A missense mutation in the non-neural G-protein alpha-subunit isoforms modulates susceptibility to obesity.

OBJECTIVE: The Gnas transcription unit located within an imprinting region encodes several proteins, including the G-protein alpha-subunit, Gsalpha, its isoform XLalphas and their variant truncated neural forms GsalphaN1 and XLN1. Gsalpha and GsalphaN1 are expressed predominantly from the maternally derived allele in some tissues, whereas XLalphas and XLN1 are expressed exclusively from the paternally derived allele. The relative contribution of full-length Gsalpha and XLalphas, and truncated forms GsalphaN1 and XLN1 to phenotype is unknown. The edematous-small point mutation (Oed-Sml) in exon 6 of Gnas lies downstream of GsalphaN1 and XLN1, but affects full-length Gsalpha and XLalphas, allowing us to address the role of full-length Gsalpha and XLalphas. The aim of this study was therefore to determine the metabolic phenotypes of Oed and Sml mice, and to correlate phenotypes with affected transcripts. METHODS: Mice were fed standard or high-fat diets and weighed regularly. Fat mass was determined by DEXA analysis. Indirect calorimetry was used to measure metabolic rate. Glucose was measured in tolerance tests and biochemical parameters in fasted plasma samples. Histological analysis of fat and liver was carried out post mortem. RESULTS: Oed mice are obese on either diet and have a reduced metabolic rate. Sml mice are lean and are resistant to a high-fat diet and have an increased metabolic rate. CONCLUSION: Adult Oed and Sml mice have opposite metabolic phenotypes. On maternal inheritance, the obese Oed phenotype can be attributed to non-functional full-length Gsalpha. In contrast, on paternal inheritance, Sml mice were small and resistant to the development of obesity on a high-fat diet, effects that can be attributed to mutant XLalphas. Thus, the neural isoforms, GsalphaN1 and XLN1, do not appear to play a role in these metabolic phenotypes.

Analysis of breeding and pathology helps refine management practices of a large-scale N'-ethyl-N'-nitrosourea mouse mutagenesis programme.

N'-ethyl-N'-nitrosourea (ENU) is a powerful germline mutagen used in conjunction with phenotype-driven screens to generate novel mouse mutants. ENU also induces genetic lesions in somatic cells and dosage requires optimization between maximum germline mutation rate versus induced sterility and tumourigenesis that compromise the welfare and fecundity of the ENU-treated males. Here, we present our experience with BALB/cAnNCrl and C57BL/6J mice in terms of the pathology induced by ENU and its impact on breeding. In both mouse strains, morbidity and mortality rises with ENU dose. In more than 75% of C57BL/6J males, morbidity and mortality were attributable to the development of malignant T-lymphoblastic lymphoma. Approximately 50% of ENU-treated BALB/cAnNCrl males develop early malignant T-lymphoblastic lymphoma, but the cohort that survives develops late-onset lung carcinoma. Within strains, the latency of these clinically important tumour(s) was not dosage-dependent, but the proportion of mice developing tumours and consequently removed from the breeding programme increased with ENU dosage. The median number of offspring per ENU-treated C57BL/6J male in standard matings with C3H/HeH females decreased with increasing dosage. The two most important underlying causes for lower male fecundity were increased infertility in the highest dosage group and reduced numbers of litters born to the remaining fertile C57BL/6J males due to a higher incidence of morbidity. These findings have allowed us to refine breeding strategy. To maximize the number of offspring from each ENU-treated male, we now rotate productive males between two cages to expose them to more females. This optimizes the number of mutation carrying offspring while reducing the number of ENU-treated males that must be generated.

Upgrading mouse health and welfare: direct benefits of a large-scale rederivation programme.

We report the outcome of a 30-month programme to rederive 310 specific pathogen-free mouse strains to populate a new individually ventilated cage barrier facility at the Mary Lyon Centre (MLC), Medical Research Council (MRC) Harwell. The mice were rederived in a self-contained quarantine suite and embryo-recipient females were health-screened to assess microbiological status, before moving their offspring into the new facility. The MLC currently houses approximately 49,000 mice in about 9750 cages and we have 30 months of follow-up health screen data. Embryo rederivation and hysterectomy have high safety margins; however, the precaution of performing the programme in isolators facilitated the containment and decontamination of two mouse hepatitis virus (MHV) infection outbreaks. Rederivation of the colony has eliminated endemic MHV, mouse adenovirus type 2 (MAV-2), Theiler's murine encephalomyelitis virus, pinworms, intestinal protozoa, Pasteurella pneumotropica, Helicobacter spp. and mites. The improvements in microbiological status have had notable benefits for mouse health and welfare and the science at MRC Harwell. Previously important clinical entities such as sudden death associated with lactation ileus in C3H/HeH mice, early weight loss associated with inflammatory bowel disease in B6-TgN(HDexon1)61Gpb and B6TgN-(HD82Gln)81Dbo (Huntington) mice and early weight loss in male mice mutagenized with N-ethyl-N-nitrosourea have been markedly reduced or eliminated.

Structure-activity relationship analysis tools: validation and applicability in predicting carcinogens.

OncoLogic and MultiCASE (MCASE) are two structure-activity relationship (SAR) analysis software programs available to screen compounds for potential carcinogenicity. Ashby-Tennant structural alerts [Ashby, J., Tennant, R.W., 1991. Definitive relationships among chemical structure, carcinogenicity, and mutagenicity for 301 chemicals tested by the US NTP. Mutat. Res. 257, 229-306] and genetic toxicity testing may also be used to assess/predict this endpoint. Six-hundred and fifty compounds tested for carcinogenicity whose results were tabulated in the Carcinogenic Potency Database (CPDB) were used to validate and compare the predictivity of OncoLogic (Version 4.1), MCASE (Version 3.1), Ashby-Tennant structural alerts, and genetic toxicity testing, individually and in combination. The sensitivity of the methods for predicting carcinogens and the specificity for predicting non-carcinogens was examined. Potent carcinogens, defined as those with TD(50) values of less than 6.25 mg/kg bw/d, were then examined separately. It is concluded that SAR analysis programs and structural alerts perform well for compounds with low human exposure levels and have the potential to supplement the results of routinely requested genetic toxicity tests in a weight-of-evidence approach in predicting carcinogenicity, although each method of analysis has limitations regarding applicability.

A metabolomic comparison of urinary changes in type 2 diabetes in mouse, rat, and human.

Type 2 diabetes mellitus is the result of a combination of impaired insulin secretion with reduced insulin sensitivity of target tissues. There are an estimated 150 million affected individuals worldwide, of whom a large proportion remains undiagnosed because of a lack of specific symptoms early in this disorder and inadequate diagnostics. In this study, NMR-based metabolomic analysis in conjunction with multivariate statistics was applied to examine the urinary metabolic changes in two rodent models of type 2 diabetes mellitus as well as unmedicated human sufferers. The db/db mouse and obese Zucker (fa/fa) rat have autosomal recessive defects in the leptin receptor gene, causing type 2 diabetes. 1H-NMR spectra of urine were used in conjunction with uni- and multivariate statistics to identify disease-related metabolic changes in these two animal models and human sufferers. This study demonstrates metabolic similarities between the three species examined, including metabolic responses associated with general systemic stress, changes in the TCA cycle, and perturbations in nucleotide metabolism and in methylamine metabolism. All three species demonstrated profound changes in nucleotide metabolism, including that of N-methylnicotinamide and N-methyl-2-pyridone-5-carboxamide, which may provide unique biomarkers for following type 2 diabetes mellitus progression.

Thresholds as a unifying theme in regulatory toxicology.

The scientific basis for the US Food and Drug Administration (FDA) threshold of regulation is discussed in relation to its toxicological testing recommendations for food contact substances and the existing methods it employs for exposure estimation. A case is made that the FDA's threshold of regulation is a natural extension of its toxicity testing regime. The genetic toxicity tests recommended in the exposure-based toxicological testing framework for food contact substances are examined regarding their ability to predict positively carcinogens of varying potency. In addition, the computational toxicology program MULTICASE v. 3.1 is also examined for its ability to predict positively carcinogens of varying potency. It is concluded that MULTICASE can provide equivalent results to genetic toxicity tests at the lowest dietary concentrations.

The use of structure-activity relationship analysis in the food contact notification program.

Food contact substances (FCS) include polymers, paper and paperboard, and substances used in their manufacture, that do not impart a technical effect on food. Moreover, FCSs are industrial chemicals generally consumed at dietary concentrations (DC) of less than 1mg/kg food (ppm), and more commonly at less than 0.05 ppm (50 ppb), in the daily diet. As such, many industrial chemicals have been analyzed for toxicological concern, some of which may share structural similarity with FCSs or their constituents, and the majority of these studies are available in the public domain. The DCs of these compounds lend themselves to using structure-activity relationship (SAR) analysis, as the available "expert systems" and use of analogs allows for prediction and management of potential carcinogens. This paper describes the newly implemented food contact notification (FCN) program, the program by which FDA reviews FCSs for safe use, the administrative review of FCSs, the SAR tools available to FDA, and qualitative and quantitative risk assessments using SAR analysis within the regulatory framework of reviewing the safety of FCSs.

Ectopic stratified squamous epithelium in bovine jejunum.

An adult dairy cow developed acute fatal diarrhoea. Necropsy provided no aetiological or morphological cause of the diarrhoea but incidental post-mortem findings included well-circumscribed areas of jejunal squamous epithelium. The cause of this unusual heterotopia is not known, but the lesion may have been of embryological or metaplastic origin.

Structure-based thresholds of toxicological concern (TTC): guidance for application to substances present at low levels in the diet.

The threshold of toxicological concern (TTC) is a pragmatic risk assessment tool that is based on the principle of establishing a human exposure threshold value for all chemicals, below which there is a very low probability of an appreciable risk to human health. The concept that there are levels of exposure that do not cause adverse effects is inherent in setting acceptable daily intakes (ADIs) for chemicals with known toxicological profiles. The TTC principle extends this concept by proposing that a de minimis value can be identified for many chemicals, in the absence of a full toxicity database, based on their chemical structures and the known toxicity of chemicals which share similar structural characteristics. The establishment and application of widely accepted TTC values would benefit consumers, industry and regulators. By avoiding unnecessary toxicity testing and safety evaluations when human intakes are below such a threshold, application of the TTC approach would focus limited resources of time, cost, animal use and expertise on the testing and evaluation of substances with the greatest potential to pose risks to human health and thereby contribute to a reduction in the use of animals. An Expert Group of the European branch of the International Life Sciences Institute-ILSI Europe-has examined the TTC principle for its wider applicability in food safety evaluation. The Expert Group examined metabolism and accumulation, structural alerts, endocrine disrupting chemicals and specific endpoints, such as neurotoxicity, teratogenicity, developmental toxicity, allergenicity and immunotoxicity, and determined whether such properties or endpoints had to be taken into consideration specifically in a step-wise approach. The Expert Group concluded that the TTC principle can be applied for low concentrations in food of chemicals that lack toxicity data, provided that there is a sound intake estimate. The use of a decision tree to apply the TTC principle is proposed, and this paper describes the step-wise process in detail. Proteins, heavy metals and polyhalogenated-dibenzodioxins and related compounds were excluded from this approach. When assessing a chemical, a review of prior knowledge and context of use should always precede the use of the TTC decision tree. The initial step is the identification and evaluation of possible genotoxic and/or high potency carcinogens. Following this step, non-genotoxic substances are evaluated in a sequence of steps related to the concerns that would be associated with increasing intakes. For organophosphates a TTC of 18microg per person per day (0.3 microg/kg bw/day) is proposed, and when the compound is not an OP, the TTC values for the Cramer structural classes III, II and I, with their respective TTC levels (e.g. 1800, 540 and 90 microg per person per day; or 30, 9 and 1.5 microg/kg bw /day), would be applied sequentially. All other endpoints or properties were shown to have a distribution of no observed effect levels (NOELs) similar to the distribution of NOELs for general toxicity endpoints in Cramer classes I, II and III. The document was discussed with a wider audience during a workshop held in March 2003 (see list of workshop participants).

Expression, purification and characterization of cytochrome P450 Biol: a novel P450 involved in biotin synthesis in Bacillus subtilis.

The bioI gene has been sub-cloned and over-expressed in Escherichia coli, and the protein purified to homogeneity. The protein is a cytochrome P450, as indicated by its visible spectrum (low-spin haem iron Soret band at 419 nm) and by the characteristic carbon monoxide-induced shift of the Soret band to 448 nm in the reduced form. N-terminal amino acid sequencing and mass spectrometry indicate that the initiator methionine is removed from cytochrome P450 BioI and that the relative molecular mass is 44,732 Da, consistent with that deduced from the gene sequence. SDS-PAGE indicates that the protein is homogeneous after column chromatography on DE-52 and hydroxyapatite, followed by FPLC on a quaternary ammonium ion-exchange column (Q-Sepharose). The purified protein is of mixed spin-state by both electronic spectroscopy and by electron paramagnetic resonance [g values=2.41, 2.24 and 1.97/1.91 (low-spin) and 8.13, 5.92 and 3.47 (high-spin)]. Magnetic circular dichroism and electron paramagnetic resonance studies indicate that P450 BioI has a cysteine-ligated b-type haem iron and the near-IR magnetic circular dichroism band suggests strongly that the sixth ligand bound to the haem iron is water. Resonance Raman spectroscopy identifies vibrational signals typical of cytochrome P450, notably the oxidation state marker v4 at 1,373 cm(-1) (indicating ferric P450 haem) and the splitting of the spin-state marker v3 into two components (1,503 cm(-1) and 1,488 cm(-1)), indicating cytochrome P450 BioI to be a mixture of high- and low-spin forms. Fatty acids were found to bind to cytochrome P450 BioI, with myristic acid (Kd=4.18+/-0.26 microM) and pentadecanoic acid (Kd=3.58+/-0.54 microM) having highest affinity. The fatty acid analogue inhibitor 12-imidazolyldodecanoic acid bound extremely tightly (Kd<1 microM), again indicating strong affinity for fatty acid chains in the P450 active site. Catalytic activity was demonstrated by reconstituting the P450 with either a soluble form of human cytochrome P450 reductase, or a Bacillus subtilis ferredoxin and E. coli ferredoxin reductase. Substrate hydroxylation at the omega-terminal position was demonstrated by turnover of the chromophoric fatty acid para-nitrophenoxydodecanoic acid, and by separation of product from the reaction of P450 BioI with myristic acid.

Preliminary characterisation of esterase and platelet-activating factor (PAF)-acetylhydrolase activities from cat flea (Ctenocephalides felis) salivary glands.

Naphthyl esterase and platelet-activating factor (PAF)-acetylhydrolase activities were detected in the salivary glands of the cat flea, Ctenocephalides felis. Salivary naphthyl esterase activity is disgorged during exploratory probing. Whole extracts of salivary glands contain esterase activity against the short-chain naphthyl esters alpha-naphthyl acetate (approximately 210pmol/min/gland pair; 10.0micromol/min/mg specific activity; K(m) approximately 59microM) and beta-naphthyl acetate (approximately 110pmol/min/gland pair; 5.2micromol/min/mg specific activity; K(m) approximately 132microM). Salivary gland extracts have PAF-acetylhydrolase activity (approximately 5pmol/min/gland pair; 0.24micromol/min/mg specific activity) but do not have detectable acetylcholinesterase activity. Native-PAGE and IEF resolve three and six salivary gland naphthyl esterase bands, respectively, and both patterns are different from carcass esterases. Salivary gland naphthyl esterase activity binds reversibly to Concanavalin A, and enzymatic deglycosylation with glycopeptidase F produced a new, fast-migrating salivary gland naphthyl esterase band on Native-PAGE. Renaturation of esterase activity after SDS-PAGE gave approximately 56kDa, approximately 57kDa and approximately 58kDa naphthyl-esterase-positive bands. On gel filtration naphthyl esterase and PAF-acetylhydrolase activities co-elute as a single peak with an apparent molecular weight of approximately 59kDa. This partially purified pool of enzyme had esterase activity against a series of short-chain alpha- and beta-naphthyl esters. The heterogeneity of salivary gland esterases, their relationship to PAF-acetylhydrolase, and the possible physiological functions of salivary gland PAF-acetylhydrolase activity are discussed.

Anaemia, diarrhoea and opportunistic infections in Fell ponies.

This report summarises clinical and pathological observations on Fell pony foals with a range of signs that included ill thrift, anaemia, respiratory infection, glossal hyperkeratosis and diarrhoea. Some of the foals had normochromic, normocytic anaemia and some had low levels of plasma proteins, including immunoglobulin G. Antibiotic and supportive treatment was ineffective and all affected foals died or were killed on humane grounds. Postmortem examination of 12 foals and tissues from 2 other foals revealed a range of lesions that included glossal hyperkeratosis, typhlocolitis, intestinal cryptosporidiosis, granulomatous enteritis, proliferative and necrotising bronchiolitis consistent with adenovirus infection; lesions similar to those in the respiratory tract were present in the salivary gland and pancreas of individual foals. Lymphoid tissue was judged to be smaller than expected. These observations suggest the possibility of opportunistic infections secondary to some form of undefined immunocompromised state.

Resection of the common digital extensor tendon in a gelding.

A four-year-old gelding was lame owing to a chronic septic common digital extensor tendon and sheath. The horse had been treated by open surgical lavage but the sepsis had recurred after three months. Physical, ultrasonographic, cytological and histological examinations confirmed chronic septic tenosynovitis and tendonitis. The entire intrathecal component of the common digital extensor tendon was resected under general anaesthesia and the synovial lining of the sheath was ablated. Postoperatively the horse regained good limb function and became sound.

A tiered approach to threshold of regulation.

This paper presents methods for extending the principle of a single "threshold of regulation" to a range of dietary concentrations between 0.5 and 15 parts per billion by using structure-activity relationships, genotoxicity, and short-term toxicity data. The database used to develop the FDA's threshold of regulation was examined to determine whether structural parameters or the result of certain short-term toxicity tests could be used to define a subset of less potent substances that supports higher threshold levels. In addition, results of reproductive toxicity tests for 3306 compounds and other multidose toxicity tests for 2542 compounds were compared with the database of carcinogenic potencies to establish that carcinogenic endpoints are the most conservative toxicity endpoint for establishing thresholds of regulation.

Genitourinary dysplasia in a cat.

A six-month-old kitten had congenital urethral sphincter mechanism incompetence due to urethral hypoplasia and associated uterine hypoplasia and vaginal aplasia. Diagnosis was based on radiographic examination, surgical exploration and histological examination of the lower urinary tract. Surgical correction resulted in a marked clinical improvement. The cat became fully continent following treatment with phenylpropanolamine.

Heterotopic pancreas at a site of colon stricture and ulceration in a guineapig.

A one-month-old female Dunkin Hartley guineapig presented with a thin hair coat and distended abdomen. Post-mortem findings after euthanasia were a protein-rich modified peritoneal transudate and heterotopic pancreas at a site of colon stricture and ulceration. Complications of pancreatic heterotopia are well recognized in man but not in animals. The finding in this individual is probably unrelated to sudden deaths in other animals in the group.

Characterization of apyrase activity from the salivary glands of the cat flea Ctenocephalides felis.

Apyrase activity (ATP diphosphohydrolase, EC 3.6.1.5) was detected in salivary glands of the cat flea Ctenocephalides felis. Whole extracts of salivary glands contain approximately 21 ng of protein, 145 U/mg ADP'ase and 158 U/mg ATP'ase activity; AMP is not hydrolysed by salivary gland extracts. DEAE-Sepharose CL-6B anion exchange chromatography, and Cibacron Blue affinity chromatography each give a single coincident peak of ADP/ATP'ase activity. Biogel P-100 gel filtration of salivary gland homogenates made in buffer containing Triton and protease inhibitors, separated enzymatic activity into 57 kD and 44 kD peaks of ADP/ATP'ase activity. Partially purified ADP/ATP'ases are dependent on divalent cations and activation increases between 0.125 mM and 5.0 mM calcium. At 5 mM, magnesium is almost equally effective as calcium in activating ADP/ATP'ase but manganese and zinc are less so, and EDTA abolishes activity. ADP/ATP'ases have a pH optima of 7-9. The Km for ADP hydrolysis by whole extracts and partially purified enzyme is approximately 66 microM ADP. The co-purification of ADP'ase and ATP'ase activity by three physiochemical techniques and parallelism between ADP and ATP hydrolysis under varying conditions of pH and activating cation indicates enzymatic activity is attributable to true apyrase(s).

Heterotopic neural tissue in the pharynx of a 7-week-old kitten.

A 7-week-old male kitten had a pharyngeal mass (1 x 2 cm) causing displacement of the tongue. The surgically resected tissue was seen to be a moderately discrete subepithelial mass comprising islands of neuroglia and neurons separated by dense collagenous connective tissue. It is not known whether this mass retained any connection with the brain. Histochemical and immunohistochemical examination confirmed the presence of neurons and a pleocellular glial population, supporting a diagnosis of heterotopic neural tissue. The cat remained well 20 months after surgical treatment. Heterotopic neural tissue is well-recognized in man but has not been described in animals.