Re-evaluation of the low-dose dexamethasone suppression test in dogs.

OBJECTIVES: This study aims to re-evaluate the low-dose dexamethasone suppression test 8-hour cortisol cut-point for the diagnosis of hypercortisolism in dogs using a solid-phase, competitive chemiluminescent enzyme immunoassay. MATERIALS AND METHODS: Twenty-seven client-owned dogs with naturally occurring hypercortisolism and 30 healthy control dogs were prospectively recruited. Performance of the low-dose dexamethasone suppression test was assessed using sensitivity, specificity and a receiver operating characteristic curve compared to a clinical diagnosis of hypercortisolism including response to treatment. RESULTS: Twenty-four dogs were diagnosed with pituitary-dependent hypercortisolism and three with adrenal-dependent hypercortisolism. In 30 healthy control dogs, 8-hour post-dexamethasone cortisol concentrations ranged from 5.5 to 39 nmol/L. A receiver operating characteristic curve curve constructed from the 8-hour post-dexamethasone cortisol concentrations of hypercortisolism and control dogs demonstrated that the most discriminatory cut-point was more than 39 nmol/L with sensitivity of 85.2% (95% confidence interval, 67.5% to 94.1%) and specificity of 100% (95% confidence interval, 88.7% to 100.0%) and an area under the curve of 0.963. CLINICAL SIGNIFICANCE: The optimal cut-point of more than 36 nmol/L proposed by this study is similar to the currently accepted 8-hour cortisol concentration cut-point for diagnosing hypercortisolism when using a solid-phase, competitive chemiluminescent enzyme immunoassay.

Changes in characteristics of inmates with latent tuberculosis infection.

OBJECTIVES: Health and social characteristics place prisoners at high risk for progression from latent tuberculosis infection (LTBI) to tuberculosis (TB), but completion of LTBI therapy is low with many patients lost to follow-up after release. Despite decreases in active TB, demographic characteristics of active cases have remained relatively unchanged. This study investigated whether characteristics have changed in inmates diagnosed with LTBI in San Francisco, CA, USA. STUDY DESIGN: Cross-sectional. METHODS: Data from baseline interviews of randomized trials conducted in 1998-1999 and 2004-2007 were compared. RESULTS: In both time periods, most subjects with LTBI (>60%) were Latinos, while the proportion in both the jail and San Francisco remained at 15-20%. Overall, the prisoners interviewed in 2004-2007 were less likely to have been on medication for LTBI previously, and expressed more likelihood of finishing their medication compared with those interviewed in 1998-1999. In 2004-2007, the foreign-born subjects were more likely to prefer English to Spanish, to have been in stable housing and to have been employed before jail compared with 1998-1999, while no such changes were seen between the two time periods for US-born subjects. CONCLUSIONS: The pool of TB-infected individuals coming from a jail is not static, and understanding the changes over time is of importance for targeted programmes. Given the high infection rate and the predominance of foreign-born individuals who may have received bacillus Calmette-Guérin vaccination, screening with interferon-gamma release assay may be beneficial to identify those with true infection.

Parental dietary effect on embryological development response to toxicants with the sea urchin Arbacia punctulata.

The role of echinoid parental nutrition in early-life stage toxicity is not well understood. Arbacia punctulata were fed either a fresh diet consisting of organic lettuce and carrots or a dry feed. Embryos from parents fed the dry feed exhibited lower sensitivity to copper, whereas the opposite occurred with 1,3,5-trinitrobenzene and sodium dodecyl sulfate (SDS). EC(50) values for the dry and fresh feed treatments, respectively, were 41.0 and 29.9 microg/L for copper, 0.5 and 1.8 mg/L for 1,3,5-trinitrobenzene, and 3.5 and 5.6 mg/L for SDS. The data suggests that nutritional standardization for sea urchins in ecotoxicological laboratories needs to be addressed and further investigated.

A cell-free assay system for beta-catenin signaling that recapitulates direct inductive events in the early xenopus laevis embryo.

In vertebrate embryos, signaling via the beta-catenin protein is known to play an essential role in the induction of the dorsal axis. In its signaling capacity, beta-catenin acts directly to affect target gene transcription, in concert with transcription factors of the TCF/LEF family. We have developed a cell-free in vitro assay for beta-catenin signaling activity that utilizes transcriptionally active nuclei and cytoplasm from cleavage-blocked Xenopus laevis embryos. Under these assay conditions, we demonstrate that either addition of beta-catenin protein or upstream activation of the beta-catenin signaling pathway can induce the expression of developmentally relevant target genes. Addition of exogenous beta-catenin protein induced expression of Siamois, XTwin, Xnr3, and Cerberus mRNAs in a protein synthesis independent manner, whereas a panel of other Spemann organizer-specific genes did not respond to beta-catenin. Lithium induction of the beta-catenin signaling pathway, which is thought to cause beta-catenin accumulation by inhibiting its proteasome-dependent degradation, caused increased expression of Siamois in a protein synthesis independent fashion. This result suggests that beta-catenin derived from a preexisting pool can be activated to signal, and that accumulation of this activated form does not require ongoing synthesis. Furthermore, activation of the signaling pathway with lithium did not detectably alter cytoplasmic beta-catenin levels and was insensitive to inhibition of the proteasome- dependent degradation pathway. Taken together, these results suggest that activation of beta-catenin signaling by lithium in this system may occur through a distinct activation mechanism that does not require modulation of levels through regulation of proteasomal degradation.

Volatilization of high molecular weight DNA by pulsed laser ablation of frozen aqueous solutions.

DNA has been volatilized by pulsed laser ablation of a thin film of a frozen aqueous DNA solution. The target film was irradiated in vacuum by a pulsed laser at power densities sufficient to ablate the ice matrix. The expanding ablated water vapor entrained embedded DNA molecules and expelled them into the gas phase. Ejection of DNA molecules as large as 410 kilodaltons was verified by collection of the ablation products and subsequent mass analysis by polyacrylamide gel electrophoresis with autoradiographic detection.

Field safety and efficacy of protamine zinc recombinant human insulin for treatment of diabetes mellitus in cats.

BACKGROUND: This study describes the efficacy of a new protamine zinc recombinant human insulin (PZIR) preparation for treating diabetic cats. OBJECTIVE: To evaluate effects of PZIR on control of glycemia in cats with newly diagnosed or poorly controlled diabetes mellitus. ANIMALS: One hundred and thirty-three diabetic cats 120 newly diagnosed and 13 previously treated. METHODS: Prospective, uncontrolled clinical trial. Cats were treated with PZIR twice daily for 45 days. Control of glycemia was assessed on days 7, 14, 30, and 45 by evaluation of change in water consumption, frequency of urination, appetite, and body weight, serum fructosamine concentration, and blood glucose concentrations determined 1, 3, 5, 7, and 9 hours after administration of PZIR. Adjustments in dosage of PZIR were made as needed to control glycemia. RESULTS: PZIR administration resulted in a significant decrease in 9-hour mean blood glucose (199+/-114 versus 417+/-83 mg/dL, X+/-SD, P<.001) and serum fructosamine (375+/-117 versus 505+/-96 micromol/L, P<.001) concentration and a significant increase in mean body weight (5.9+/-1.4 versus 5.4+/-1.5 kg, P=.017) in 133 diabetic cats at day 45 compared with day 0, respectively. By day 45, polyuria and polydipsia had improved in 79% (105 of 133), 89% (118 of 133) had a good body condition, and 9-hour mean blood glucose concentration, serum fructosamine concentration, or both had improved in 84% (112 of 133) of the cats compared with day 0. Hypoglycemia (<80 mg/dL) was identified in 151 of 678, 9-hour serial blood glucose determinations and in 85 of 133 diabetic cats. Hypoglycemia causing clinical signs was confirmed in 2 diabetic cats. CONCLUSIONS AND CLINICAL RELEVANCE: PZIR is effective for controlling glycemia in diabetic cats and can be used as an initial treatment or as an alternative treatment in diabetic cats that do not respond to treatment with other insulin preparations.

Predictive factors and the effect of phenoxybenzamine on outcome in dogs undergoing adrenalectomy for pheochromocytoma.

BACKGROUND: Some studies in dogs undergoing adrenalectomy for pheochromocytoma suggest that anesthetic complications and perioperative mortality are common. In humans, surgical outcome has improved with the use of phenoxybenzamine (PBZ) before adrenalectomy. HYPOTHESIS: Dogs treated with PBZ before adrenalectomy have increased survival compared with untreated dogs. ANIMALS: Forty-eight dogs that underwent adrenalectomy for pheochromocytoma. METHODS: A retrospective medical record review for dogs that underwent adrenalectomy for pheochromocytoma at a veterinary medical teaching hospital over the period from January 1986 through December 2005. RESULTS: Twenty-three of 48 dogs were pretreated with PBZ (median dosage: 0.6 mg/kg PO q12h) for a median duration of 20 days before adrenalectomy. Duration of anesthesia and surgery, percentage of dogs with pheochromocytoma involving the right versus left adrenal gland, size of tumor, and presence of vascular invasion were similar for PBZ-treated and untreated dogs. Thirty-three (69%) of 48 dogs survived adrenalectomy in the perioperative period. PBZ-treated dogs had a significantly (P = .014) decreased mortality rate compared with untreated dogs (13 versus 48%, respectively). Additional significant prognostic factors for improved survival included younger age (P = .028), lack of intraoperative arrhythmias (P = .0075), and decreased surgical time (P = .0089). CONCLUSIONS AND CLINICAL IMPORTANCE: Results from this retrospective study support treatment with PBZ before surgical removal of pheochromocytoma in dogs.

Frequency and risk factors for urinary tract infection in cats with diabetes mellitus.

BACKGROUND: Identification and control of infections are important in the management of diabetic cats. Urinary tract infections have not been well characterized in diabetic cats. This retrospective study was performed to review and characterize urinary tract infections in diabetic cats. HYPOTHESIS: Urinary tract infections are common in diabetic cats. ANIMALS: A review was made of the medical records of 141 diabetic cats that had had urine obtained for culture by antepubic cystocentesis and that had not been treated with antibiotics, undergone urinary tract catheterization or urinary tract surgery within 2 weeks of urine collection or had urethral obstruction at the time of urine collection. METHODS: A review of medical records. RESULTS: Urinary tract infection was identified in 18 of 141 diabetic cats. Escherichia coli was the most common isolate (67%). Female cats were at increased risk (prevalence odds ratios [POR], 3.7; 95% confidence interval [CI], 1.3 to 10.2; P = .013). Clinical signs of lower urinary tract disease and findings on urine sediment examination were good predictors of positive urine cultures. CONCLUSIONS AND CLINICAL IMPORTANCE: Urinary tract infections are common in diabetic cats regardless of status of diabetic control, suggesting routine monitoring with urine sediment exams or urine culture is warranted.

Measurements of growth hormone and insulin-like growth factor 1 in cats with diabetes mellitus.

Serum concentrations of insulin-like growth factor 1 (IGF-1) and growth hormone were measured in 25 cats with untreated diabetes mellitus (11 of which were used for follow-up measurements, one to three, four to eight, nine to 12 and 13 to 16 weeks after their treatment with insulin began), 14 diabetic cats that had previously been treated with insulin, and seven diabetic cats that also had hypersomatotropism, two of which had not previously been treated with insulin; 18 healthy cats were used as controls. In the untreated diabetic cats the concentration of IGF-1 ranged from 13.0 to 433.0 ng/ml (median 170.5 ng/ml), which was significantly lower than the concentrations in the control cats (196.0 to 791.0 ng/ml, median 452.0 ng/ml). Their IGF-1 concentrations increased significantly when they were treated with insulin and after four to eight weeks were not different from those in the control cats. In the diabetic cats that had previously been treated with insulin the IGF-1 concentrations were 33.0 to 476.0 ng/ml (median 316.0 ng/ml), which was significantly lower than the concentrations in the control cats, but significantly higher than in the untreated diabetic cats. The IGF-1 concentrations in the two previously untreated diabetic cats with hypersomatotropism were low and low-normal but increased markedly after treatment with insulin. In the five previously treated cats with hypersomatotropism the concentration of IGF-1 was above the normal range. The concentrations of growth hormone in the treated and untreated diabetic cats without hypersomatotropisms were not significantly different and there was an overlap in its concentrations in the diabetic cats with and without hypersomatotropism.

Recognition of transition metal ions by peptides. Identification of specific metal-binding peptides in proteolytic digest maps by UV laser desorption time-of-flight mass spectrometry.

Metal-binding peptides in proteolytic digest maps have been identified by matrix-assisted UV laser desorption time-of-flight mass spectrometry (LDTOF-MS). The plasma and milk metal transport protein chosen to demonstrate this process, histidine-rich glycoprotein (HRG), was purified and then digested with trypsin; the cleavage products were analyzed by LDTOF-MS with dihydroxybenzoic acid as the matrix. The selective interaction of specific peptides with one or more Cu atoms was observed when Cu(II) ions were added to the digest mixture. At least one specific metal-binding peptide was identified by computerized sequence analysis using the molecular mass data and available cDNA sequence. These results demonstrate the first direct observation by mass spectrometry of differential peptide-metal ion interactions in protein digest maps. The ability to evaluate peptide-metal ion interactions, including stoichiometry, with less than 1 pmol of sample improves significantly our ability to identify metal binding domains in metal-binding proteins.

Comparative aspects of Cushing's syndrome in dogs and cats.

Naturally occurring hyperadrenocorticism (Cushing's syndrome) is extremely common in dogs, with an incidence far greater than that in humans. The incidence of the syndrome in cats, much less frequently diagnosed than in dogs, probably is similar to that in humans. Many features of canine hyperadrenocorticism are strikingly similar to those in humans; however, several alterations in dogs are unique and not observed in other species. Clinical features and hospital testing abnormalities are reviewed in this article.

Mass spectrometric methods for evaluating point mutations.

Two methods for internally calibrating spectra resulting from the matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry analysis of partially digested proteins are described. Partial digestion of proteins results in a large number of ion signals present in the MALDI-TOF mass spectrum, which in turn represent a significant over-sampling of each amino acid present in the analyte. This over-sampling allows ion signals of undisputed origin to be used as internal calibrants for the evaluation of fragments suspected to contain point mutations. Correlated with the correct amino acid sequence, the mass values of all ion signals (calibrants and analytes) are observed to fall into a single low-error data set. Conversely, empirically derived data applied to an incorrect sequence split the data into subsets of different errors. The methods take advantage of the self-consistent nature of data generated during the enzymatic mass mapping of proteins using MALDI-TOF, and they aid in the rapid, sensitive and accurate evaluation of point mutations present in proteins.

Analysis of human urine protein biomarkers via biomolecular interaction analysis mass spectrometry.

Biomolecular interaction analysis mass spectrometry (BIA/MS) is a two-dimensional chip-based analytical technique geared toward quantitative and qualitative analysis of small volumes of biological samples. Interactions between surface-immobilized ligands and solute-borne analytes are quantitatively viewed in real time through surface plasmon resonance sensing, followed by qualitative matrix-assisted laser desorption/ionization time-of-flight MS analysis of the analyte(s) affinity-retained on the sensor surface. In this work, BIA/MS was used in the detection of a number of protein biomarkers from human urine. Small volumes of human urine were analyzed for cystatin C, beta(2)-microglobulin, urinary protein 1, and retinol-binding protein (RBP). Multiaffinity sensor surfaces were created to simultaneously and rapidly detect all four proteins in a single BIA/MS analysis on a two-flow cell sensor chip configuration. Furthermore, RBP was analyzed separately from both urine and plasma samples. Results indicate that BIA/MS can be used successfully in rapid screening of a number of urinary proteins indicated as putative biological markers for renal dysfunction.

Electrospray ionization and matrix-assisted laser desorption ionization mass spectrometry. Emerging technologies in biomedical sciences.

Tremendous progress in biomedical sciences has been made possible in part by recent advances in bioanalytical methods, in particular biological mass spectrometry. Since the introduction of electrospray ionization mass spectrometry (ESI-MS) in 1984 and matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) in 1988, the field of bioanalytical mass spectrometry has seen rapid growth. In concert with separation techniques such as capillary electrophoresis and high performance liquid chromatography, mass spectrometry allows characterization of a large array of small organic molecules, peptides, proteins, oligonucleotides, and RNA fragments. Thus, substantially more expedient and definitive determination of molecular weight is now possible by mass spectrometric analysis. In this commentary, general descriptions of ESI- and MALDI-MS are presented. Furthermore, several recent developments and applications in addressing difficult biological problems are discussed.

Detection and characterization of DNA adducts at the femtomole level by desorption ionization mass spectrometry.

Current methodologies for the detection and isolation of carcinogen-DNA adducts have advanced beyond the capabilities of the methods used to elucidate their structures. This difficulty seriously limits the potential use of DNA-carcinogen adducts in human dosimetry. We have investigated two general strategies for the analysis of model arylamine-nucleoside adducts using desorption ionization mass spectrometry (MS). Using fast atom bombardment MS-MS with constant neutral loss scans, we can identify the protonated molecule of derivatized adducts in samples as small as 1 pmole, and then apply daughter ion MS-MS scans to obtain structure-specific fragmentation. Using this strategy we have differentiated adducts having the same carcinogen and different bases [e.g., N-(deoxyadenosin-8-yl)-4-aminobiphenyl and N-(deoxyguanosin-8-yl)-4- aminobiphenyl] or the same base and different carcinogens [e.g., N-(deoxyguanosin-8-yl)-4- aminobiphenyl and N-(deoxyguanosin-8-yl)-2-aminofluorene]. In the second approach we used laser desorption time-of-flight MS to obtain spectra from adduct samples as small as 20 fmole. These data indicate that MS can be used for the analysis of very low (picomole-femtomole) levels of nucleoside adducts, including isomers, and that desorption ionization MS and MS-MS have significant potential for applications in human dosimetry.

Beta-catenin directly induces expression of the Siamois gene, and can initiate signaling indirectly via a membrane-tethered form.

Beta-catenin is shown to directly induce the expression of siamois (a homeobox-containing gene involved in axial patterning) in a cell-autonomous, protein synthesis independent manner. Siamois can thus be considered a direct target of beta-catenin signaling in Xenopus. Expression of a portion of the armadillo repeat region of beta-catenin via a membrane-tethered fusion protein is shown to give similar levels of siamois induction and axis duplication as a free, untethered form. Reduction of endogenous free beta-catenin levels by overexpression of C-cadherin leads to complete inhibition of signaling by the membrane-tethered repeat region. Since the membrane-tethered repeat region is unlikely to be bound up by C-cadherin, these results show that the membrane-tethered beta-catenin relies on endogenous beta-catenin for signaling. We propose that the membrane-tethered construct acts by titrating a cytoplasmic inhibitor of beta-catenin signaling.

Analysis of native proteins from biological fluids by biomolecular interaction analysis mass spectrometry (BIA/MS): exploring the limit of detection, identification of non-specific binding and detection of multi-protein complexes.

Biomolecular interaction analysis mass spectrometry (BIA/MS) is a two-dimensional analytical technique that quantitatively and qualitatively detects analytes of interests. In the first dimension, surface plasmon resonance (SPR) is utilized for detection of biomolecules in their native environment. Because SPR detection is non-destructive, analyte(s) retained on the SPR-active sensor surface can be analyzed in a second dimension using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. The qualitative nature of the MALDI-TOF MS analysis complements the quantitative character of SPR sensing and overcomes the shortcomings of the SPR detection stemming from the inability to differentiate and characterize multi-protein complexes and non-specific binding. In this work, the benefit of performing MS analysis following SPR sensing is established. Retrieval and detection of four markers present in biological fluids (cystatin C, beta-2-microglobulin, urinary protein 1 and retinol binding protein) was explored to demonstrate the effectiveness of BIA/MS in simultaneous detection of clinically related biomarkers and delineation of non-specific binding. Furthermore, the BIA/MS limit of detection at very low SPR responses was investigated. Finally, detection of in-vivo assembled protein complexes was achieved for the first time using BIA/MS.

Multitoxin biosensor-mass spectrometry analysis: a new approach for rapid, real-time, sensitive analysis of staphylococcal toxins in food.

Biomolecular interaction analysis mass spectrometry (BIA-MS) was applied to detection of bacterial toxins in food samples. This two-step approach utilizes surface plasmon resonance (SPR) to detect the binding of the toxin(s) to antibodies immobilized on a surface of a sensor chip. SPR detection is then followed by identification of the bound toxin(s) by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Staphylococcal enterotoxin B (SEB) was readily detected in milk and mushroom samples at levels of 1 ng/ml. In addition, non-specific binding of food components to the immobilized antibody and to the sensor chip surface was detected. To evaluate the applicability of BIA-MS in the analysis of materials containing multiple toxic components, sample containing both SEB and toxic-shock syndrome toxin-1 was analyzed. Both toxins were successfully and simultaneously detected through the utilization of multiaffinity sensor chip surfaces.

Models for the pharmacokinetics and pharmacodynamics of insulin in alloxan-induced diabetic dogs.

A combined pharmacokinetic/pharmacodynamic model was proposed to describe the pharmacokinetics of intravenously administered regular insulin (0.55 units/kg) in alloxan-induced diabetic dogs. Serum insulin concentrations were described by either a one- or two-compartment open model, in which a hypothetical effect compartment was linked to the central pharmacokinetic compartment, or in which the effect compartment was linked to the peripheral compartment. Response, as measured by percent change in glucose concentration from adjusted basal plasma concentrations, was modeled using the sigmoidal Emax effect model, a linear effects model, a log-linear effects model, and a gamma-linear effects model, using the insulin pharmacokinetic parameters to describe the amount in the hypothetical effect compartment. The results indicated that insulin pharmacokinetics are usually described by a two-compartment open model. Response to insulin was predicted more accurately in half of the dogs using the gamma-linear effects model in which the effect compartment was linked to the central compartment. In the other half of the dogs the best model was the sigmoidal Emax model in which the effect compartment was linked to the central pharmacokinetic compartment. The parameters in the latter model were correlated with each other and the confidence limits of the parameter estimates were larger than the parameters of the gamma-linear effects model. These models should be further investigated, but may offer an alternative method for distinguishing rapid insulin metabolism from insulin resistance.

Occipito-cervico-thoracic spine fusion in a patient with occipito-cervical dislocation and survival.

A 16-year-old man with occipito-cervical dislocation and survival was treated at Rancho Los Amigos Medical Center. The patient had ligamentous instability between the occiput and the cervical spine. His neurologic level was complete at the C1 level, and he was dependent on a mechanical respirator. Sternocleidomastoid and other neck musculature were not functional. A fusion of the occiput to cervical spine and cervical spine to thoracic spine was performed to obviate the need for external neck support. The patient went on to fuse and is now independently mobile with a tongue-switch driven wheelchair with a respirator trailer 2 years after injury. The patient has no need for external support as a result of his fusion.