The '3Rs' model and the concept of alternatives in animal research: a questionnaire survey

It has been 45 years since Russell and Burch first proposed the concept of the '3Rs', yet it remains unclear how those individuals involved in animal research view and implement these concepts. The authors used a questionnaire survey to determine how well-known experts judged issues related to the 3Rs.     

DNA binding ligands targeting drug-resistant Gram-positive bacteria. Part 1: Internal benzimidazole derivatives

Novel DNA minor-groove binding ligands with a promising antibacterial profile are described. Apart from excellent in vitro potency against multiple Gram-positive bacterial strains such as methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus faecalis (VRE), and penicillin-intermediate Streptococcus pneumoniae (PISP), a small subset of compounds was active against Gram-negative bacteria such as Escherichia coli (E. coli).     

Signal detection behavior in humans and rats: a comparison with matched tasks

Animal models of human cognitive processes are essential for studying the neurobiological mechanisms of these processes and for developing therapies for intoxication and neurodegenerative diseases. A discrete-trial signal detection task was developed for assessing sustained attention in rats; a previous study showed that rats perform as predicted from the human sustained attention literature. In this study, we measured the behavior of humans in a task formally homologous to the task for rats, varying two of the three parameters previously shown to affect performance in rats. Signal quality was manipulated by varying the increment in the intensity of a lamp. Trial rate was varied among values of 4, 7, and 10 trials/min. Accuracy of signal detection was quantified by the proportion of correct detections of the signal (P(hit)) and the proportion of false alarms (P(fa), i.e. incorrect responses on non-signal trials). As with rats, P(hit) in humans increased with increasing signal intensity whereas P(fa) did not. Like rats, humans were sensitive to the trial rate, though the change in behavior depended on the sex of the subject. These data show that visual signal detection behavior in rats and humans is controlled similarly by two important parameters, and suggest that this task assesses similar processes of sustained attention in the two species.     

Understanding resistance to beta-lactams and beta-lactamase inhibitors in the SHV beta-lactamase: lessons from the mutagenesis of SER-130

Bacterial resistance to beta-lactam/beta-lactamase inhibitor combinations by single amino acid mutations in class A beta-lactamases threatens our most potent clinical antibiotics. In TEM-1 and SHV-1, the common class A beta-lactamases, alterations at Ser-130 confer resistance to inactivation by the beta-lactamase inhibitors, clavulanic acid, and tazobactam. By using site-saturation mutagenesis, we sought to determine the amino acid substitutions at Ser-130 in SHV-1 beta-lactamase that result in resistance to these inhibitors. Antibiotic susceptibility testing revealed that ampicillin and ampicillin/clavulanic acid resistance was observed only for the S130G beta-lactamase expressed in Escherichia coli. Kinetic analysis of the S130G beta-lactamase demonstrated a significant elevation in apparent Km and a reduction in kcat/Km for ampicillin. Marked increases in the dissociation constant for the preacylation complex, KI, of clavulanic acid (SHV-1, 0.14 microm; S130G, 46.5 microm) and tazobactam (SHV-1, 0.07 microm; S130G, 4.2 microm) were observed. In contrast, the k(inact)s of S130G and SHV-1 differed by only 17% for clavulanic acid and 40% for tazobactam. Progressive inactivation studies showed that the inhibitor to enzyme ratios required to inactivate SHV-1 and S130G were similar. Our observations demonstrate that enzymatic activity is preserved despite amino acid substitutions that significantly alter the apparent affinity of the active site for beta-lactams and beta-lactamase inhibitors. These results underscore the mechanistic versatility of class A beta-lactamases and have implications for the design of novel beta-lactamase inhibitors.     

Ghrelin can bind to a species of high density lipoprotein associated with paraoxonase

Ghrelin is a 28-residue peptide hormone that is principally released from the stomach during fasting and prior to eating. Two forms are present in human plasma: the unmodified peptide and a less abundant acylated version, in which octanoic acid is attached to the third residue, a serine, via an ester linkage. The acylated form of ghrelin acts as a ligand for the growth hormone secretagogue receptor and can stimulate the release of growth hormone from the pituitary gland. It also initiates behavioral and metabolic adaptations to fasting. Here we show that an immobilized form of ghrelin specifically binds a species of high density lipoprotein associated with the plasma esterase, paraoxonase, and clusterin. Both free ghrelin and paraoxon, a substrate for paraoxonase, can inhibit this interaction. An endogenous species of ghrelin is found to co-purify with high density lipoprotein during density gradient centrifugation and subsequent gel filtration. This interaction links the orexigenic peptide hormone ghrelin to lipid transport and metabolism. Furthermore, the interaction of the esterified hormone ghrelin with a species of HDL containing an esterase suggests a possible mechanism for the conversion of ghrelin to des-acyl ghrelin.     

Controlling the mealworm Alphitobius diaperinus (Coleoptera: Tenebrionidae) in broiler and turkey houses: field trials with a combined insecticide treatment: insect growth regulator and pyrethroid

Trials were conducted during one year under field conditions to control the lesser mealworm, Alphitobius diaperinus (Panzer), in broiler and turkey houses. The tested combined treatment included an adulticidal compound (pyrethroid: cyfluthrin) and a larvicidal compound (insect growth regulator [IGR]: triflumuron). The combined insecticide treatment greatly reduced the adult and larval stocks throughout the different broiler growing periods, and control of A. diaperinus populations was achieved by the end of the second treatment. Control of the insect population in a turkey house was not similar. A reestablishment of the insect population was observed during the second turkey growing period in summer. Building characteristics and management practices of the breeding system (duration of the breeding period, management of the litter) interact with the combined insecticide treatment and lead to a different efficiency.     

Analysis of the Sarcocystis neurona microneme protein SnMIC10: protein characteristics and expression during intracellular development

Sarcocystis neurona, an apicomplexan parasite, is the primary causative agent of equine protozoal myeloencephalitis. Like other members of the Apicomplexa, S. neurona zoites possess secretory organelles that contain proteins necessary for host cell invasion and intracellular survival. From a collection of S. neurona expressed sequence tags, we identified a sequence encoding a putative microneme protein based on similarity to Toxoplasma gondii MIC10 (TgMIC10). Pairwise sequence alignments of SnMIC10 to TgMIC10 and NcMIC10 from Neospora caninum revealed approximately 33% identity to both orthologues. The open reading frame of the S. neurona gene encodes a 255 amino acid protein with a predicted 39-residue signal peptide. Like TgMIC10 and NcMIC10, SnMIC10 is predicted to be hydrophilic, highly alpha-helical in structure, and devoid of identifiable adhesive domains. Antibodies raised against recombinant SnMIC10 recognised a protein band with an apparent molecular weight of 24 kDa in Western blots of S. neurona merozoites, consistent with the size predicted for SnMIC10. In vitro secretion assays demonstrated that this protein is secreted by extracellular merozoites in a temperature-dependent manner. Indirect immunofluorescence analysis of SnMIC10 showed a polar labelling pattern, which is consistent with the apical position of the micronemes, and immunoelectron microscopy provided definitive localisation of the protein to these secretory organelles. Further analysis of SnMIC10 in intracellular parasites revealed that expression of this protein is temporally regulated during endopolygeny, supporting the view that micronemes are only needed during host cell invasion. Collectively, the data indicate that SnMIC10 is a microneme protein that is part of the excreted/secreted antigen fraction of S. neurona. Identification and characterisation of additional S. neurona microneme antigens and comparisons to orthologues in other Apicomplexa could provide further insight into the functions that these proteins serve during invasion of host cells.     

The roles of aromaticity in the structure of DNA and its intercalation complexes with quinones

The X-ray crystallographic coordinate data of a 56 DNA double helical oligomers were examined, using the molecular modeling program STR3DI32.EXE, in order to ascertain the aromatic statuses of the Watson-Crick hydrogen bonded base pairs. Several oligomers that were intercalated with anthraquinoid molecules (like the daunomycin and nogalamycin aglycones) were also included in the study in order to evaluate the aromatic statuses of the intercalated entities. This study revealed that the base pairs were aromatic in their Watson-Crick hydrogen bonded double helices, whereas they are known to be non-aromatic in situations in which they are not involved in Watson-Crick hydrogen bonding. The resonance energy gained by the aromatization of these bases, while engaged in Watson-Crick hydrogen bonding, must contribute to the stability of these DNA double helices. The anthraquinoid intercalates were revealed to be in their radical anion form, having received an electron from one of the bases between which these intercalates were sited. These anthraquinoid intercalates are therefore "held" in position by ionic - charge transfer - interactions, as well as hydrogen bonding due to their glycosidic entities. These observations are also relevant to investigations of the electrical conductivity of DNA double helices that are similarly intercalated.     

Central mechanisms of acute ANG II modulation of arterial baroreflex control of renal sympathetic nerve activity

Short-term intravenous infusion of angiotensin II (ANG II) into conscious rabbits reduces the range of renal sympathetic nerve activity (RSNA) by attenuating reflex disinhibition of RSNA. This action of ANG II to attenuate the arterial baroreflex range is exaggerated when ANG II is directed into the vertebral circulation, which suggests a mechanism involving the central nervous system. Because an intact area postrema (AP) is required for ANG II to attenuate arterial baroreflex-mediated bradycardia and is also required for maintenance of ANG II-dependent hypertension, we hypothesized that attenuation of maximum RSNA during infusion of ANG II involves the AP. In conscious AP-lesioned (APX) and AP-intact rabbits, we compared the effect of a 5-min intravenous infusion of ANG II (10 and 20 ng x kg(-1) x min(-1)) on the relationship between mean arterial blood pressure (MAP) and RSNA. Intravenous infusion of ANG II into AP-intact rabbits resulted in a dose-related attenuation of maximum RSNA observed at low MAP. In contrast, ANG II had no effect on maximum RSNA in APX rabbits. To further localize the central site of ANG II action, its effect on the arterial baroreflex was assessed after a midcollicular decerebration. Decerebration did not alter arterial baroreflex control of RSNA compared with the control state, but as in APX, ANG II did not attenuate the maximum RSNA observed at low MAP. The results of this study indicate that central actions of peripheral ANG II to attenuate reflex disinhibition of RSNA not only involve the AP, but may also involve a neural interaction rostral to the level of decerebration.