Determination of clindamycin in human plasma by high-performance liquid chromatography using coupled columns

A rapid automated method has been developed for the determination of clindamycin, a lincosamide antibiotic, in human plasma. Coupled column HPLC was used after precipitation of plasma proteins with a saturated ammonium sulfate solution. As a first step, the drug and internal standard were trapped on a precolumn of LiChrospher 60RP-select B. A reversed-phase Nucleosil 100 C18 HD column then separated drug and internal standard from each other and from remaining plasma components. The assay was validated in the range 0.2–10.0 μg ml⁻¹ plasma. The results obtained for accuracy, intra- and inter-day precision complied very well with the generally accepted criteria for bioanalytical assays.     

Chemoselective Nitro Group Reduction and Reductive Dechlorination Initiate Degradation of 2-Chloro-5-Nitrophenol by Ralstonia eutropha JMP134

Ralstonia eutropha JMP134 utilizes 2-chloro-5-nitrophenol as a sole source of nitrogen, carbon, and energy. The initial steps for degradation of 2-chloro-5-nitrophenol are analogous to those of 3-nitrophenol degradation in R. eutropha JMP134. 2-Chloro-5-nitrophenol is initially reduced to 2-chloro-5-hydroxylaminophenol, which is subject to an enzymatic Bamberger rearrangement yielding 2-amino-5-chlorohydroquinone. The chlorine of 2-amino-5-chlorohydroquinone is removed by a reductive mechanism, and aminohydroquinone is formed. 2-Chloro-5-nitrophenol and 3-nitrophenol induce the expression of 3-nitrophenol nitroreductase, of 3-hydroxylaminophenol mutase, and of the dechlorinating activity. 3-Nitrophenol nitroreductase catalyzes chemoselective reduction of aromatic nitro groups to hydroxylamino groups in the presence of NADPH. 3-Nitrophenol nitroreductase is active with a variety of mono-, di-, and trinitroaromatic compounds, demonstrating a relaxed substrate specificity of the enzyme. Nitrosobenzene serves as a substrate for the enzyme and is converted faster than nitrobenzene.     

Heterogeneity of breast cancer associations with five susceptibility loci by clinical and pathological characteristics

A three-stage genome-wide association study recently identified single nucleotide polymorphisms (SNPs) in five loci (fibroblast growth receptor 2 (FGFR2), trinucleotide repeat containing 9 (TNRC9), mitogen-activated protein kinase 3 K1 (MAP3K1), 8q24, and lymphocyte-specific protein 1 (LSP1)) associated with breast cancer risk. We investigated whether the associations between these SNPs and breast cancer risk varied by clinically important tumor characteristics in up to 23,039 invasive breast cancer cases and 26,273 controls from 20 studies. We also evaluated their influence on overall survival in 13,527 cases from 13 studies. All participants were of European or Asian origin. rs2981582 in FGFR2 was more strongly related to ER-positive (per-allele OR (95%CI)=1.31 (1.27–1.36)) than ER-negative (1.08 (1.03–1.14)) disease (P for heterogeneity=10⁻¹³). This SNP was also more strongly related to PR-positive, low grade and node positive tumors (P=10⁻⁵, 10⁻⁸, 0.013, respectively). The association for rs13281615 in 8q24 was stronger for ER-positive, PR-positive, and low grade tumors (P=0.001, 0.011 and 10⁻⁴, respectively). The differences in the associations between SNPs in FGFR2 and 8q24 and risk by ER and grade remained significant after permutation adjustment for multiple comparisons and after adjustment for other tumor characteristics. Three SNPs (rs2981582, rs3803662, and rs889312) showed weak but significant associations with ER-negative disease, the strongest association being for rs3803662 in TNRC9 (1.14 (1.09–1.21)). rs13281615 in 8q24 was associated with an improvement in survival after diagnosis (per-allele HR=0.90 (0.83–0.97). The association was attenuated and non-significant after adjusting for known prognostic factors. Our findings show that common genetic variants influence the pathological subtype of breast cancer and provide further support for the hypothesis that ER-positive and ER-negative disease are biologically distinct. Understanding the etiologic heterogeneity of breast cancer may ultimately result in improvements in prevention, early detection, and treatment.     

Quantitation of psilocin in human plasma by high-performance liquid chromatography and electrochemical detection: comparison of liquid–liquid extraction with automated on-line solid-phase extraction

Two modifications of the HPLC–ED method with respect to extraction procedure used have been developed for psilocin, the active metabolite of psilocybin, in human plasma using either liquid–liquid extraction (LLE) or automated on-line solid-phase extraction (on-line SPE). Each type of the sample preparation required a different HPLC system followed by electrochemical detection at 650 to 675 mV. The limit of quantitation of both modifications was 10 ng/ml psilocin. There was no significant difference observable between the LLE and the on-line SPE in terms of method standard deviation (LLE 1.82%, on-line SPE 1.13%) and the analytical results. However, the advantages of on-line SPE in addition to different selectivity were less manual effort, smaller plasma volumes of 400 μl (LLE 2 ml) and a recovery of psilocin in human plasma of nearly 100% (LLE 88%). In contrast to a previous procedure both methods were rapid, simple and reliable and yielded high plasma recoveries. They were used successfully in the quantitation of psilocin in plasma samples obtained from healthy volunteers after p.o. administration of 0.2 mg psilocybin per kg body mass. Plasma concentration curves and pharmacokinetic parameters were calculated.     

Exploiting the complexity of the genome and transcriptome using pharmacogenomics towards personalized medicine

A report of the 8th annual Pharmacogenomics and Personalized Therapy meeting, Cold Spring Harbor Laboratory, USA, 17-21 November 2010.     

Mutations in the VHL tumor suppressor gene and associated lesions in families with von Hippel-Lindau disease from central Europe

von Hippel-Lindau (VHL) disease is a dominantly inherited familial cancer syndrome predisposing to retinal, cerebellar and spinal hemangioblastoma, renal cell carcinoma (RCC), pheochromocytoma and pancreatic tumors. Clinically two types of the disease can be distinguished: VHL type 1 (without pheochromocytoma) and VHL type 2 (with pheochromocytoma). We report VHL germline mutations and trends in phenotypic variation in families from central Europe. We identified 28 mutations in 53/65 (81.5%) families with 18 (64%) mutations being unique to this population. Whereas types and distribution of mutations as well as a strong correlation of missense mutations with the VHL 2 phenotype were similar to those identified in other populations, these families have provided new insights into the molecular basis for variability in the VHL 2 phenotype. Seven different missense mutations in exons 1 and 3 varied in their biological consequences from a minimal VHL 2 phenotype with pheochromocytoma only to a full VHL 2 phenotype with RCC and pancreatic lesion. These findings contribute to a better understanding of the fundamental mechanisms of VHL disease and its phenotypic variability. Further, we have provided rapid VHL screening for the families in central Europe, which has resulted in improved diagnosis and clinical management. Received: 10 November 1995 / Revised: 1 March 1996     

Fixed archival tissue

The labeling of oligonucleotide probes using a fluorescein-labeled nucleotide is described. The reaction is characterized by careful control of the nucleotide and probe molar ratio in order to produce a tail that gives good detection sensitivity without compromising hybridization stringency control of the probe sequence. The labeling reaction can be easily monitored for incorporation of the fluorescent label and the probes can be used in many applications.     

Loss of heterozygosity in a gene coding for a thyroid hormone receptor in lung cancers

The ERBA beta gene codes for a DNA-binding thyroid hormone receptor (THR) and maps to chromosome 3p21-p25, overlapping a 3p deletion characterizing small-cell lung carcinoma (SCLC). A DNA clone detecting an RFLP at the ERBA beta locus has been used to probe a large number of lung tumors. Virtually all SCLC had lost heterozygosity, showing that the 3p deletion in SCLC includes this gene. A substantial but smaller proportion of non-small-cell carcinomas had lost heterozygosity at ERBA beta. Among all non-small-cell tumors some had lost heterozygosity at the proximal locus DNF15S2 (band 3p21) but not at ERBA beta, whereas none were found where the reverse was true. Therefore, the locus which plays a role in non-small-cell tumorigenesis probably lies closer to DNF15S2 than to ERBA beta and is almost certainly not the latter.