Comparison of ionised calcium measured using a portable analyser to a reference method in healthy dogs.

OBJECTIVES: To compare the ionised calcium measured on a portable analyser (iSTAT, Abbott) to a reference method. MATERIALS AND METHODS: Blood samples from 39 apparently healthy dogs were analysed in duplicate using a portable analyser and a reference method (Radiometer ABL800 FLEX). Bland-Altman plots and Passing-Bablok regression were used to assess constant and proportional bias between the two instruments. A within-assay percentage coefficient of variation and total error (TE) was calculated for both analysers. The reference interval was calculated for the portable analyser using the robust method with confidence interval bootstrapping. RESULTS: The Bland-Altman plot showed a -0.036 mmol/L difference between the two instruments (95% confidence limit -0.08 to 0.01 mmol/L; limits of agreement -0.07 to 0.006 mmol/L). Neither the Bland-Altman plot nor the Passing-Bablock regression (slope -0.03; 95% confidence interval -0.08 to 0.19 and intercept 1; 95% confidence interval 0.83 to 1.2) showed significant proportional bias. The coefficient of variation for the portable analyser was 1.08%, compared to 0.78% for the reference method with a total error of 3.5% for the portable analyser. The estimated population-based reference interval for ionised calcium using the portable analyser is 1.23 to 1.42 mmol/L. CLINICAL SIGNIFICANCE: For the healthy dogs in this study, compared to the reference method, the portable analyser showed no significant bias for measurement of ionised calcium. Further studies including hyper and hypocalcaemic dogs are required to determine clinical impact of the use of this analyser.

Molecular tagging of a senescence gene by introgression mapping of a stay-green mutation from Festuca pratensis.

* Intergeneric hybrids between Lolium multiflorum and Festuca pratensis (Lm/Fp) and their derivatives exhibit a unique combination of genetic and cytogenetic characteristics: chromosomes undergo a high frequency of homoeologous recombination at meiosis; the chromosomes of the two species can easily be discriminated by genomic in situ hybridization (GISH); recombination occurs along the entire length of homoeologous bivalents; a high frequency of marker polymorphism is observed between the two species. * This combination of characters has been used to transfer and isolate a F. pratensis chromosome segment carrying a mutant 'stay-green' gene conferring a disrupted leaf senescence phenotype into L. multiflorum. * The genetic location within the introgressed F. pratensis segment of the senescence gene has been mapped using amplified fragment length polymorphisms (AFLPs), and F. pratensis-specific AFLP markers closely flanking the green gene have been cloned. * The use of these cloned sequences as markers for the stay-green locus in marker-assisted selection programmes has been tested. The potential application of Lm/Fp introgressions as a tool for the map-based cloning of introgressed Fp genes is discussed.

Introgression-mapping of genes for drought resistance transferred from Festuca arundinacea var. glaucescens into Lolium multiflorum.

Procedures for the transfer of genes for drought resistance from Festuca glaucescens (2n=4x=28) into Lolium multiflorum (2n=2x=14) are described. Following the initial hybridisation of a synthetic autotetraploid of L. multiflorum (2n=4x=28) with F. glaucescens, the F1 hybrid was backcrossed twice onto diploid L. multiflorum (2n=2x=14) to produce a diploid Lolium genotype with a single F. glaucescens introgression located distally on the nucleolar organiser region arm of chromosome 3. The transmission of F. glaucescens-derived amplified fragment length polymorphisms and a sequence-tagged-site (STS) marker was monitored throughout the breeding programme. Those genotypes of a mapping population of backcross 3 that survived combined severe drought and heat stress all contained the F. glaucescens-derived markers. The STS marker provided a prototype for a PCR-based system for high-throughput screening during cultivar development for the presence of the F. glaucescens-derived genes for drought resistance. The frequency of intergeneric recombination between L. multiflorum and F. glaucescens is described. During the initial stages of the breeding programme, preferential intraspecific chromosome pairing between Lolium homologues and Festuca homoeologues dominated with low frequencies of intergeneric chromosome associations. However, these increased in the backcross 1 due to the absence of opportunities for intraspecific chromosome pairing between homoeologous Festuca chromosomes following the loss of half of the Festuca chromosomes. Once transferred to Lolium, F. glaucescens sequences recombined with Lolium at high frequencies, thereby enabling the loss of potentially deleterious gene combinations that might reduce the forage quality of Lolium.

Introgression of crown rust (Puccinia coronata) resistance from meadow fescue (Festuca pratensis) into Italian ryegrass (Lolium multiflorum) and physical mapping of the locus.

Resistance was found in the meadow fescue (Festuca pratensis) to crown rust (Puccinia coronata), originating from ryegrasses (Lolium spp). A backcrossing programme successfully transferred this resistance into diploid Italian ryegrass (Lolium multiflorum) and genomic in situ hybridisation (GISH) was used to identify the introgressed fescue chromosome segment. The resistant (R) plants in two BC3 lines all carried an introgressed segment on a single chromosome, which in one of the lines was confined to the short arm of the chromosome. Susceptible (S) plants either contained no introgressed chromosome segment or a segment which was physically smaller than the segments in resistant plants. Using GISH the resistance locus could be physically mapped to the midpoint of a short arm. Segregation ratios of the progeny of BC3 plants, when crossed as R x S and R x R, were in agreement with the hypothesis that the resistance was controlled by a single gene or very closely linked genes. No R plants were produced by crossing S x S plants.

Notch signaling in development and disease.

Notch receptors and ligands were first identified in flies and worms, where they were shown to regulate cell proliferation, cell differentiation, and, in particular, binary cell fate decisions in a variety of developmental contexts. The first mammalian Notch homolog was discovered to be a partner in a chromosomal translocation in a subset of human T-cell leukemias. Subsequent studies in mice and humans have shown that Notch signaling plays essential roles at multiple stages of hematopoiesis, and also regulates the development or homeostasis of cells in many tissues and organs. Thus, it is not surprising that mutations which disrupt Notch signaling cause a wide range of cancers and developmental disorders. Perhaps because it is so widely used, Notch signaling is subject to many unusual forms of regulation. In this review, we will first outline key aspects of Notch signaling and its regulation by endocytosis, glycosylation, and ubiquitination. We will then overview recent literature elucidating how Notch regulates cell-lineage decisions in a variety of developmental contexts. Finally, we will describe the roles of dysregulated Notch signaling in causing several types of cancer and other pathologies.

Gross chromosome rearrangements are occurring in an accession of the grass Lolium rigidum.

Chromosome structure was analysed at mitosis in root tip meristems of eight genotypes of Lolium rigidum. FISH revealed changed positions in the rDNA sites indicating extensive chromosome rearrangements; indeed no two genotypes were the same. In one genotype, there were differences between cells within individual root tips. The changed positions of the rDNA sites appear to be reflections of chromosome translocations and this was confirmed by the presence of quadrivalents at metaphase I of meiosis. Possible mechanisms are discussed for this exceptional level of chromosome instability.

A mitochondrial uncoupling artifact can be caused by expression of uncoupling protein 1 in yeast.

Uncoupling protein 1 (UCP1) from mouse was expressed in yeast and the specific (GDP-inhibitable) and artifactual (GDP-insensitive) effects on mitochondrial uncoupling were assessed. UCP1 provides a GDP-inhibitable model system to help interpret the uncoupling effects of high expression in yeast of other members of the mitochondrial carrier protein family, such as the UCP1 homologues UCP2 and UCP3. Yeast expressing UCP1 at modest levels (approx. 1 microg/mg of mitochondrial protein) showed no growth defect, normal rates of chemically uncoupled respiration and an increased non-phosphorylating proton conductance that was completely GDP-sensitive. The catalytic-centre activity of UCP1 in these yeast mitochondria was similar to that in mammalian brown-adipose-tissue mitochondria. However, yeast expressing UCP1 at higher levels (approx. 11 microg/mg of mitochondrial protein) showed a growth defect. Their mitochondria had depressed chemically uncoupled respiration rates and an increased proton conductance that was partly GDP-insensitive. Thus, although UCP1 shows native behaviour at modest levels of expression in yeast, higher levels (or rates) of expression can lead to an uncoupling that is not a physiological property of the native protein and is therefore artifactual. This observation might be important in the interpretation of results from experiments in which the functions of UCP1 homologues are verified by their ability to uncouple yeast mitochondria.

Genetic and physical analysis of a single Festuca pratensis chromosome segment substitution in Lolium perenne.

Molecular marker analysis and genomic in situ hybridisation (GISH) were used to examine the process of chromosome segment introgression in BC2 diploid hybrids (2n=2x=14) between Lolium perenne and Festuca pratensis. Two genotypes having what appeared to be the same, single, introgressed chromosome segment of F. pratensis in the L. perenne background were crossed with diploid L. perenne to produce a recombinant series for the introgressed region. Physical and genetic analysis of this series showed that, while recombination seemed to be possible at all points along the chromosome arm, the rate of recombination varied depending on relative position: more recombination was detected in the interstitial region as compared with the centromeric or telomeric regions. The implications of these results for the use of GISH and molecular marker analysis in the measurement of linkage drag in backcross breeding programmes is discussed.

Physiological levels of mammalian uncoupling protein 2 do not uncouple yeast mitochondria.

We assessed the ability of human uncoupling protein 2 (UCP2) to uncouple mitochondrial oxidative phosphorylation when expressed in yeast at physiological and supraphysiological levels. We used three different inducible UCP2 expression constructs to achieve mitochondrial UCP2 expression levels in yeast of 33, 283, and 4100 ng of UCP2/mg of mitochondrial protein. Yeast mitochondria expressing UCP2 at 33 or 283 ng/mg showed no increase in proton conductance, even in the presence of various putative effectors, including palmitate and all-trans-retinoic acid. Only when UCP2 expression in yeast mitochondria was increased to 4 microg/mg, more than an order of magnitude greater than the highest known physiological concentration, was proton conductance increased. This increased proton conductance was not abolished by GDP. At this high level of UCP2 expression, an inhibition of substrate oxidation was observed, which cannot be readily explained by an uncoupling activity of UCP2. Quantitatively, even the uncoupling seen at 4 microgram/mg was insufficient to account for the basal proton conductance of mammalian mitochondria. These observations suggest that uncoupling of yeast mitochondria by UCP2 is an overexpression artifact leading to compromised mitochondrial integrity.

Mitochondrial uncoupling as a target for drug development for the treatment of obesity.

Mitochondrial proton cycling is responsible for a significant proportion of basal or standard metabolic rate, so further uncoupling of mitochondria may be a good way to increase energy expenditure and represents a good pharmacological target for the treatment of obesity. Uncoupling by 2,4-dinitrophenol has been used in this way in the past with notable success, and some of the effects of thyroid hormone treatment to induce weight loss may also be due to uncoupling. Diet can alter the pattern of phospholipid fatty acyl groups in the mitochondrial membrane, and this may be a route to uncoupling in vivo. Energy expenditure can be increased by stimulating the activity of uncoupling protein 1 (UCP1) in brown adipocytes either directly or through beta 3-adrenoceptor agonists. UCP2 in a number of tissues, UCP3 in skeletal muscle and the adenine nucleotide translocase have also been proposed as possible drug targets. Specific uncoupling of muscle or brown adipocyte mitochondria remains an attractive target for the development of antiobesity drugs.

Uncoupling protein 2 from carp and zebrafish, ectothermic vertebrates.

Uncoupling protein 1 (UCP1) is of demonstrated importance in mammalian thermogenesis, and early hypotheses regarding the functions of the newly discovered UCP homologues, UCP2, UCP3 and others, have focused largely on their potential roles in thermogenesis. Here we report the amino acid sequences of two new UCPs from ectothermic vertebrates. UCPs from two fish species, the zebrafish (Danio rerio) and carp (Cyprinus carpio), were identified in expressed sequence tag databases at the European Molecular Biology Laboratory. cDNAs from a C. carpio 'peritoneal exudate cell' cDNA library and from a D. rerio 'day 0 fin regeneration' cDNA library were obtained and fully sequenced. Each cDNA encodes a 310 amino acid protein with an average 82% sequence identity to mammalian UCP2s. The fish UCP2s are about 70% identical to mammalian UCP3s, and 60% identical to mammalian UCP1s. Carp and zebrafish are ectotherms--they do not raise their body temperatures above ambient by producing excess heat. The presence of UCP2 in these fish thus suggests the protein may have function(s) not related to thermogenesis.

The significance and mechanism of mitochondrial proton conductance.

There is a futile cycle of pump and leak of protons across the mitochondrial inner membrane. The contribution of the proton cycle to standard metabolic rate is significant, particularly in skeletal muscle, and it accounts for 20% or more of the resting respiration of a rat. The mechanism of the proton leak is uncertain: basal proton conductance is not a simple biophysical leak across the unmodified phospholipid bilayer. Equally, the evidence that it is catalysed by homologues of the brown adipose uncoupling protein, UCP1, is weak. The yeast genome contains no clear UCP homologue but yeast mitochondria have normal basal proton conductance. UCP1 catalyses a regulated inducible proton conductance in brown adipose tissue and the possibility remains open that UCP2 and UCP3 have a similar role in other tissues, although this has yet to be demonstrated.

Mitochondrial proton leak and the uncoupling proteins.

An energetically significant leak of protons occurs across the mitochondrial inner membranes of eukaryotic cells. This seemingly wasteful proton leak accounts for at least 20% of the standard metabolic rate of a rat. There is evidence that it makes a similar contribution to standard metabolic rate in a lizard. Proton conductance of the mitochondrial inner membrane can be considered as having two components: a basal component present in all mitochondria, and an augmentative component, which may occur in tissues of mammals and perhaps of some other animals. The uncoupling protein of brown adipose tissue, UCP1, is a clear example of such an augmentative component. The newly discovered UCP1 homologs, UCP2, UCP3, and brain mitochondrial carrier protein 1 (BMCP1) may participate in the augmentative component of proton leak. However, they do not appear to catalyze the basal leak, as this is observed in mitochondria from cells which apparently lack these proteins. Whereas UCP1 plays an important role in thermogenesis, the evidence that UCP2 and UCP3 do likewise remains equivocal.

Venous interruption for pulmonary embolism: the illustrative case of Richard M. Nixon.

This politically prominent patient was seen in consultation on October 26, 1974 because of chronic venous thrombosis and a recent pulmonary embolism. His problems had begun in 1965 when he developed venous thrombosis in the left leg after a length trip by air. His treatment had been sporadic and his compliance with treatment less than satisfactory. Because of detailed phlebography demonstrating (1) no clots in the veins of the right leg, (2) extensive loose lying clot filling the superficial, deep, and external iliac veins on the left, and (3) because of prior difficulties with patient compliance unilateral interruption of the left external iliac vein above the top of the clot was proposed. Despite some postoperative complications, the patient made a full recovery and lived 19 years on warfarin therapy before death from unrelated causes. He suffered no significant edema or other postphlebitic symptoms in the affected leg. The history of the use of venous interruption under these circumstances is reviewed to justify the operation that was performed.

Physical mapping of ribosomal DNA sites in Festuca arundinacea and related species by in situ hybridization.

UNLABELLED: The positions of the 18S-5.8S-26S and 5S rRNA genes have been physically mapped on the chromosomes of diploid, tetraploid, and hexaploid Festuca species by in situ hybridization. The number and position of the rDNA sites in the species were compared. The results confirm some of the earlier phylogenetic studies of these species but suggest that some structural rearrangements have occurred and that sites have been lost during polyploidization. KEYWORDS: Festuca, in situ hybridization, phylogeny, physical mapping, rDNA.

Comparison of ribosomal DNA sites in Lolium species by fluorescence in situ hybridization.

The position of the 18S-5.8S-26S and 5S rRNA genes have been physically mapped on the chromosomes of seven Lolium taxa. 18S-5.8S-26S sites were seen on two pairs of chromosomes in the inbreeding taxa. In the outbreeding taxa six sites were found in the L. multiflorum, seven in L. perenne and nine in L. rigidum var. rigidum. Two 5S sites were found in each of the taxa. In the inbreeders, the 5S sites were found adjacent to the 18S-5.8S-26S sites on chromosome 2. In L. multifiorum and L.perenne the 5S sites were on the short arm of chromosome 3. However, in L. rigidum var. rigidum the 5S rDNA site was found in either of the two positions.

Labelling telomeres of cereals, grasses and clover by primed in situ DNA labelling.

Primed in situ DNA labelling (PRINS) labels the telomeres of Avena, Triticum, Secale, Hordeum, Lolium, Festuca and Trifolium when primers are used that correspond to the repeat unit of Arabidopsis telomeres. There are interstitial sites labelled in a Lolium x Festuca hybrid.

Patient-controlled lumbar epidural fentanyl compared with patient-controlled intravenous fentanyl for post-thoracotomy pain.

Thirty-four patients undergoing thoracotomy were entered into a randomized, double-blind, placebo-controlled study to compare the effects of patient-controlled, lumbar epidural (PCA-E) fentanyl with patient-controlled intravenous (PCA-i.v.) fentanyl with respect to drug requirements, analgesic efficacy and respiratory function. Prior to chest closure patients received fentanyl 2 micrograms.kg-1 by the epidural or i.v. route. In the recovery room further doses of epidural or i.v. fentanyl, 50 micrograms, were administered by the patients who controlled two PCA pumps. Background fentanyl infusion rates were increased by 10 micrograms.hr-1 each time the patient administered a drug bolus and were decreased by 10 micrograms.hr-1 whenever visual analogue scale (VAS) pain scores were less than 2 on a maximum 10 scale. Twenty-nine patients completed the study. Patients in the PCA-E group (n = 14) required less total fentanyl than those in the PCA-i.v. (n = 15) group (1857 +/- 693 micrograms vs 2573 +/- 890 micrograms respectively, P less than 0.05). Fentanyl infusion rates were lower in the PCA-E group at most measurement times. There were no differences between groups in respiratory rates, PaCO2, VAS pain scores or changes in pulmonary function as measured by FVC and FEV1. It is concluded that satisfactory patient-controlled analgesia can be achieved with both epidural and i.v. fentanyl after thoracotomy but that fentanyl requirements are less when given via the epidural route. This supports a direct spinal cord site of action for lumbar epidural fentanyl.

Genetic variation in the incidence of pipped eggs in turkeys selected for low and high semen ejaculate volume.

Genetic variation in embryonic mortality, expressed as embryos that pip their eggshell but do not hatch, was investigated in turkeys selected for low and high semen ejaculate volume (SEV). Through five generations (Generations 10 to 14, inclusive) mean heritability estimates for pipped eggs were .21 and .08 in the low- and high-SEV lines, respectively. Estimates of sire, dam, and within-hatch components of variance suggest greater environmental and maternal effects than genetic influences on the incidence of pipped eggs in turkeys.