Nociception after intraperitoneal injection of a sodium pentobarbitone formulation with and without lidocaine in rats quantified by expression of neuronal c-fos in the spinal cord--a preliminary study

After a search on Medline, it appears that intraperitoneal injection of sodium pentobarbitone is often used for anaesthesia and euthanasia of rodents. In the present pilot study in rats, spinal nociception after intraperitoneal injection of sodium pentobarbitone, with and without lidocaine, was examined by estimation of the number of c-fos-expressing neurones in the spinal dorsal horn. One group of rats received an intraperitoneal injection of 0.4 mL/kg sodium pentobarbitone (100 mg/mL; n=4). Another group of rats received a similar intraperitoneal injection of sodium pentobarbitone formulated with lidocaine 10 mg/mL (n=4); a control group received a similar intraperitoneal injection of 0.9% saline (n=4). After 3 h, the animals were re-anaesthetized and perfused with 4% formaldehyde, and the spinal cord was collected and processed by immunohistochemistry for stereological quantification of the number of neurones with c-fos-like immunoreactivity (FLI). Intraperitoneal injection of the sodium pentobarbitone formulation caused a significantly increased number of neurones with FLI in the spinal cord (3930+/-247; mean+/-SEM; P<0.001) compared with the saline control group (765+/-131). The lidocaine added to the sodium pentobarbitone formulation significantly reduced the number to 2716+/-393 (P<0.05). In conclusion, intraperitoneal injection of sodium pentobarbitone caused a significant increase in nociception which was lowered by adding lidocaine to the formulation, although it was still significantly higher than the control level. Further studies are needed with the aim of optimizing the lidocaine concentration and also to examine the effect of the combination of lidocaine with a long-acting local anaesthetic agent, e.g. bupivacaine.     

Are all bony fishes oxygen regulators? Evidence for oxygen regulation in a putative oxygen conformer,the swamp eel Synbranchus marmoratus

This study investigated the oxygen consumption of the putative oxygen conformer marbled swamp eel Synbranchus marmoratus during progressive hypoxia. Earlier studies have not reached an agreement on whether S. marmoratus is a conformer or a regulator. Our results support the view that S. marmoratus is an oxygen regulator, like most bony fishes.     

The in vitro survival of human monosomies and trisomies as embryonic stem cells

Chromosomal aneuploidies are responsible for severe human genetic diseases. Aiming at creating models for such disorders, we have generated human embryonic stem cell (hESC) lines from pre-implantation genetic screened (PGS) embryos. The overall analysis of more than 400 aneuploid PGS embryos showed a similar risk of occurrence of monosomy or trisomy for any specific chromosome. However, the generation of hESCs from these embryos revealed a clear bias against monosomies in autosomes. Moreover, only specific trisomies showed a high chance of survival as hESC lines, enabling us to present another categorization of human aneuploidies. Our data suggest that chromosomal haploinsufficiency leads to lethality at very early stages of human development.     

Pre-analytical and biological variability in circulating interleukin 6 in healthy subjects and patients with rheumatoid arthritis.

Interleukin (IL)-6, a key player in the inflammatory response, may be a useful biomarker in rheumatoid arthritis (RA). The aim was to determine analytical variability, a reference interval in healthy subjects, and long- and short-term variation in serum and plasma IL-6 in healthy subjects and RA patients. An enzyme-linked immunosorbent assay from R&D was used for determination of serum and plasma IL-6. The IL-6 concentration did not depend on the type of anticoagulant used or the 3-h time delay between sampling and processing or repeated freeze-thaw cycles. The median plasma and serum IL-6 in 318 healthy subjects were 1.3 pg ml(-1) (range 0.33-26) and 1.4 pg ml(-1) (range 0.25-23), respectively. The median coefficient of variation in plasma IL-6 in 27 healthy subjects during 1 month, and repeated after 6 and 12 months were 27%, 31% and 26%, respectively. No significant long-term changes were observed in serum IL-6 over a 3-year period (14%, p = 0.33). Exercise (cycling) increased serum IL-6 in healthy subjects but not in RA patients. In conclusion, circulating IL-6 is stable regarding sample handling and shows little variation over time. Changes in IL-6 concentrations > 60% (2 times the biological variation) are likely to reflect changes in disease activity and not only pre-analytical or normal biological variability.     

Stem cells and Parkinson's disease: toward a treatment, not a cure

Early clinical trials designed to treat Parkinson's disease by transplantation of fetal tissue containing dopamine neuron precursors yielded promising results, but the approach retains several limitations. Multiple recent papers describe longer-term outcomes in these patients, and two additional studies offer novel approaches that may lead to autologous sources of transplantable dopamine neurons.     

Increased antibacterial activity of 15-residue murine lactoferricin derivatives.

LFM W8 is a synthetic 15-residue lactoferricin derivative (H2N-EKCLRWQWEMRKVGG-COOH), corresponding to residues 16-30 of the mature murine lactoferrin protein except that the asparagine residue in position 8 of the native peptide is replaced with tryptophan. We have previously reported that the two tryptophan residues in positions 6 and 8 are of crucial importance for the antibacterial activity of many lactoferricin derivatives but, despite fulfilling this requirement, LFM W8 is inactive against Escherichia coli and Staphylococcus aureus. In order to solve this puzzle, a quantitative structure-antibacterial activity relationship study of synthetic LFM W8 derivatives was performed by replacing the glutamate residues in positions 1 and 9 with arginine or alanine, and the valine residue in position 13 with tyrosine. The results from the study were analyzed using multivariate data analysis. The derived mathematical model clustered the peptides into distinct groups which reflected their antibacterial activities, pointed out correlations between different structural parameters, highlighted the structural parameters that were important for antibacterial activity, and enabled us to predict the activity of a 15-residue bovine lactoferricin derivative. The results showed that net charge and micelle affinity, as determined from the ratio of alpha-helicity in sodium dodecyl sulfate micelles and in 1,1,1,3,3,3-hexafluoro-2-propanol, were the most important structural parameters affecting antibacterial activity. The most active derivative, LFM R1,9 W8 Y13, displayed a minimal inhibitory concentration of 10 and 12 microM against E. coli and S. aureus, respectively. This represented more than 50-fold and 40-fold increases in antibacterial activity, respectively, compared with LFM W8.     

Respirometry increases cortisol levels in rainbow trout Oncorhynchus mykiss: implications for measurements of metabolic rate

This study aimed to assess the extent to which chasing, handling and confining Oncorhynchus mykiss to a small respirometer chamber during respirometric experiments is stressful and affects metabolic measurements. The study observed increased cortisol levels in animals tested using a chase protocol and subsequent intermittent‐flow respirometry, suggesting that this procedural treatment may stress animals.     

Binding of Thermomyces (Humicola) lanuginosa lipase to the mixed micelles of cis-parinaric acid/NaTDC.

The binding of Thermomyces lanuginosa lipase and its mutants [TLL(S146A), TLL(W89L), TLL(W117F, W221H, W260H)] to the mixed micelles of cis-parinaric acid/sodium taurodeoxycholate at pH 5.0 led to the quenching of the intrinsic tryptophan fluorescence emission (300-380 nm) and to a simultaneous increase in the cis-parinaric acid fluorescence emission (380-500 nm). These findings were used to characterize the Thermomyces lanuginosa lipase/cis-parinaric acid interactions occurring in the presence of sodium taurodeoxycholate.The fluorescence resonance energy transfer and Stern-Volmer quenching constant values obtained were correlated with the accessibility of the tryptophan residues to the cis-parinaric acid and with the lid opening ability of Thermomyces lanuginosa lipase (and its mutants). TLL(S146A) was found to have the highest fluorescence resonance energy transfer. In addition, a TLL(S146A)/oleic acid complex was crystallised and its three-dimensional structure was solved. Surprisingly, two possible binding modes (sn-1 and antisn1) were found to exist between oleic acid and the catalytic cleft of the open conformation of TLL(S146A). Both binding modes involved an interaction with tryptophan 89 of the lipase lid, in agreement with fluorescence resonance energy transfer experiments.As a consequence, we concluded that TLL(S146A) mutant is not an appropriate substitute for the wild-type Thermomyces lanuginosa lipase for mimicking the interaction between the wild-type enzyme and lipids.     

Combined Dietary and Pharmacological Weight Management in Obese Hypopituitary Patients

Objective: The high prevalence of obesity and cardiovascular risk factors in hypopituitarism affirms the need for effective weight loss intervention. In this study, we investigated the combined effect of sibutramine, diet, and exercise in obese hypopituitary patients (HPs). Research Methods and Procedures: In an open‐label prospective intervention trial, 14 obese well‐substituted nondiabetic HPs and 14 matched simple obese controls were allocated to 11‐month treatment with sibutramine (10 to 15 mg), diet (600 kcal/d deficit), and exercise. Anthropometric indices and body composition (obtained from DXA scan) were assessed monthly for the first 5 months and thereafter every second month for the next 6 months. Results: Mean (±SD) weight loss at 11 months was 11.3 ± 4.8 kg in patients vs. 10.7 ± 4.7 kg in controls. The HPs exhibited the same improvements in body composition, waist circumference, blood lipids, and fasting glucose as the simple obese. In a multivariate model, baseline weight, duration of growth hormone replacement therapy, and duration of pituitary disease explained 79% (p = 0.001) of the variation in weight loss at 4 months in the HPs. Only baseline weight and waist circumference could predict weight loss at 11 months. Discussion: HPs are not resistant to weight loss therapy. Almost all will achieve at least 5% weight loss, and 60% can lose >10% weight within 11 months. However, the long‐term effect on risk factors associated with type 2 diabetes and cardiovascular disease as well as on mortality needs to be established.     

Characterisation of G418-induced metabolic load in recombinant CHO and BHK cells: effect on the activity and expression of central metabolic enzymes

In a previous article (Yallop and Svendsen 2001), recombinant CHO and BHK cell lines, expressing the human glucagon receptor and the gastric inhibitory peptide receptor, respectively, showed reduced growth rates and altered nutrient utilisation when grown with increasing concentrations of G418. This response was associated with an increased expression of the neo ^r protein, while expression of the recombinant membrane receptors remained unaltered. The metabolic response was characterised in both cell lines by an increase in the specific rate of glutamine utilisation and in CHO cells by a decrease in the yield of lactate from glucose, suggesting a change in the flux of glucose through central metabolism. The aim of this study was to further elucidate these metabolic changes by determining the activity and relative expression of key enzymes involved in glucose and glutamine metabolism. For both CHO and BHK cells, there was an increase in the activity of glutaminase, glutamate dehydrogenase and glutamine synthetase, suggesting an increased flux through the glutaminolysis pathway. The activity of glucose-6-phosphate dehydrogenase and pyruvate carboxylase in CHO cells was also increased whilst lactate dehydrogenase activity remained unaltered, suggesting an increased flux to the pentose phosphate pathway and TCA cycle, respectively. The activity of these enzymes in BHK cells was unchanged. Quantitative RT-PCR showed that expression levels of glutaminase and pyruvate carboxylase were the same with and without G418, indicating that the differences in activities were likely due to post-translational modifications. This revised version was published online in July 2006 with corrections to the Cover Date.