Synthesis and antiviral activity of phosphonoacetic and phosphonoformic acid esters of 5-bromo-2'-deoxyuridine and related pyrimidine nucleosides and acyclonucleosides

Phosphonoacetic acid (PAA, 1) was coupled with various acyclonucleosides, 2'-deoxyuridines, cytidines, and arabinosyluracils, with 2,4,6-triisopropylbenzenesulfonyl chloride (TPS) or dicyclohexylcarbodiimide (DCCI) as condensing agents, to give a range of phosphonate esters. The carboxylic ester linkage of PAA to the 5'-position of 5-bromo-2'-deoxyuridine (BUdR, 3) was achieved via the mixed anhydride formed from (diethylphosphono)acetic acid and trifluoroacetic anhydride. Phosphonoformic acid (PFA, 2) was coupled with BUdR by using the DCCI method to give the phosphonate ester. Of these compounds only phosphonate esters in the 2'-deoxyuridine series showed significant activity against herpes simplex virus types 1 and 2. The BUdR-PAA derivative and the BUdR-PFA derivative were highly active, especially the latter, which was more active than the parent nucleoside BUdR against the type 2 virus. The active compounds may exert their effects by extracellular or intracellular hydrolysis to the corresponding antiviral agents, but an intrinsic component of antiviral activity may also be involved.     

Missense mutations in the human glutathione synthetase gene result in severe metabolic acidosis, 5-oxoprolinuria, hemolytic anemia and neurological dysfunction

Severe glutathione synthetase (GS) deficiency is a rare genetic disorder with neonatal onset. The enzymatic block of the gamma-glutamyl cycle leads to a generalized glutathione deficiency. Clinically affected patients present with severe metabolic acidosis, 5- oxoprolinuria, increased rate of hemolysis and defective function of the central nervous system. The disorder is inherited in an autosomal recessive mode and, until recently, the molecular basis has remained unknown. We have sequenced 18 GS alleles associated with enzyme deficiency and we detected missense mutations by direct sequencing of cDNAs and genomic DNA. In total, 13 different mutations were identified. Four patients were found to be compound heterozygotes and two individuals were apparently homozygous. Reduced enzymatic activities were demonstrated in recombinant protein expressed from cDNAs in four cases with different missense mutations. The results from biochemical analysis of patient specimens, supported by the properties of the expressed mutant proteins, indicate that a residual activity is present in affected individuals. Our results suggest that complete loss of function of both GS alleles is probably lethal. It is postulated that missense mutations will account for the phenotype in the majority of patients with severe GS deficiency.      

Mobile Application for Promoting Gluten-Free Diet Self-Management in Adolescents with Celiac Disease: Proof-of-Concept Study

Celiac disease (CD) is a chronic disease treated by maintaining and managing a lifelong restrictive gluten-free diet. The purpose of this study was to develop a mobile application, Plan My C-Day, to promote self-management skills among youth with CD during adolescence—a time when decreased adherence often occurs—and examine its usability among adolescents with CD. Plan My C-Day contains three simulations of activities involving eating out and actions to take when preparing for these events. It was developed and pilot tested by 13 adolescents with CD. Application use and user perception data were collected and analyzed. Participants chose 160 actions within the simulations. For over 75% of participants, the time to complete the simulation decreased from the first to the third (last) simulation by an average of 50%. The average reported usability perception was 3.71 on a scale of 1 to 5, with system ease of use and ease of learning obtaining the highest scores. This study demonstrated that the Plan My C-Day mobile application’s self-management content, features, and functions operated well and that the simulations were easy to understand and complete. Further development will include the option to add self-created activities and adaptation to different languages and cultures.     

Effect of de novo purine synthesis inhibitors on 5-fluorouracil metabolism and cytotoxicity

Methotrexate pretreatment of L1210 cells had been shown previously by us to cause an enhancement of the intracellular accumulation of 5-fluorouracil and of the formation of 5-fluorouracil nucleotides which was correlated with synergistic cytotoxicity. This effect of methotrexate was associated with increases in 5-phosphoribosyl-1-pyrophosphate, the cofactor required for the conversion of 5-fluorouracil to 5-fluorouridine-5'-monophosphate (FUMP). Because these influences on 5-fluorouracil metabolism were most likely mediated by the activity of methotrexate as an inhibitor of purine synthesis, the effects of other agents that inhibit purine synthesis were examined. An inhibitor of amidophosphoribosyltransferase, 6-methylmercaptopurine ribonucleoside, the glutamine antagonists, azaserine and 6-diazo-5-oxo-L-norleucine (DON), and the L-aspartate analogue inhibitor of adenylsuccinate synthetase, L-alanosine, all reduced the incorporation of [1-14C]glycine into adenine and guanine bases isolated from nucleic acids. Each drug also resulted in intracellular elevations of 5-phosphoribosyl-1-pyrophosphate that were 15- to 25-fold greater than control levels. These alterations in de novo purine nucleotide synthesis were associated with enhanced intracellular 5-fluorouracil accumulation and synergistic cytotoxicity.     

Rapid and reliable detection of previous freezing of cerebral tissue by computed tomography and magnetic resonance imaging

Due to slowing or even inhibition of postmortem processes, freezing may make an estimation of the time-since-death very difficult. This is also true in previously frozen and subsequently thawed bodies. Knowledge of prior freezing is important, as it may lead to a different assessment of the time since death. Twelve pig heads were frozen at ?20 °C, and 6 heads were either kept at room temperature (approximately 20 °C) or in a cooling cell (approximately 5 °C). The frozen brains and cadavers were thawed at either room temperature or in a cooling cell. All specimens underwent repeated CT and MRI scanning until the brains were sampled for histological examination. Two radiologists assessed the images and two pathologists reviewed the histological slides with regard to thawing artifacts and putrefaction. All raters were blinded regarding whether the samples had been frozen, for how long and how they had been thawed. Imaging revealed distinct, tiny bubble-like artifacts only in previously frozen specimens. Histology also revealed artifacts only seen in such cases, namely very distinct, columnar bubbles in the cerebral cortex. All raters successfully identified previously unfrozen brains (100% specificity) and nearly all previously frozen brains. Our results suggest that initial post-mortem imaging can be of enormous importance in everyday forensic practice by identifying possible cases of previous freezing – cases that would therefore warrant closer scrutiny and thus raise caution regarding the time of death.