Comparative analysis of ACTH and corticosterone sampling methods in rats

A frequently debated question for studies involving the measurement of stress hormones in rodents is the optimal method for collecting blood with minimal stress to the animal. Some investigators prefer the implantation of indwelling catheters to allow for frequent sampling. Others argue that the implantation of a catheter creates a chronic stress to the animal that confounds stress hormone measures and therefore rely on tail vein sampling. Moreover, some investigators measure hormones in trunk blood samples obtained after anesthesia, a practice that may itself raise hormone levels. To address these controversies, we 1) compared plasma ACTH and corticosterone (Cort) concentrations in pre- and poststress rat blood samples obtained via previously implanted vena cava catheters, tail vein nicks, or clipping the tip off the tail and 2) compared plasma ACTH and Cort in rat blood samples obtained by decapitation with and without anesthesia. Rats sampled via indwelling catheters displayed lower prestress ACTH levels than those sampled by tail vein nick if the time to acquire samples was not limited; however, elevated basal ACTH was not observed in samples obtained by tail clip or tail nick when the samples were obtained within 3 min. Baseline Cort levels were similar in all groups. After restraint stress, the profile of the plasma ACTH and Cort responses was not affected by sampling method. Decapitation with prior administration of CO2 or pentobarbital sodium increased plasma ACTH levels approximately 13- and 2-fold, respectively, when compared with decapitation without anesthesia. These data indicate that tail vein nicking, tail clipping, or indwelling venous catheters can be used for obtaining plasma for ACTH and Cort during acute stress studies without confounding the measurements. However, the elevation in basal ACTH seen in the tail vein nick group at baseline suggests that sampling needs to be completed rapidly (<3 min) to avoid the initiation of the pituitary stress response. Death by CO2 and pentobarbital sodium injection before trunk blood collection cause significant stress to animals, as reflected in the elevated plasma ACTH levels. These results support the use of either chronic vascular cannulas or sampling from a tail vein. However, collection of blood under pentobarbital sodium or CO2 anesthesia is likely to confound the results of stress studies when ACTH is an important endpoint.     

Weight loss through ileal transposition is accompanied by increased ileal hormone secretion and synthesis in rats

Bariatric surgeries, such as gastric bypass, result in dramatic and sustained weight loss that is usually attributed to a combination of gastric volume restriction and intestinal malabsorption. However, studies parceling out the contribution of enhanced intestinal stimulation in the absence of these two mechanisms have received little attention. Previous studies have demonstrated that patients who received intestinal bypass or Roux-en-Y surgery have increased release of gastrointestinal hormones. One possible mechanism for this increase is the rapid transit of nutrients into the intestine after eating. To determine whether there is increased secretion of anorectic peptides produced in the distal small intestine when this portion of the gut is given greater exposure to nutrients, we preformed ileal transpositions (IT) in rats. In this procedure, an isolated segment of ileum is transposed to the jejunum, resulting in an intestinal tract of normal length but an alteration in the normal distribution of endocrine cells along the gut. Rats with IT lost more weight (P < 0.05) and consumed less food (P < 0.05) than control rats with intestinal transections and reanastomosis without transposition. Weight loss in the IT rats was not due to malabsorption of nutrients. However, transposition of distal gut to a proximal location caused increased synthesis and release of the anorectic ileal hormones glucagon-like peptide-1 (GLP-1) and peptide YY (PYY; P < 0.01). The association of weight loss with increased release of GLP-1 and PYY suggests that procedures that promote gastrointestinal endocrine function can reduce energy intake. These findings support the importance of evaluating the contribution of gastrointestinal hormones to the weight loss seen with bariatric surgery.     

Diseases and mortality in free-ranging brown bear (Ursus arctos), gray wolf (Canis lupus), and wolverine (Gulo gulo) in Sweden

Ninety-eight brown bears (Ursus arctos), 20 gray wolves (Canis lupus), and 27 wolverines (Gulo gulo), all free-ranging, were submitted to the National Veterinary Institute, Uppsala, Sweden, during 1987-2001 for investigation of diseases and causes of mortality. The most common cause of natural death in brown bears was infanticide. Infanticide also was observed in wolverines but not in wolves. Traumatic injuries, originating from road or railway accidents, were the most common cause of death in wolves and occurred occasionally in brown bears. Most wolverines were submitted as forensic cases in which illegal hunting/poaching was suspected. Sarcoptic mange was observed in several wolves but not in brown bears or wolverines. Sarcoptic mange most likely was acquired from infected red foxes (Vulpes vulpes) that were killed by wolves. Other parasites and infectious diseases were only found sporadically.     

Fallot's tetralogy in a European brown bear (Ursus arctos)

A congenital heart malformation was diagnosed in a free-ranging adult female European brown bear (Ursus arctos) found dead due to intraspecific violence. At necropsy, the heart had all four features of Fallot's tetralogy. No further malformations were identified in the animal, which was of normal size and weight for its age, in normal body condition, and had probably borne young. The heart condition was considered to have contributed to death during an attack by another bear.     

Epizootiologic investigations of selected infectious disease agents in free-ranging Eurasian lynx from Sweden

Serum samples from 106 Eurasian lynx (Lynx lynx) from across Sweden, found dead or shot by hunters in 1993-99, were investigated for presence of antibodies to feline parvovirus (FPV), feline coronavirus, feline calicivirus, feline herpesvirus, feline immunodeficiency virus, Francisella tularensis, and Anaplasma phagocytophila, and for feline leukemia virus antigen. In addition, tissue samples from 22 lynx submitted in 1999 were analyzed by real-time polymerase chain reaction (PCR) to detect nucleic acids specific for viral agents and A. phagocytophila. Except for FPV antibodies in one lynx and A. phagocytophila in four lynx, all serology was negative. All PCR results also were negative. It was concluded that free-ranging Swedish lynx do not have frequent contact with the infectious agents considered in this study.     

Gene delivery with low molecular weight linear polyethylenimines

BACKGROUND: Linear polyethylenimine (LPEI) with a molecular weight (MW) of 22 kDa has been described as having a superior ability to induce gene transfer compared to its branched form. However, the transfection efficiency of the polymer cannot be enhanced beyond a certain limit due to cytotoxicity. We explored the potential of utilizing LPEIs with MWs ranging from 1.0 to 9.5 kDa to overcome this limitation. METHODS: Polyplexes of plasmid DNA encoding for the enhanced green fluorescent protein (EGFP) and various LPEIs were compared concerning their transfection efficiency and cytotoxicity in CHO-K1 and HeLa cells by flow cytometry. The involvement of endolysosomes in LPEI-mediated gene transfer was investigated by applying the proton pump inhibitor bafilomycin A1 and the lysosomotropic agent sucrose. Confocal laser scanning microscopy was applied to assess the size and shape of polyplexes under cell culture conditions, to detect their endolysosomal localization and to observe their translocation to the nucleus. RESULTS: The transfection efficiency could be altered by varying the MW and the amount of the polymer available for polyplex formation. The highest transfection efficiency (about 44%), i.e. the fraction of EGFP-positive cells, was obtained with LPEI 5.6 kDa, while the cytotoxicity remained low. The colocalization of polyplexes and endolysosomes was observed, and it appeared that the larger polyplexes escaped from the acidic organelles particularly quickly. For LPEI 5.0 and 9.0 kDa, the number of cells and nuclei that had taken up DNA after 6 hours was similar, as determined by flow cytometry. CONCLUSIONS: Our study suggests that LPEIs with low MWs are promising candidates for non-viral gene delivery, because they are more efficient and substantially less toxic than their higher MW counterparts.     

Change of the donor substrate specificity of Clostridium difficile toxin B by site-directed mutagenesis

The large cytotoxins of Clostridia species glycosylate and thereby inactivate small GTPases of the Rho family. Clostridium difficile toxins A and B and Clostridium sordellii lethal toxin use UDP-glucose as the donor for glucosylation of Rho/Ras GTPases. In contrast, alpha-toxin from Clostridium novyi N-acetylglucosaminylates Rho GTPases by using UDP-N-acetylglucosamine as a donor substrate. Based on the crystal structure of C. difficile toxin B, we studied the sugar donor specificity of the toxins by site-directed mutagenesis. The changing of Ile-383 and Gln-385 in toxin B to serine and alanine, respectively, largely increased the acceptance of UDP-N-acetylglucosamine as a sugar donor for modification of RhoA. The K(m) value was reduced from 960 to 26 mum for the double mutant. Accordingly, the potential of the double mutant of toxin B to hydrolyze UDP-N-acetylglucosamine was higher than that for UDP-glucose. The changing of Ile-383 and Gln-385 in the lethal toxin of C. sordellii allowed modification of Ras in the presence of UDP-N-acetyl-glucosamine and reduced the acceptance of UDP-glucose as a donor for glycosylation. Vice versa, the changing of the equivalent residues in C. novyi alpha-toxin from Ser-385 and Ala-387 to isoleucine and glutamine, respectively, reversed the donor specificity of the toxin from UDP-N-acetylglucosamine to UDP-glucose. These data demonstrate that two amino acid residues are crucial for the co-substrate specificity of clostridial glycosylating toxins.     

Characterization of the NifS-like domain of ABA3 from Arabidopsis thaliana provides insight into the mechanism of molybdenum cofactor sulfuration

The molybdenum cofactor sulfurase ABA3 from Arabidopsis thaliana specifically regulates the activity of the molybdenum enzymes aldehyde oxidase and xanthine dehydrogenase by converting their molybdenum cofactor from the desulfo-form into the sulfo-form. ABA3 is a two-domain protein with an NH2-terminal domain sharing significant similarities to NifS proteins that catalyze the decomposition of l-cysteine to l-alanine and elemental sulfur for iron-sulfur cluster synthesis. Although different in its physiological function, the mechanism of ABA3 for sulfur mobilization was found to be similar to NifS proteins. The protein binds a pyridoxal phosphate cofactor and a substrate-derived persulfide intermediate, and site-directed mutagenesis of strictly conserved binding sites for the cofactor and the persulfide demonstrated that they are essential for molybdenum cofactor sulfurase activity. In vitro, the NifS-like domain of ABA3 activates aldehyde oxidase and xanthine dehydrogenase in the absence of the C-terminal domain, but in vivo, the C-terminal domain is required for proper activation of both target enzymes. In addition to its cysteine desulfurase activity, ABA3-NifS also exhibits selenocysteine lyase activity. Although l-selenocysteine is unlikely to be a natural substrate for ABA3, it is decomposed more efficiently than l-cysteine. Besides mitochondrial AtNFS1 and plastidial AtNFS2, which are both proposed to be involved in iron-sulfur cluster formation, ABA3 is proposed to be a third and cytosolic NifS-like cysteine desulfurase in A. thaliana. However, the sulfur transferase activity of ABA3 is used for post-translational activation of molybdenum enzymes rather than for iron-sulfur cluster assembly.