Monitoring the effects of drug treatment in rat models of disease by serum protein analysis

In this review we list from literature investigations on rat serum proteins using electrophoretic techniques in connection with drug testing. From our own research work, we provide annotated two-dimensional maps of rat serum proteins under control and experimental conditions. Emphasis is on species-specific components and on the effects of acute and chronic inflammation. We discuss our project of structural proteomics on rat serum as a minimally invasive approach to pharmacological investigation, and we outline a typical experimental plan for drug testing according to the above guidelines. We then report in detail on the results of our trials of anti-inflammatory drugs on adjuvant arthritis, an animal model of disease resembling in many aspects human rheumatoid arthritis. We demonstrate a correlation between biochemical parameters and therapeutic findings and outline the advantages of the chosen methodological approach, which proved also sensitive in revealing "side effects" of the test drugs. In an appendix we describe our experimental protocol when performing two-dimensional electrophoresis of rat serum.     

Correlation between loss of pro-chiral hydrogen and E,Z geometry in isoprenoid biosynthesis

Biosynthesis of several mono- and sesqui-terpenes that possess E or Z double bonds, or which are generally considered to be derived from precursors possessing such geometries, involved loss of the pro-4S hydrogen of mevalonate in the construction of the double bond. These results confirm and extend previous observations. A recent claim to have newly discovered such a stereochemical correlation is rejected.     

Enzymic aspects of the biosynthesis monoterpenes in plants

The biosynthesis of monoterpenes in higher plants is reviewed, with particular emphasis on recent studies of the enzymology of biosynthesis.     

Preclinical models for neuroblastoma: establishing a baseline for treatment

Background

Preclinical models of pediatric cancers are essential for testing newchemotherapeutic combinations for clinical trials. The most widely usedgenetic model for preclinical testing of neuroblastoma is the TH-MYCN mouse.This neuroblastoma-prone mouse recapitulates many of the features of humanneuroblastoma. Limitations of this model include the low frequency of bonemarrow metastasis, the lack of information on whether the gene expressionpatterns in this system parallels human neuroblastomas, the relatively slowrate of tumor formation and variability in tumor penetrance on differentgenetic backgrounds. As an alternative, preclinical studies are frequentlyperformed using human cell lines xenografted into immunocompromised mice,either as flank implant or orthtotopically. Drawbacks of this system includethe use of cell lines that have been in culture for years, the inappropriatemicroenvironment of the flank or difficult, time consuming surgery fororthotopic transplants and the absence of an intact immune system.

Principal Findings

Here we characterize and optimize both systems to increase their utility forpreclinical studies. We show that TH-MYCN mice develop tumors in theparaspinal ganglia, but not in the adrenal, with cellular and geneexpression patterns similar to human NB. In addition, we present a newultrasound guided, minimally invasive orthotopic xenograft method. Thisinjection technique is rapid, provides accurate targeting of the injectedcells and leads to efficient engraftment. We also demonstrate that tumorscan be detected, monitored and quantified prior to visualization usingultrasound, MRI and bioluminescence. Finally we develop and test a“standard of care” chemotherapy regimen. This protocol, which isbased on current treatments for neuroblastoma, provides a baseline forcomparison of new therapeutic agents.

Significance

The studies suggest that use of both the TH-NMYC model of neuroblastoma andthe orthotopic xenograft model provide the optimal combination for testingnew chemotherapies for this devastating childhood cancer.     

Thermoregulation and roost selection by reproductive female big brown bats (Eptesicus fuscus) roosting in rock crevices

A free-ranging maternity colony of big brown bats Eptesicus fuscus roosting in rock crevices along the South Saskatchewan River in south-eastern Alberta, Canada, was studied to understand better the discrepancy that exists in the literature regarding torpor use by reproductive female bats. Using radio-telemetry, thermoregulatory patterns and roost microclimate were recorded for pregnant, lactating and post-lactating females. Relative torpor use is described in several ways: the proportion of days on which torpor was used, depth, minimum body temperature, time spent in torpor, and a comprehensive torpor unit (degree-min). Pregnant and lactating female E. fuscus used torpor to the same extent overall (degree-min), but pregnant bats used torpor less frequently and with more time in deep torpor. Torpor was used to the greatest extent after weaning (post-lactation). Evidence is presented that the cost:benefit ratio for deep and prolonged periods of torpor may be highest during lactation. Microclimates of rock-crevice roosts mirrored the use of torpor throughout reproduction by bats. Lactation roosts (deeper, larger opening size) were more thermally stable and remained warmer at night compared to the shallow roosts used by pregnant and post-lactating females. It is shown that conclusions about relative use of torpor can differ depending on the units of comparison, necessitating measurement of all aspects of torpor (depth, duration and frequency). Comprehensive measurements, individual-based normothermic temperatures, and a definition of torpor that accounts for all energy savings, allow a more accurate depiction of patterns and facilitates inter-study comparisons.     

Identification of proteins undergoing glutathionylation in oxidatively stressed hepatocytes and hepatoma cells

Protein glutathionylation is a post-translational modification consisting of the formation of a mixed disulfide between protein cysteines and glutathione (GSH). To identify proteins undergoing glutathionylation in primary rat hepatocytes and in human HepG2 hepatoma cells, we radiolabeled the intracellular GSH pool with L-[(35)S] cysteine. Cells were then exposed to oxidative stress. Proteins were separated by two-dimensional gel electrophoresis under nonreducing conditions, and glutathionylated proteins were located by autoradiography and identified by mass spectrometry after tryptic digestion. Several proteins previously not known to undergo glutathionylation were thus recognized. Among the identified proteins some are the same or belong to the same functional class as those we have already identified in a previous paper on T cell blasts (actin, nucleophosmin, phosphogluconolactonase, myosin, profilin, cyclophilin A, stress 70 protein, ubiquitin in HepG2 cells and actin, peroxiredoxin 5, cytochrome C oxidase, heat shock cognate 70 in hepatocytes) while others are newly recognized (Ran specific GTPase activating protein, histidine triad nucleotide binding protein 2 in HepG2 cells and enoyl CoA hydratase in hepatocytes). The technique described proved equally applicable to a variety of cell types.