Treatment-time-dependent difference of ketamine pharmacological response and toxicity in rats

Circadian rhythms impact many physiological functions that may affect drug pharmacological response. Ketamine is a dissociative agent commonly used for surgical anesthesia in rats. The aim of the present study was to analyze the central nervous system (CNS) depression and lethality of ketamine injected intraperitoneally at different times during the 24 h. The study was conducted in October 2001, spring in the Southern hemisphere. Female prepuberal Sprague-Dawley rats synchronized to a 12h light:12h dark cycle (light, 07:00h-19:00h) were studied. Ketamine (40 mg/kg) was administered to one of six different clock-time treatment groups (n=6-7 rats each). Duration of latency period, ataxia, loss of righting reflex (LRR), post-LRR ataxia, and total pharmacological response were determined by visual assessment. To investigate acute toxicity, ketamine lethal dose 50 (148.0 mg/kg) was also administered as a single injection to six different treatment-time groups of rats. Significant temporal differences and circadian rhythms were detected in drug-induced post-LRR ataxia and total pharmacological response duration. The longest pharmacological response occurred in rats injected during the light (rest) phase and the shortest response in the dark (activity) phase. No circadian rhythm was detected in acute toxicity. The study findings indicate that the duration of CNS depression of ketamine in rats exhibits circadian rhythmic variation.     

The regulation of genes and genomes by small RNAs

A recent Keystone Symposium on 'MicroRNAs and siRNAs: Biological Functions and Mechanisms' was organized by David Bartel and Shiv Grewal (and was held in conjunction with 'RNAi for Target Validation and as a Therapeutic', organized by Stephen Friend and John Maraganore). The 'MicroRNAs and siRNAs' meeting brought together scientists working on diverse biological aspects of small regulatory RNAs, including microRNAs, small interfering RNAs (siRNAs) and Piwi-interacting RNAs (piRNAs and rasiRNAs). Among the themes discussed were the diversity of small regulatory RNAs and their developmental functions, their biogenesis, the identification of their regulatory targets, their mechanisms of action, and their roles in the elaboration of multicellular complexity.     

Effect of self-association on the structural organization of partially folded proteins: inactivated actin

The propensity to associate or aggregate is one of the characteristic properties of many nonnative proteins. The aggregation of proteins is responsible for a number of human diseases and is a significant problem in biotechnology. Despite this, little is currently known about the effect of self-association on the structural properties and conformational stability of partially folded protein molecules. G-actin is shown to form equilibrium unfolding intermediate in the vicinity of 1.5 M guanidinium chloride (GdmCl). Refolding from the GdmCl unfolded state is terminated at the stage of formation of the same intermediate state. An analogous form, known as inactivated actin, can be obtained by heat treatment, or at moderate urea concentration, or by the release of Ca(2+). In all cases actin forms specific associates comprising partially folded protein molecules. The structural properties and conformational stability of inactivated actin were studied over a wide range of protein concentrations, and it was established that the process of self-association is rather specific. We have also shown that inactivated actin, being denatured, is characterized by a relatively rigid microenvironment of aromatic residues and exhibits a considerable limitation in the internal mobility of tryptophans. This means that specific self-association can play an important structure-forming role for the partially folded protein molecules.     

Application of Giemsa banding to orchid karyotype analysis

A method for obtaining orchid chromosome squash preparations from ovular tissues and a Giemsa C-band technique are described. Jointly applied, they result in well-defined chromosome banding patterns. Preliminary tests with two species of the genusCephalanthera show that Giemsa banding is also well suited for orchids. Besides aiding in chromosome identification and karyotype analysis, it should prove valuable in studies of chromosomal variation and karyotype evolution of this large family.     

Physiologically Based Pharmacokinetic Modeling Is Essential in 90Y-Labeled Anti-CD66 Radioimmunotherapy

IntroductionRadioimmunotherapy (RIT) with ⁹⁰Y-labeled anti-CD66 antibody is used to selectively irradiate the red marrow (RM) before blood stem cell transplantation of acute leukemia patients. To calculate the activity to administer, time-integrated activity coefficients are required. These are estimated prior to therapy using gamma camera and serum measurements after injection of ¹¹¹In labeled anti-CD66 antibody. Equal pre-therapeutic and therapeutic biodistributions are usually assumed to calculate the coefficients. However, additional measurements during therapy had shown that this assumption had to be abandoned. A physiologically based pharmacokinetic (PBPK) model was developed to allow the prediction of therapeutic time-integrated activity coefficients in eight patients.AimsThe aims of the study were to demonstrate using a larger patient group 1) the need to perform patient-specific dosimetry in ⁹⁰Y-labeled anti-CD66 RIT, 2) that pre-therapeutic and therapeutic biodistributions differ, and most importantly 3) that this difference in biodistributions can be accurately predicted using a refined model.ResultsVariability of the RM time-integrated activity coefficients ((37.3±7.5) h) indicates the need for patient-specific dosimetry. The relative differences between pre-therapeutic and therapeutic serum time-activity curves were (-25±16)%. The prediction accuracy of these differences using the refined PBPK models was (-3±20)%.ConclusionIndividual treatment is needed due to biological differences between patients in RIT with ⁹⁰Y-labeled anti-CD66 antibody. Differences in pre-therapeutic and therapeutic biokinetics are predominantly caused by different degrees of saturation due to different amounts of administered antibody. These differences could be predicted using the PBPK models.     

Enriched Bone Marrow Derived Disseminated Neuroblastoma Cells Can Be a Reliable Source for Gene Expression Studies—A Validation Study

Background

Metastases in the bone marrow (BM) in form of disseminated tumor cells (DTCs) are frequent events at diagnosis and also at relapse in high-risk neuroblastoma patients. The frequently highly diluted occurrence of DTCs requires adequate enrichment strategies to enable their detailed characterization. However, to avoid methodical artifacts we tested whether pre-analytical processing steps—including transport duration, temperature and, importantly, tumor cell enrichment techniques—are confounding factors for gene expression analysis in DTCs.

Methods

LAN-1 neuroblastoma cells were spiked into tumor free BM and/or peripheral blood and: i) kept at room temperature or at 4°C for 24, 48 and 72 hours; ii) frozen down at -80°C and thawed; iii) enriched via magnetic beads. The effect on the gene expression signature of LAN-1 cells was analyzed by qPCR arrays and gene expression microarrays.

Results

Neither storage at –80°C in DMSO and subsequent thawing nor enrichment of spiked-in neuroblastoma cells changed the expression of the analyzed genes significantly. Whereas storage at 4°C altered the expression of analyzed genes (14.3%) only at the 72h-timepoint in comparison to the 0h-timepoint, storage at room temperature had a much more profound effect on gene expression by affecting 20% at 24h, 26% at 48h and 43% at 72h of the analyzed genes.

Conclusion

Using neuroblastoma as a model, we show that tumor cell enrichment by magnetic bead separation has virtually no effect on gene expression in DTCs. However, transport time and temperature can influence the expression profile remarkably. Thus, the expression profile of routinely collected BM samples can be analyzed without concern as long as the transport conditions are monitored.