Buchbesprechungen

Ohne Zusammenfassung

---

Book reviews

     

Buchbesprechungen

Ohne Zusammenfassung     

Buchbesprechungen

Ohne Zusammenfassung     

Neuroprotection against CCl4 induced brain damage with crocin in Wistar rats

Owing to its lipophilic property, carbon tetrachloride (CCl₄) is rapidly absorbed by both the liver and brain. We investigated the protective effects of crocin against brain damage caused by CCl₄. Fifty rats were divided into five groups of ten: control, corn oil, crocin, CCl₄ and CCl₄ + crocin. CCl₄ administration decreased glutathione (GSH) and total antioxidant status (TAS) levels, and catalase (CAT) activity, while significant increases were observed in malondialdehyde (MDA) and total oxidant status (TOS) levels and superoxide dismutase (SOD) activity. The cerebral cortex nuclear lamina developed a spongy appearance, neuronal degeneration was observed in the hippocampus, and heterochromatic and pyknotic neurons with increased cytoplasmic eosinophilia were observed in the hippocampus after CCl₄ treatment. Because crocin exhibits strong antioxidant properties, crocin treatment increased GSH and TAS levels and CAT activities, and decreased MDA and TOS levels and SOD activity; significant improvements also were observed in histologic architecture. We found that crocin administration nearly eliminated CCl₄ induced brain damage by preventing oxidative stress.     

Lipophilic prodrugs of a triazole-containing colchicine analogue in liposomes: Biological effects on human tumor cells

Colchicine site binders—blockers of tubulin polymerization—are potential antimitotic agents for anticancer therapy. To reduce their systemic toxicity and improve biodistribution, encapsulation in nanosized liposomes may be employed. Liposomes present a convenient means for preparation of injectable for-mulations of hydrophobic compounds, however colchicine as such is known to leak through the lipid bilayer. In this study, newly synthesized triazole-containing analogues of colchicine and allocolchicine, and their palmitic and oleic esters (lipophilic prodrugs) were tested for anti-proliferative activity and apoptosis-inducing potential. In contrast to colchicine conjugates, whose activities ranged with those of colchicine, allocolchicine derivatives exhibited drastically lower effects and were discarded. Liposomes of about 100 nm in diameter composed of egg phosphatidylcholine-yeast phosphatidylinositol-palmitic or oleic prodrug, 8: 1: 1, by mol, were prepared by standard extrusion technique and tested in a panel of four human tumor cell lines. Liposome formulations preserved the biological activities of the parent colchicinoid the most towards human epithelial tumor cells. Moreover, liposomal form of the oleoyl bearing colchicinoid inhibited cell proliferation more efficiently than free lipophilic prodrug. Due to substantial loading capacity of the liposomes, the dispersions contain sufficient concentration of the active agent to test wide dose range in experiments on systemic administration to animals.     

Identification and Monitoring of Host Cell Proteins by Mass Spectrometry Combined with High Performance Immunochemistry Testing

Biotherapeutics are often produced in non-human host cells like Escherichia coli, yeast, and various mammalian cell lines. A major focus of any therapeutic protein purification process is to reduce host cell proteins to an acceptable low level. In this study, various E. coli host cell proteins were identified at different purifications steps by HPLC fractionation, SDS-PAGE analysis, and tryptic peptide mapping combined with online liquid chromatography mass spectrometry (LC-MS). However, no host cell proteins could be verified by direct LC-MS analysis of final drug substance material. In contrast, the application of affinity enrichment chromatography prior to comprehensive LC-MS was adequate to identify several low abundant host cell proteins at the final drug substance level. Bacterial alkaline phosphatase (BAP) was identified as being the most abundant host cell protein at several purification steps. Thus, we firstly established two different assays for enzymatic and immunological BAP monitoring using the cobas® technology. By using this strategy we were able to demonstrate an almost complete removal of BAP enzymatic activity by the established therapeutic protein purification process. In summary, the impact of fermentation, purification, and formulation conditions on host cell protein removal and biological activity can be conducted by monitoring process-specific host cell proteins in a GMP-compatible and high-throughput (> 1000 samples/day) manner.     

Predominance of Methanolobus spp. and Methanoculleus spp. in the Archaeal Communities of Saline Gas Field Formation Fluids

The microbial communities in sulfate-rich, saline formation fluids of a natural gas reservoir in Lower Saxony, Germany were investigated to enhance the knowledge about microbial communities in potential carbon dioxide sequestration sites. This investigation of the initial state of the deep subsurface microbiota is necessary to predict their influence on the long-term stability and storage capacity of such sites. While the bacterial 16S rDNA gene library was comprised of sequences affiliating with the Firmicutes, the Alphaproteobacteria, the Gammaproteobacteria and the Thermotogales, the archaeal 16S rDNA libraries were simply dominated by two phylotypes related to the genera Methanolobus and Methanoculleus. The monitoring of the archaeal communities in different formation fluid samples by T-RFLP and Real-Time-PCR indicated that these two methanogenic genera dominated at all, whereas the proportion of the two groups varied. Thus, methylotrophic and autotrophic methanogenesis seems to be of importance in the reservoir fluids, dependent on the provided reduction equivalents and substrates and it also may influence the fate of CO₂ in the subsurface.     

Paraoxonase-1 is a major determinant of clopidogrel efficacy

Clinical efficacy of the antiplatelet drug clopidogrel is hampered by its variable biotransformation into the active metabolite. The variability in the clinical response to clopidogrel treatment has been attributed to genetic factors, but the specific genes and mechanisms underlying clopidogrel bioactivation remain unclear. Using in vitro metabolomic profiling techniques, we identified paraoxonase-1 (PON1) as the crucial enzyme for clopidogrel bioactivation, with its common Q192R polymorphism determining the rate of active metabolite formation. We tested the clinical relevance of the PON1 Q192R genotype in a population of individuals with coronary artery disease who underwent stent implantation and received clopidogrel therapy. PON1 QQ192 homozygous individuals showed a considerably higher risk than RR192 homozygous individuals of stent thrombosis, lower PON1 plasma activity, lower plasma concentrations of active metabolite and lower platelet inhibition. Thus, we identified PON1 as a key factor for the bioactivation and clinical activity of clopidogrel. These findings have therapeutic implications and may be exploited to prospectively assess the clinical efficacy of clopidogrel.     

Online immunoaffinity liquid chromatography/tandem mass spectrometry determination of a type II collagen peptide biomarker in rat urine: Investigation of the impact of collision-induced dissociation fluctuation on peptide quantitation

Proteolytic fragments of type II collagen, a major component of joint tissue, have recently been identified as biomarkers for osteoarthritis, a progressive disease associated with cartilage degeneration. A liquid chromatography/tandem mass spectrometry (MS/MS) assay that utilizes online immunoaffinity chromatography and column switching was developed in our laboratory for the neoepitope of type II collagen (NET2C). During method development, peptide collision-induced dissociation (CID) was found to be a significant source of assay variation, which exceeded 10% CV, despite the fact that a stable-isotope-labeled (SIL) internal standard was used to minimize imprecision. This phenomenon was studied in detail using peptides and associated SIL internal standards of varying lengths and amino acid compositions. Variability in peptide CID necessitated the monitoring of multiple MS/MS transitions to obtain acceptable assay precision. The assay was subsequently validated to measure NET2C concentrations in rat urine over the range of 0.1 to 10 ng/mL. The interday accuracy and precision ranged from 3.9 to 13.1 (%CV) and 10.7 to 5.3 (%RE), respectively, across the range of validated concentrations. A specific application of the assay is presented in which the role of estrogen deficiency in the development and progression of osteoarthritis was investigated. In this study, the effect of estrogen on lowering NET2C concentrations in urine in ovariectomized rats was demonstrated.