Effective use of preparative chiral HPLC in a preclinical drug synthesis

Access to a key 3-aryl-delta-lactone intermediate in enantiopure form using preparative chiral chromatography allowed expedited preparation of an important drug discovery target. A preclinical drug discovery strategy that combines rapid route discovery with effective use of preparative chiral chromatography can result in significant savings of both time and labor.     

Descriptive urological record of chimpanzees (Pan troglodytes) in the wild and limitations associated with using multi-reagent dipstick test strips

Ten urine chemistry parameters were measured on 74 voided urine samples from 34 wild chimpanzees (Pan troglodytes). Multi-reagent urine dipstick tests were performed and results determined using colorimetric scales. Urine pH measured between 8 and 9 units in 91% of the chimpanzees. Test pads detected protein, erythrocytes, leukocyte esterase activity, and nitrites, ketones and bilirubin in 47, 32, 29, and <10% of the chimpanzees, respectively. No apparent association between positive test results for blood in adult females and reproductive status was found. Overall, 17 of the 34 chimpanzees had positive urine test results for protein, hemoglobin, erythrocytes, leukocytes, nitrites, ketones, and/or bilirubin. Dipstick urinalysis alone is an unreliable method for assessing health and physiological status of wild chimpanzees. However, if combined with other diagnostics it could prove to be a valuable health-monitoring tool. Limitations associated with this methodology need to be considered when interpreting urinary dipstick test results.     

Preferential escape of subdominant CD8+ T cells during negative selection results in an altered antiviral T cell hierarchy

Negative selection is designed to purge the immune system of high-avidity, self-reactive T cells and thereby protect the host from overt autoimmunity. In this in vivo viral infection model, we show that there is a previously unappreciated dichotomy involved in negative selection in which high-avidity CD8(+) T cells specific for a dominant epitope are eliminated, whereas T cells specific for a subdominant epitope on the same protein preferentially escape deletion. Although this resulted in significant skewing of immunodominance and a substantial depletion of the most promiscuous T cells, thymic and/or peripheral deletion of high-avidity CD8(+) T cells was not accompanied by any major change in the TCR V beta gene family usage or an absolute deletion of a single preferred complementarity-determining region 3 length polymorphism. This suggests that negative selection allows high-avidity CD8(+) T cells specific for subdominant or cryptic epitopes to persist while effectively deleting high-avidity T cells specific for dominant epitopes. By allowing the escape of subdominant T cells, this process still preserves a relatively broad peripheral TCR repertoire that can actively participate in antiviral and/or autoreactive immune responses.     

Proteasome-cytochrome c interactions: a model system for investigation of proteasome host-guest interactions

Owing to its high thermal stability and structural simplicity, the archaebacterium Thermoplasma Acidophilum 20S proteasome was selected for mechanistic studies in this work. This oligomeric enzyme complex consists of a barrel-shaped 20S core (approximately 700kDa) comprised of four stacked seven-membered rings with a alpha(7)beta(7)beta(7)alpha(7) subunit structure situated around a 7-fold symmetry axis. The hollow interior of the proteasome has three large interconnected chambers with narrow (13 A diameter) entrances from solution located at either end of the barrel. The 14 beta-subunit proteolytic sites are located on the inner surface of the central chamber. Herein, we demonstrate that unfolded horse heart ferricytochrome c (Cyt c) is a novel chromophoric probe for investigation of the mechanism of proteasome action. Under conditions of temperature and denaturant which unfold Cyt c but do not alter the thermophilic proteasome, Cyt c is extensively cleaved by the proteasome. Ten peptides were isolated and sequenced from the proteasome digest. Analysis of the cleavage products established that unfolded Cyt c and its covalently attached heme prosthetic group are translocated to the central chamber where proteolysis occurs. In the presence of site-specific inhibitors of the proteasome, we demonstrate that unfolded cytochrome c can be sequestered inside the proteasome complex. Upon cooling, a quasistable host-guest complex is formed. Analysis of the complex via UV/visible spectroscopy and mass spectrometry gave evidence that the sequestered Cyt c is essentially intact within the inhibited proteasome. High-performance liquid chromatography data show that (1) complexes with an apparent stoichiometry of approximately one Cyt c per proteasome can be formed and (2) when inhibition is removed from the complex, a rapid turnover of the sequestered Cyt c occurs.     

Using simulation and budget models to scale-up nitrogen leaching from field to region in Canada

Efforts are underway at Agriculture and Agri-Food Canada (AAFC) to develop an integrated, nationally applicable, socioeconomic/biophysical modeling capability in order to predict the environmental impacts of policy and program scenarios. This paper outlines our Decision Support System (DSS), which integrates the IROWCN (Indicator of the Risk of Water Contamination by Nitrogen) index with the agricultural policy model CRAM (Canadian Regional Agricultural Model) and presents an outline of our methodology to provide independent assessments of the IROWCN results through the use of nitrogen (N) simulation models in select, data-rich areas. Three field-level models--DSSAT, N_ABLE, and EPIC--were evaluated using local measured data. The results show that all three dynamic models can be used to simulate biomass, grain yield, and soil N dynamics at the field level; but the accuracy of the models differ, suggesting that models need to be calibrated using local measured data before they are used in Canada. Further simulation of IROWCN in a maize field using N_ABLE showed that soil-mineral N levels are highly affected by the amount of fertilizer N applied and the time of year, meaning that fertilizer and manure N applications and weather data are crucial for improving IROWCN. Methods of scaling-up simulated IROWCN from field-level to soil-landscape polygons and CRAM regions are discussed.     

Regulation of MTP expression in developing swine

To define the developmental expression of microsomal triglyceride transfer protein (MTP) large subunit mRNA and protein, samples of small intestine and liver were collected from 40-day gestation fetal, 2-day-old newborn, 3-week-old suckling, and 2-month-old weanling swine. In fetal animals, MTP mRNA expression was high in intestine and liver. Postnatally, jejunal expression paralleled the intake of a high-fat breast milk diet and declined after weaning. Ileal expression was comparable with that of jejunum in 2-day-old animals, but declined to low levels afterward. Hepatic expression declined postnatally and remained low. MTP protein expression generally paralleled mRNA expression, except in fetal intestine in which no 97 kDa protein was detected. In 2-day-old piglets, a high-triacylglycerol diet increased jejunal and ileal MTP mRNA levels, as compared to a low-triacylglycerol diet. To test the roles of glucocorticoids and fatty acids in MTP regulation, a newborn swine enterocyte cell line (IPEC-1) was used. Except at day 2 of differentiation, dexamethasone did not influence MTP expression. Fatty acids either up-regulated or down-regulated MTP expression, depending on the specific fatty acid and duration of exposure. Although programmed genetic cues regulate MTP expression during development, clearly the amount and fatty acid composition of dietary lipid also play regulatory roles.     

Comparison of tissue culture and animal models for assessment of Cryptospridium parvum infection

The current increased interest for using tissue culture as a surrogate for mouse infection to assess Cryptospridium viability suggests that a comparison of the two models is essential for data interpretation. Therefore, a need remains for a statistical comparison that can demonstrate if infection and inactivation predicted by new tissue culture models are comparable with those predicted by animal models. Data from a total of 31 dose-response trials using both tissue culture and mouse models to assess C. parvum infectivity were compared. The dose needed to infect 50% of the tissue cultures (ID(50)) was also compared to each ID(50) in mice. Average ID(50)s developed using the logit dose-response method for tissue culture and mice were 8 and 107, respectively, suggesting that tissue culture was more sensitive to infection. However, correlation (r) between tissue culture and mouse infectivity was statistically significant (0.9167 [95% CI=0.8428 to 0.9594, p<0.0001]). Comparison of oocyst disinfection by UV and chlorine dioxide showed no significant difference between inactivation predicted by tissue culture and mouse models (p=0.8893; t=0.0141; n=21). These results demonstrate that tissue culture can successfully be used to measure C. parvum infection and can be used for determining inactivation in disinfection studies.     

Increase in tannin consumption by sifaka (<Emphasis Type="Italic">Propithecus verreauxi verreauxi</Emphasis>) females during the birth season: a case for self-medication in prosimians?

In this study we report preliminary data on the consumption of tannin-rich plants by sifakas (Propithecus verreauxi verreauxi) living in the Kirindy forest, western Madagascar. Sifakas spent most of their time feeding on only a few plant species. The tannin intake during the period between the pregnancy and birth season was significantly higher in pregnant females or females with lactating infants than in non-reproductive females and males. These periparturient females secured a larger proportion of condensed tannins by short feeding bouts on plants not included in the group's limited preferred food species. The measured increase in tannin intake is puzzling in light of the fact that tannins are commonly known for their protein-binding properties. Since protein demands are highest in pregnant and lactating females, possible medicinal benefits of tannin ingestion are considered. Tannin consumption is associated with an increase in body weight and stimulation of milk secretion. Veterinarians administer tannins as an astringent, anti-hemorrhagic and anti-abortive. Their high potential as an alternative anthelminthic has also recently been recognized. Thus, when viewed as self-medicating behavior, controlled increase in tannin intake could have multiple prophylactic advantages for females during the periparturient period. The high selectivity in their plant choice, and the presence of unusual feeding habits by a particular group of individuals (females with infants) limited in time (birth season), suggests that an increase in tannin ingestion may be a self-medicating behavior with multiple directly adaptive benefits to female reproduction. Electronic Publication     

Extraordinarily low bone mineral density in an old female chimpanzee (<Emphasis Type="Italic">Pan troglodytes schweinfurthii</Emphasis>) from the Mahale Mountains National Park

We examined bone mineral density (BMD) of the femoral neck and lumbar vertebrae of four chimpanzee skeletons from Mahale Mountains National Park, Tanzania, and four captive ones, with a dual energy X-ray absorptiometer. The BMD of Wansombo, an old female chimpanzee from Mahale , was remarkably lower than the mean of the other six younger adult female chimpanzees and categorized as osteoporosis. Posture, locomotion, and trunk-sacral anatomy of chimpanzees may have prevented fractures in Wansombo, whose BMD was below human osteoporosis criteria. Electronic Publication     

Phenylbutyrates as potent,orally bioavailable vitronectin receptor (integrin alphavbeta3) antagonists

In our continuing efforts to identify small molecule vitronectin receptor antagonists, we have discovered a series of phenylbutyrate derivatives, exemplified by 16, which have good potency and excellent oral bioavailability (approximately 100% in rats). This new series is derived conceptually from opening of the seven-membered ring of SB-265123.