RNA and DNA synthesis during its replication in the erythrocyte..Adenosine is a major purine nucleoside of human blood, but the adenosine kinase activity of P. falciparum is very low (9). Adenosine deaminase is very active in the erythrocyte and the parasite; hence deamination and subsequent phosphorolysis of the product inosine by PNP 1 yield hypoxanthine, a major purine precursor for purine salvage (Fig. 1). There is no adenosine phosphorylase activity in humans, and adenine is present at very low concentrations (10). Hypoxanthine is converted to IMP by hypoxanthine phosphoribosyltransferase activity, present in large amounts in P. falciparum (11). This path makes hypoxanthine the common precursor for all purine nucleotides in the parasite (12). Depleting Plasmodium of hypoxanthine by the addition of xanthine oxidase to culture medium prevents parasite growth (13,14). This mechanism has also evolved in vivo in the cape buffalo, where resistance to Trypanosoma brucei infections has resulted from an active serum xanthine oxidase (15).In mammals, hypoxanthine is formed from nucleosides only through phosphorolysis of inosine, the reaction catalyzed by PNP. Here we test the hypothesis that inhibition of purine nucleoside phosphorylase in infected erythrocytes will inhibit P. falciparum growth by preventing hypoxanthine production. This goal is possible through the use of transition state analogue inhibitors we have developed against both human and P. falciparum PNPs (16,17). The immucillins are purine nucleoside analogue inhibitors containing a 1-(9-deazapurin-9-yl)-1,4-dideoxy-4-iminoribitol moiety (Fig. 2). Inhibition of both human and P. falciparum PNP by immucillins prevents the utilization of inosine and deoxyinosine as hypoxanthine sources. P. falciparum parasites cultured in human erythrocytes are killed by immucillins but can be rescued by hypoxanthine and not ...

A major unresolved issue in the field of secretory granule biogenesis is the extent to which the aggregation of granule content proteins is responsible for the sorting of regulated from constitutively secreted proteins. The aggregation process is postulated to take place in the trans-Golgi network and immature secretory granules as the proteins encounter mildly acidic pH and high calcium concentrations. We have developed in vitro assays that reconstitute the precipitation out of solution of secretory granule content proteins of anterior pituitary gland and adrenal medulla. In the assays, all of the major granule content polypeptides form a precipitate as the pH is titrated below 6.5, and this precipitate can be recovered in the pellet fraction after centrifugation. Addition of calcium is required for the aggregation of chromaffin granule content. In contrast to the proteins secreted by the regulated pathway, the constitutively secreted proteins IgG, albumin, and angiotensinogen, when added to the assays, remain predominantly in the supernatant. Among the individual proteins tested, prolactin is found to aggregate homophilically under these conditions and can drive the co-aggregation of other proteins, such as the chromogranins. Soluble forms of granule membrane proteins, including dopamine beta-hydroxylase and peptidyl glycine alpha-amidating enzyme also co-aggregated with granule content proteins. The results are consistent with the idea that spontaneous aggregation of proteins occurring under ionic conditions similar to those at the sites of granule formation is a property restricted to those proteins packaged in secretory granules. In addition, the association of luminal domains of membrane proteins with content proteins in vitro raises the possibility that analogous interactions between membrane-bound and content proteins also occur during granule formation in intact cells.

Immucillins are logically designed transition-state analogue inhibitors of mammalian purine nucleoside phosphorylase (PNP) that induce purine-less death of Plasmodium falciparum in cultured erythrocytes (Kicska, G. A., Tyler, P. C., Evans, G. B., Furneaux, R. H., Schramm, V. L., and Kim, K. (2002) J. Biol. Chem. 277, 3226 -3231). PNP is present at high levels in human erythrocytes and in P. falciparum, but the Plasmodium enzyme has not been characterized. A search of the P. falciparum genome data base yielded an open reading frame similar to the PNP from Escherichia coli. PNP from P. falciparum (P. falciparum PNP) was cloned, overexpressed in E. coli, purified, and characterized. The primary amino acid sequence has 26% identity with E. coli PNP, has 20% identity with human PNP, and is phylogenetically unique among known PNPs with equal genetic distance between PNPs and uridine phosphorylases. Recombinant P. falciparum PNP is catalytically active for inosine and guanosine but is less active for uridine. The immucillins are powerful inhibitors of P. falciparum PNP. Immucillin-H is a slow onset tight binding inhibitor with a K i * value of 0.6 nM. Eight related immucillins are also powerful inhibitors with dissociation constants from 0.9 to 20 nM. The K m /K i * value for immucillin-H is 9000, making this inhibitor the most powerful yet reported for P. falciparum PNP. The PNP from P. falciparum differs from the human enzyme by a lower K m for inosine, decreased preference for deoxyguanosine, and reduced affinity for the immucillins, with the exception of 5-deoxy-immucillin-H. These properties of P. falciparum PNP are consistent with a metabolic role in purine salvage and provide an explanation for the antibiotic effect of the immucillins on P. falciparum cultured in human erythrocytes.

The Coronavirus Disease 2019 pandemic initially presented in the United States in the greater Seattle area, and has rapidly progressed across the nation in the past 2 months, with the United States having the highest number of cases in the world. Radiology departments play a critical role in policy and guideline development both for the department and for the institutions, specifically in planning diagnostic screening, triage, and management of patients. In addition, radiology workflows, volumes and access must be optimized in preparation for the expected COVID-19 patient surges. This article discusses the processes that have been implemented at the University of Washington in managing the COVID-19 pandemic as well in preparing for patient surges, which may provide important guidance for other radiology departments who are in the early stages of preparation and management. Essentials Radiology policy goals are to reduce COVID-19-related morbidity and mortality through early diagnosis, appropriate treatment and prevention of disease dissemination.  Imaging currently is not routinely used to screen for COVID-19 unless access to RT-PCR results for COVID-19 is limited.  Postponing elective imaging and procedures will preserve resources and hospital beds, while also limiting patient population exposures.  Determination of time-sensitivity of procedures and imaging tests is by consensus with input from radiologists, patients, and/or ordering clinicians.  Radiology departments must prepare for patient surges through streamlined approaches to imaging that will limit exposures to healthcare workers and patients.