Nuclease-resistant ribozymes decrease stromelysin mRNA levels in rabbit synovium following exogenous delivery to the knee joint.

Catalytic RNA molecules, or ribozymes, have generated significant interest as potential therapeutic agents for controlling gene expression. Although ribozymes have been shown to work in vitro and in cellular assays, there are no reports that demonstrate the efficacy of synthetic, stabilized ribozymes delivered in vivo. We are currently utilizing the rabbit model of interleukin 1-induced arthritis to assess the localization, stability, and efficacy of exogenous antistromelysin hammerhead ribozymes. The matrix metalloproteinase stromelysin is believed to be a key mediator in arthritic diseases. It seems likely therefore that inhibiting stromelysin would be a valid therapeutic approach for arthritis. We found that following intraarticular administration ribozymes were taken up by cells in the synovial lining, were stable in the synovium, and reduced synovial interleukin 1 alpha-induced stromelysin mRNA. This effect was demonstrated with ribozymes containing various chemical modifications that impart nuclease resistance and that recognize several distinct sites on the message. Catalytically inactive ribozymes were ineffective, thus suggesting a cleavage-mediated mechanism of action. These results suggest that ribozymes may be useful in the treatment of arthritic diseases characterized by dysregulation of metalloproteinase expression.

Cloning of a sodium channel alpha subunit from rabbit Schwann cells.

Overlapping cDNA clones spanning the entire coding region of a Na-channel alpha subunit were isolated from cultured Schwann cells from rabbits. The coding region predicts a polypeptide (Nas) of 1984 amino acids exhibiting several features characteristic of Na-channel alpha subunits isolated from other tissues. Sequence comparisons showed that the Nas alpha subunit resembles most the family of Na channels isolated from brain (approximately 80% amino acid identity) and is least similar (approximately 55% amino acid identity) to the atypical Na channel expressed in human heart and the partial rat cDNA, NaG. As for the brain II and III isoforms, two variants of Nas exist that appear to arise by alternative splicing. The results of reverse transcriptase-polymerase chain reaction experiments suggest that expression of Nas transcripts is restricted to cells in the peripheral and central nervous systems. Expression was detected in cultured Schwann cells, sciatic nerve, brain, and spinal cord but not in skeletal or cardiac muscle, liver, kidney, or lung.

Rabbit monoclonal antibodies: generating a fusion partner to produce rabbit-rabbit hybridomas.

During the last 15 years several laboratories have attempted to generate rabbit monoclonal antibodies, mainly because rabbits recognize antigens and epitopes that are not immunogenic in mice or rats, two species from which monoclonal antibodies are usually generated. Monoclonal antibodies from rabbits could not be generated, however, because a plasmacytoma fusion partner was not available. To obtain a rabbit plasmacytoma cell line that could be used as a fusion partner we generated transgenic rabbits carrying two transgenes, c-myc and v-abl. These rabbits developed plasmacytomas, and we obtained several plasmacytoma cell lines from which we isolated hypoxanthine/aminopterin/thymidine-sensitive clones. One of these clones, when fused with spleen cells of immunized rabbits, produced stable hybridomas that secreted antibodies specific for the immunogen. The hybridomas can be cloned and propagated in nude mice, and they can be frozen without change in their ability to secrete specific monoclonal antibodies. These rabbit-rabbit hybridomas will be useful not only for production of monoclonal antibodies but also for studies of immunoglobulin gene rearrangements and isotype switching.

Transfection of the human heme oxygenase gene into rabbit coronary microvessel endothelial cells: protective effect against heme and hemoglobin toxicity.

Heme oxygenase (HO) is a stress protein and has been suggested to participate in defense mechanisms against agents that may induce oxidative injury such as metals, endotoxin, heme/hemoglobin, and various cytokines. Overexpression of HO in cells might therefore protect against oxidative stress produced by certain of these agents, specifically heme and hemoglobin, by catalyzing their degradation to bilirubin, which itself has antioxidant properties. We report here the successful in vitro transfection of rabbit coronary microvessel endothelial cells with a functioning gene encoding the human HO enzyme. A plasmid containing the cytomegalovirus promoter and the human HO cDNA complexed to cationic liposomes (Lipofectin) was used to transfect rabbit endothelial cells. Cells transfected with human HO exhibited an approximately 3.0-fold increase in enzyme activity and expressed a severalfold induction of human HO mRNA as compared with endogenous rabbit HO mRNA. Transfected and nontransfected cells expressed factor VIII antigen and exhibited similar acetylated low-density lipoprotein uptake (two important features that characterize endothelial cells) with > 85% of cells staining positive for each marker. Moreover, cells transfected with the human HO gene acquired substantial resistance to toxicity produced by exposure to recombinant hemoglobin and heme as compared with nontransfected cells. The protective effect of HO overexpression against heme/hemoglobin toxicity in endothelial cells shown in these studies provides direct evidence that the inductive response of human HO to such injurious stimuli represents an important tissue adaptive mechanism for moderating the severity of cell damage produced by these blood components.

Inactivation of the superior cerebellar peduncle blocks expression but not acquisition of the rabbit's classically conditioned eye-blink response.

The localization of sites of memory formation within the mammalian brain has proven to be a formidable task even for simple forms of learning and memory. Recent studies have demonstrated that reversibly inactivating a localized region of cerebellum, including the dorsal anterior interpositus nucleus, completely prevents acquisition of the conditioned eye-blink response with no effect upon subsequent learning without inactivation. This result indicates that the memory trace for this type of learning is located either (i) within this inactivated region of cerebellum or (ii) within some structure(s) efferent from the cerebellum to which output from the interpositus nucleus ultimately projects. To distinguish between these possibilities, two groups of rabbits were conditioned (by using two conditioning stimuli) while the output fibers of the interpositus (the superior cerebellar peduncle) were reversibly blocked with microinjections of the sodium channel blocker tetrodotoxin. Rabbits performed no conditioned responses during this inactivation training. However, training after inactivation revealed that the rabbits (trained with either conditioned stimulus) had fully learned the response during the previous inactivation training. Cerebellar output, therefore, does not appear to be essential for acquisition of the learned response. This result, coupled with the fact that inactivation of the appropriate region of cerebellum completely prevents learning, provides compelling evidence supporting the hypothesis that the essential memory trace for the classically conditioned eye-blink response is localized within the cerebellum.

Pancreatic digestive enzyme secretion in the rabbit: rapid cyclic variations in enzyme composition.

The role and mechanism of nonparallel pancreatic secretion of digestive enzymes, in which enzyme proportions change in rapidly regulated fashion, remain controversial. Secretion was collected from male 2.2-kg New Zealand rabbits in 5-min intervals for 3 h under basal conditions or constant stimulation with cholecystokinin (CCK; 0.1 microgram per kg per h i.v.) or methacholine chloride (MCh; 40 micrograms per kg per h i.v.). Both CCK and MCh produced an 8-fold stimulation of protein output. Enzymes were separated by SDS/PAGE and quantitated by densitometry of Coomassie blue-stained gels. Under both basal conditions and constant MCh infusion, rapid neurosecretory-like 12-min cyclic changes occurred in the proportions of amylase, lipase I, chymotrypsinogen, and trypsinogen. During constant infusion their percentages changed as much as 10-fold, and their ratios cycled by as much as 30-fold. The mean percentage for the entire infusion period for lipase I declined > 25% with CCK or MCh, for amylase it rose approximately 30%, and for chymotrypsinogen and trypsinogen it doubled (for all, P < 0.05). CCK and MCh elicited subtly but significantly different mean enzyme percentages and enzyme ratios (P < 0.05) for amylase, chymotrypsinogen, and trypsinogen; these differences were also confirmed by regression and correlation analyses. The changes in enzyme percentages and ratios were explicitly consistent with secretagogue-caused shifts in the intrapancreatic enzyme secretory sources. Nonparallel secretion of digestive enzymes occurs routinely, even during constant stimulation, and is due to cyclic neurosecretory-like secretion from heterogeneous intrapancreatic sources.

Main ecological gradients and landscape matrix affect wild rabbit Oryctolagus cuniculus (Linnaeus, 1758) (Mammalia: Leporidae) abundance in a coastal region (South-East Spain)

Landscape analysis with transects, in the Marina Baja area (province of Alicante, Spain), has contributed to establish the influence of different landscape matrices and some environmental gradients on wild rabbit Oryctolagus cuniculus (Linnaeus, 1758) (Mammalia: Leporidae) abundance (kilometric abundance index, KAI). Transects (n = 396) were developed to estimate the abundance of this species in the study area from 2006 to 2008.Our analysis shows that rabbits have preferences for a specific land use matrix (irrigated: KAI = 3.47 ± 1.14 rabbits/km). They prefer the coastal area (KAI = 3.82 ± 1.71 rabbits/km), which coincides with thermo-Mediterranean (a bioclimatic belt with a tempered winter and a hot and dry summer with high human density), as opposed to areas in the interior (continental climate with lower human occupation). Their preference for the southern area of the region was also noted (KAI = 8.22 ± 3.90 rabbits/km), which coincides with the upper semi-arid area, as opposed to the northern and intermediate areas (the north of the region coinciding with the upper dry and the intermediate area with the lower dry). On the other hand, we found that the number of rabbits increased during the 3-year study period, with the highest abundance (KAI = 2.71 ± 1.30 rabbits/km) inMay. Thus, this study will enable more precise knowledge of the ecological factors (habitat variables) that intervene in the distribution of wild rabbit populations in a poorly studied area.

The Economic Basis of Attitudes towards the European Community: Familiarity Breeds Content. Institute for Advanced Studies Political Science Series, 12 April 1994

The purpose of this paper is to analyse the economic basis of mass support for, and opposition to the European Community. In other words the main question is: to what extent, and in what ways, is popular support or opposition to the EC dependent upon economic circumstances and considerations? Behind this research question lies the matter of the legitimacy of the EC in the eyes of citizens across Europe. In some respects the results of this study are frustrating and inconclusive. In other respects, however, the results suggest some clear generalizations and conclusions. First, we have found rather little evidence that the EC or European unification are evaluated in primarily economic terms. Secondly, support seems to be associated more strongly with social and attitudinal variables of a non-economic kind. Thirdly, the figures suggest that diffuse and somewhat idealistic reasons for supporting unification and EC membership tend to outweigh more specific reasons. Fourthly, a solid foundation of inertia, custom, and national tradition seem to maintain support and make it grow.

Masking odour when regrouping rabbit does: Effect on aggression, stress and lesions.

Regrouping female rabbits (Oryctolagus cuniculus) in group housing systems is a common management practice in Swiss rabbit breeding which may, however, induce agonistic interactions resulting in social stress and severe lesions. On farms using artificial insemination, does are usually kept singly for 12 days after parturition to avoid pseudopregnancy and fighting for nests. The integration of new group members usually occurs after this isolation phase. This study was conducted with 128 gravid does of the Hycole hybrid, housed in pens covering a floor area of 5.7 m2 that was bedded with straw and furnished with elevated areas, hiding places and eight compartments with nest boxes. In the experiment, the fur of 16 groups of 8 does each was sprayed with either alcohol or vinegar to mask the pre-existing group odours, or with water (control groups) shortly before regrouping. Lesion scores, stress parameters (body temperature and blood glucose level) and behaviour were assessed before and after the isolation phase. Effects of treatment and time on all collected parameters were analysed using mixed models. On the second day after regrouping 43% of the does showed new lesions. In the first five days after regrouping, new lesions occurred in 60% of the does; 32% had severe lesions. After regrouping, more agonistic interactions were observed and body temperature and blood glucose levels were higher than before regrouping (P<0.001 each). Body temperature increased less in groups treated with vinegar compared to the other two treatments on the first day after regrouping (P=0.017). In all other parameters no influence of the treatment with alcohol or vinegar was found. These findings suggest that masking the group odours with alcohol or vinegar had little effect on lesions, stress and agonistic interactions. Therefore, alternative management procedures need to be developed to reduce lesions and stress caused by aggressive behaviour.

Girl holding treat for rabbit / McKenzie Hosp. Schools

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