Introducing a gatekeeping system for amyloid status assessment in mild cognitive impairment.

BACKGROUND: In patients with mild cognitive impairment (MCI), enhanced cerebral amyloid-ß plaque burden is a high-risk factor to develop dementia with Alzheimer's disease (AD). Not all patients have immediate access to the assessment of amyloid status (A-status) via gold standard methods. It may therefore be of interest to find suitable biomarkers to preselect patients benefitting most from additional workup of the A-status. In this study, we propose a machine learning-based gatekeeping system for the prediction of A-status on the grounds of pre-existing information on APOE-genotype 18F-FDG PET, age, and sex. METHODS: Three hundred and forty-two MCI patients were used to train different machine learning classifiers to predict A-status majority classes among APOE-ε4 non-carriers (APOE4-nc; majority class: amyloid negative (Aß-)) and carriers (APOE4-c; majority class: amyloid positive (Aß +)) from 18F-FDG-PET, age, and sex. Classifiers were tested on two different datasets. Finally, frequencies of progression to dementia were compared between gold standard and predicted A-status. RESULTS: Aß- in APOE4-nc and Aß + in APOE4-c were predicted with a precision of 87% and a recall of 79% and 51%, respectively. Predicted A-status and gold standard A-status were at least equally indicative of risk of progression to dementia. CONCLUSION: We developed an algorithm allowing approximation of A-status in MCI with good reliability using APOE-genotype, 18F-FDG PET, age, and sex information. The algorithm could enable better estimation of individual risk for developing AD based on existing biomarker information, and support efficient selection of patients who would benefit most from further etiological clarification. Further potential utility in clinical routine and clinical trials is discussed.

Conditioning Regimens for Hematopoietic Cell Transplantation in Primary Immunodeficiency.

PURPOSE OF REVIEW: Hematopoietic cell transplantation (HCT) is an established curative treatment for children with primary immunodeficiencies. This article reviews the latest developments in conditioning regimens for primary immunodeficiency (PID). It focuses on data regarding transplant outcomes according to newer reduced toxicity conditioning regimens used in HCT for PID. RECENT FINDINGS: Conventional myeloablative conditioning regimens are associated with significant acute toxicities, transplant-related mortality, and late effects such as infertility. Reduced toxicity conditioning regimens have had significant positive impacts on HCT outcome, and there are now well-established strategies in children with PID. Treosulfan has emerged as a promising preparative agent. Use of a peripheral stem cell source has been shown to be associated with better donor chimerism in patients receiving reduced toxicity conditioning. Minimal conditioning regimens using monoclonal antibodies are in clinical trials with promising results thus far. Reduced toxicity conditioning has emerged as standard of care for PID and has resulted in improved transplant survival for patients with significant comorbidities.

Effects of the sodium-glucose cotransporter 2 (SGLT2) inhibitor velagliflozin, a new drug with therapeutic potential to treat diabetes in cats.

Sodium-glucose cotransporter 2 (SGLT2) inhibitors are used in the treatment of human diabetics. They increase glucose excretion and correct hyperglycemia. We examined the investigational SGLT2 inhibitor velagliflozin in two groups of six neutered adult obese cats (equal gender distribution). Placebo (Pl) or drug (D; 1 mg/kg) was administered for 35 days. Routine blood examinations, fructosamine, beta-hydroxybutyrate (BHB), nonesterified fatty acids (NEFA), glucagon, adiponectin, and leptin were measured before and after treatment, also water intake, and urinary electrolytes, glucose, and volume. Indirect calorimetry, an intravenous glucose tolerance test (IVGTT; 0.8 g/kg) and insulin tolerance test (IVITT) were conducted. All cats tolerated treatment well. Significant changes with D included a decrease in the respiratory exchange ratio, an increase in cholesterol, a small increase in albumin, and a rise in BHB and NEFA. Glucose clearance was unaltered, although less insulin was secreted during the IVGTT (p = .056) suggesting improved insulin sensitivity. IVITT was unchanged. Treatment did not affect glucagon, leptin, or adiponectin. Water intake, urine output, urinary glucose excretion, and the glucose/creatinine ratio but not urinary electrolytes were significantly higher post-D. We conclude that velagliflozin is a promising drug, which increases urinary glucose excretion in cats and could thereby be beneficial for the treatment of hyperglycemia.

The Cause of Acute Respiratory Failure Predicts the Outcome of Noninvasive Ventilation in Immunocompromised Children.

BACKGROUND: Noninvasive ventilation (NIV) may be superior to conventional therapy in immunocompromised children with respiratory failure. METHODS: Mortality, success rate, prognostic factors and side effects of NIV for acute respiratory failure (ARF) were investigated retrospectively in 41 in children with primary immunodeficiency, after stem cell transplantation or chemotherapy for oncologic disease. RESULTS: In 11/41 (27%) children invasive ventilation was avoided and patients were discharged from ICU. In children with NIV failure ICU-mortality was 19/30 (63%). 8/11 (72%) children with NIV success had recurrence of ARF after 27 days. Only 4/11 (36%) children with first episode NIV success and 8/30 (27%) with NIV failure survived to hospital discharge. Lower FiO2, SpO2/FiO2 and blood culture positive bacterial sepsis were predictive for NIV success, while fungal sepsis or culture negative ARF were predictive for NIV failure. We observed catecholamine treatment in 14/41 (34%), pneumothorax in 2/41 (5%), mediastinal emphysema in 3/41 (7%), a life threatening nasopharyngeal hemorrhage and need for resuscitation during intubation in 5/41 (12%) NIV-episodes. CONCLUSIONS: The prognosis of ARF in immunocompromised children remains guarded independent of initial success or failure of NIV due to a high rate of recurrent ARF. Reversible causes like bacterial sepsis had a higher NIV response rate. Relevant side effects of NIV were observed.

Pharmacokinetics and pharmacodynamics of protamine zinc recombinant human insulin in healthy dogs.

Protamine zinc insulins are generally considered to be long acting, with slow absorption from subcutaneous tissue. Protamine zinc recombinant human insulin (PZIR) may be useful to treat diabetic dogs. The purpose of this study was to describe the pharmacokinetics and pharmacodynamics of PZIR in dogs. PZIR was administered subcutaneously to 10 healthy Beagles using an incomplete crossover design, at doses of 0.3 or 0.5 U/kg (each n=5), 0.8 U/kg (n=10), or 0.8 U/kg at three separate sites (n=6). Insulin and glucose concentrations were measured over 24 h. The shapes of insulin and glucose curves were variable among dogs, and the relationship between insulin dose, concentration, and glucose-lowering effect was nonlinear. For single-site 0.8 U/kg, median (range) onset of action was 3.5 h (0.5-10 h), time to glucose nadir was 14 h (5 to >24 h), and duration of action was >24 h (16 to >24 h). Mathematical model predictions of times to 50% and 90% insulin absorption, and fraction of insulin absorbed in 24 h, were not significantly different among protocols. Results confirm the tendency toward a late onset and long duration of action for PZIR in dogs. This insulin may be an alternative treatment option for diabetic dogs.

Pharmacokinetics of pioglitazone in lean and obese cats.

Pioglitazone is a thiazolidinedione insulin sensitizer that has shown efficacy in Type 2 diabetes and nonalcoholic fatty liver disease in humans. It may be useful for treatment of similar conditions in cats. The purpose of this study was to investigate the pharmacokinetics of pioglitazone in lean and obese cats, to provide a foundation for assessment of its effects on insulin sensitivity and lipid metabolism. Pioglitazone was administered intravenously (median 0.2 mg/kg) or orally (3 mg/kg) to 6 healthy lean (3.96 ± 0.56 kg) and 6 obese (6.43 ± 0.48 kg) cats, in a two by two Latin Square design with a 4-week washout period. Blood samples were collected over 24 h, and pioglitazone concentrations were measured via a validated high-performance liquid chromatography assay. Pharmacokinetic parameters were determined using two-compartmental analysis for IV data and noncompartmental analysis for oral data. After oral administration, mean bioavailability was 55%, t(1/2) was 3.5 h, T(max) was 3.6 h, C(max) was 2131 ng/mL, and AUC(0-∞) was 15 556 ng/mL · h. There were no statistically significant differences in pharmacokinetic parameters between lean and obese cats following either oral or intravenous administration. Systemic exposure to pioglitazone in cats after a 3 mg/kg oral dose approximates that observed in humans with therapeutic doses.

Hematopoietic stem cell transplantation from matched unrelated donors in chronic granulomatous disease.

We report on 12 patients with chronic granulomatous disease transplanted with hematopoietic stem cells from matched unrelated (n = 9) or matched sibling donors (n = 3). The most common infectious complication was pulmonary aspergillosis, which nine patients had previously developed. Only 5 of 12 individuals had normal lung function prior to transplantation. At a mean follow-up of 53 months 9 of the 12 patients are alive including 7 of 9 following matched unrelated donor (MUD) transplantation. One patient died from ARDS, another from systemic BK virus infection, the third from complications of chronic graft-versus-host disease. Seven of nine surviving patients have normal lung function now. HSCT from a MUD is an option worth considering when no matched family donor is available. Restricted lung function prior to HSCT does not appear to be a limiting factor for such treatment.

Triiodothyronine differentially regulates key metabolic factors in lean and obese cats.

The effect of a 2-week administration of 75microg triiodothyronine (T3) on substrate oxidation, heat production, non-esterified fatty acids, and leptin was evaluated in eight lean (three females and five males) and eight obese (five females and three males) age-matched adult neutered cats. In addition, using real-time RT-PCR, expression of muscle and adipose tissue uncoupling proteins (UCP2 and UCP3), deiodinase 1 and 2 (D1; D2), and peroxisome proliferator-activated receptor (PPAR) alpha and gamma and peroxisome-proliferator-activator receptor-gamma co-activator 1alpha (PGC1) was examined. Compared to lean cats, obese cats had increased NEFA, leptin, UCP2, and D1mRNA in muscle and UCP3mRNA levels in fat, but lower heat production, and fat PPARs and PGC1. T3 administration increased thermogenesis and NEFA in lean and obese cats, and adipose tissue PPARgamma in lean cats. It also increased muscle D1 in lean and D2 in obese cats. The increase in muscle D2 was interpreted to be reflective of the reduced serum total T4 concentration following T3 suppression of the pituitary. No effect was seen on leptin, or UCP2 and 3. This shows that T3 regulates thermogenesis but not through changes in uncoupling protein expression. It also indicates that PPARs have an important role in the pathogenesis of obesity in cats.

Development of a feline proinsulin immunoradiometric assay and a feline proinsulin enzyme-linked immunosorbent assay (ELISA): a novel application to examine beta cell function in cats.

The prevalence of feline diabetes mellitus has increased several-fold over the last three decades. In humans, progression from obesity to diabetes is marked by changes in the release of proinsulin. A specific proinsulin (FPI) assay has not been available to examine similar changes in cats. The goal of this study was to develop a proinsulin assay for the analysis of beta cell function in cats. Monoclonal antibodies were developed against recombinant FPI and used in a two-site sandwich immunoradiometric assay (IRMA) and enzyme-linked immunosorbent Assay (ELISA). The antibody pair had negligible cross-reactivity with bovine insulin and feline C-peptide. The working range was 11-667pmol/L for the IRMA and 11-1111pmol/L for the ELISA. An intravenous glucose tolerance test was performed in six long-term obese and six lean adult healthy cats and serum glucose, insulin, and FPI concentrations were determined. The proinsulin and insulin secretion pattern in response to glucose was significantly different between lean and obese cats but the pattern was similar within a group. Both groups had similar baseline proinsulin/insulin ratios; however, obese cats showed a significantly higher proinsulin/insulin ratio during the first 15min of the IVGTT and a much lower ratio during the last 30min suggesting a time-delayed adjustment to the increased insulin demand. In conclusion, we report the development and validation of an IRMA and an ELISA for FPI. This novel assay is useful to elucidate FPI secretion and can be used similar to a C-petide assay to evaluate residual beta cell function in cats.

Dyslipidemia in obese cats.

Obesity is an important endocrine disorder in cats and is a risk factor for diabetes similar to humans. The goal of this study was to examine the effect of long-term obesity and different diets (high protein, and high carbohydrate supplemented with saturated fatty acids or n-3 polyunsaturated fatty acids) on plasma lipids in the fasted and fed states in 12 lean (LEAN) and 12 obese (OBESE) cats with ultracentrifugation, and nuclear magnetic resonance spectroscopy. OBESE had higher plasma non-esterified fatty acids and triglycerides, as well as very-low-density-lipoproteins (VLDL) consisting primarily of medium-sized particles. The concentration of low-density-lipoproteins (LDL) was comparable between the groups, although OBESE had mostly very small, whereas LEAN had mostly large particles. The concentration of high-density-lipoproteins (HDL) was lower in OBESE and consisted primarily of small particles. Plasma triglycerides, and triglycerides and cholesterol in all lipoproteins increased postprandially. Different diets had little effect on lipids. Our results show that long-term obese cats develop similar lipoprotein changes to humans, yet, hypertension and atherosclerosis have not been described in obese cats. This suggests that dyslipidemia alone is not sufficient to induce hypertension and atherosclerosis. Other anti-atherogenic factors may be present in the obese, dyslipidemic cat.

Variation of binocular-vertical fusion amplitude with convergence.

PURPOSE: The maximum binocular vertical disparity that can be fused with disparity vergence (vertical-fusion amplitude or VFA), varies with convergence angle. VFA is larger for convergence responses to near than to far viewing distances; however, the clinical norms for changes in VFA with convergence have not been established. VFA at several convergence angles was measured to obtain a quantitative description of the changes in VFA with convergence. METHODS: Fifty-six adults took part in the study. Horizontal and vertical disparity stimuli were presented on a computer monitor by using the red-green anaglyphic technique. Stimulus to convergence was altered either by changing horizontal disparity on the computer monitor (experiment I: nine horizontal disparities: 1.2-22.5 PD [Delta]) or by changing the binocular viewing distance (experiment II: five viewing distances: 25-300 cm). Convergence was held constant during an experimental session, while vertical disparity was incremented in steps of 0.05 Delta after a subjective report of fusion, until the subject reported diplopia. The maximum vertical disparity that could be fused was defined as the VFA. RESULTS: VFA increased linearly over the range of convergence stimuli (y = 0.10x + 1.62) and intersubject variability of VFA increased marginally with the amount of convergence. Linear regression equations with similar slopes and y-intercepts were observed in experiments I and II. CONCLUSIONS: The results of the experiments provide a quantitative description of a linear relationship between VFA and convergence. The linear regression equation could be used in a clinical setting to establish norms and to screen for vertical vergence abnormalities.

Obesity increases free thyroxine proportionally to nonesterified fatty acid concentrations in adult neutered female cats.

The obese cat is a model for the study of the progression toward type 2 diabetes. In this study, the impact of obesity on the hypothalamic-pituitary-thyroid axis was examined in 21 domestic shorthair cats before and after the development of obesity, which significantly increased body mass index (BMI), % body fat (BF), and girth (P<0.0001 for all). Serum total thyroxine (TT(4)), tri-iodothyronine, free T(4) (FT(4)) by direct dialysis, nonesterified fatty acids (NEFA), and leptin were measured, and FT(4) fraction (FFT(4)) was calculated. Serum thyrotropin (TSH) concentrations were measured in nine animals by validating a heterologous canine TSH assay with recombinant feline TSH as a standard. FT(4), FFT(4), NEFAs, and leptin were significantly higher in obese cats. FT(4) had the strongest positive correlation with obesity indices BF, BMI, girth, NEFA, and leptin. Fatty acids oleate and palmitate were shown to inhibit T(4) binding to pooled cat serum in vitro, suggesting the possibility that this mechanism was also relevant in vivo. Serum TT(4) and TSH did not rise significantly. The implications for thyroid hormone (TH) action are not yet clear, but fatty acids have been proposed to inhibit the cellular uptake of TH and/or pituitary TH receptor binding, leading to TH resistance. Increased leptin may also alter sensitivity to negative feedback of TH. In conclusion, feline obesity is associated with a significant increase in FT(4) within the normal range; future investigation into the cellular thyroid status will be necessary to establish cause and effect in this obesity model.

Fatty acid turnover, substrate oxidation, and heat production in lean and obese cats during the euglycemic hyperinsulinemic clamp.

Simultaneous application of the euglycemic hyperinsulinemic clamp (EHC) and indirect calorimetry was used to examine heat production, fat, and glucose metabolism in lean and obese adult neutered male and female cats. The results show that in lean insulin-sensitive cats glucose oxidation predominated during fasting, whereas lipid oxidation became more prominent in obese cats. Insulin infusion during the EHC in lean cats and obese male cats led to a large increase in glucose oxidation, glycogenesis, and lipogenesis. It also led to an increase in glucose oxidation and glycogenesis in obese female cats but it was significantly less compared to lean cats and obese males. This indicates that obese females show greater metabolic inflexibility. In obese cats of either gender, insulin caused greater suppression of non-esterified fatty acids compared to lean cats suggesting that obese cats show greater fatty acid clearance than lean cats. The heat production per metabolic size was lower in obese cats than lean cats. This would perpetuate obesity unless food intake is decreased. The higher glucose oxidation rate in obese neutered male cats suggests that they are able to replete their glycogen and lipid stores at a faster rate than females in response to insulin and it implies that they gain weight more rapidly. Further studies are needed to investigate if the different response to insulin of male cats is involved in their higher risk to develop diabetes.

Early invasive versus conservative strategies for unstable angina & non-ST-elevation myocardial infarction in the stent era.

BACKGROUND: In patients with unstable angina and non-ST-elevation myocardial infarction (UA/NSTEMI) two strategies are possible: a routine invasive strategy where all patients undergo coronary angiography shortly after admission and, if indicated, coronary revascularization; or a conservative strategy where medical therapy alone is used initially with selection of patients for angiography based on clinical symptoms or investigational evidence of persistent myocardial ischemia. OBJECTIVES: To determine the benefits of an invasive compared to a conservative strategy for treating UA/NSTEMI in the stent era. SEARCH STRATEGY: The Cochrane Central Register of Controlled Trials (Issue 3 2005), MEDLINE and EMBASE were searched from 1996 to September 2005 with no language restrictions. SELECTION CRITERIA: Included studies were prospective trials comparing invasive with conservative strategies in UA/NSTEMI. DATA COLLECTION AND ANALYSIS: We identified 5 studies (7818 participants). Using intention-to-treat analysis with random effects models, summary estimates of relative risk (95% confidence interval [CI]) were determined for primary end-points of all-cause death, fatal and non-fatal myocardial infarction; all-cause death or non-fatal myocardial infarction; and refractory angina. Further analysis of included studies was undertaken based on whether glycoprotein IIb/IIIa receptor antagonists were used routinely. Heterogeneity was assessed using chi-square and variance (I(2)) methods. MAIN RESULTS: In the all-study analysis, mortality during initial hospitalization showed a trend to hazard with an invasive strategy; relative risk 1.59 (95% CI 0.96 to 2.64). Mortality and myocardial infarction assessed at 2-5 years in two trials were significantly decreased by an invasive strategy with relative risk of 0.75 (95% CI 0.62 to 0.92) and 0.75 (95% CI 0.61 to 0.91) respectively. The composite end-point of death or non-fatal myocardial infarction was significantly decreased by an invasive strategy at several time points after initial hospitalization. The incidence of early (<4 months) and intermediate (6-12 months) refractory angina were both significantly decreased by an invasive strategy; relative risk 0.47 (95% CI 0.32 to 0.68) and 0.67 (95% CI 0.55 to 0.83) respectively, as were early and intermediate rehospitalization rates with relative risk 0.60 (95% CI 0.41 to 0.88) and 0.67 (95% CI 0.61 to 0.74) respectively. The invasive strategy was associated with a two-fold increase in the relative risk of peri-procedural myocardial infarction (as variably defined) and a 1.7-fold increase in the relative risk of bleeding. AUTHORS' CONCLUSIONS: An early invasive strategy is preferable to a conservative strategy in the treatment of UA/NSTEMI.

Cloning, expression and purification of feline proinsulin.

Feline proinsulin was cloned and expressed using a bacterial expression system. It was then purified from inclusion bodies using size exclusion chromatography and further processed including reduction of the protein. Following refolding, proinsulin was purified by reversed-phase high-performance liquid chromatography (RP-HPLC). RP-HPLC and mass spectrometric analysis indicated that the proinsulin contained the correct disulfide bridging pattern. This proinsulin can be used for therapeutic and diagnostic purposes.

Cloning and sequencing of feline thyrotropin (fTSH): heterodimeric and yoked constructs.

The genes encoding the mature common glycoprotein alpha (CGA) and hormone-specific beta subunits of feline thyroid stimulating hormone (fTSH) were cloned and sequenced. The feline CGA gene was cloned from RNA extracted from the feline pituitary gland by the reverse transcription polymerase chain reaction (RT-PCR). The gene fragment that encodes mature TSHbeta was cloned from feline genomic DNA after direct polymerase chain reaction (PCR). In both cases, primers were based on the consensus sequences from TSH in other species. The resulting 510 bp PCR product for the CGA-subunit included the full coding sequence for the 96 amino acid mature subunit preceded by a 24 amino acid signal peptide. The 850 bp sequence of fTSHbeta genomic DNA consisted of two coding exons, an intron of 418 bp, and a 60 bp signal sequence. The octapeptide immunoaffinity tag FLAG was added to 3' end of the alpha gene to facilitate detection and purification. Both genes were cloned independently downstream from the EF1alpha promoter of the PEAK transfer vector to facilitate co-expression studies in PEAK cells (modified human embryonic kidney (HEK) cells). A single-chain analogue of fTSH termed yoked fTSH (yfTSH) was developed by fusing the nucleotides encoding the C-terminus of the beta-subunit fused to the N-terminus of the alpha-subunit with DNA encoding the C-terminal peptide (CTP) of human chorionic gonadotropin beta-subunit as a linker peptide. The resulting single-chain analogue encoded from N-terminus to C-terminus: beta-CTP-alpha-FLAG. The resulting DNA sequence was cloned, sequenced, ligated and recloned into expression vector PEAK. This report constitutes the first cloning and sequencing of the genes encoding the subunits of feline thyrotropin.

Assessment and mathematical modeling of glucose turnover and insulin sensitivity in lean and obese cats.

Insulin sensitivity (SI) of glucose disposal can be quantified with the euglycemic hyperinsulinemic clamp (EHC) with tracer glucose infusion. True steady state is, however, difficult to achieve, and non-steady state analysis of EHC data is preferred. This analysis requires information on glucose kinetics that can be obtained from bolus injection of cold and tracer glucose. The aim of this study was to assess glucose kinetics in cats. Mathematical modeling and non-steady state analysis was applied to assess effects of obesity on glucose turnover, glycolysis/glycogen synthesis, SI, and inhibition of endogenous glucose production (EGP) in lean cats (L) and obese cats (O). D-[3-(3)H]-glucose kinetics and 3H-H2O production were analyzed in 4 L and 4 O with three-compartment modeling. Frequently sampled insulin-modified intravenous glucose tolerance tests (FSIGT) with minimal model analysis were performed in 5L and 3 O to assess glucose kinetics and SI. EHC was performed in 10 L and 10 O with primed-constant infusion of 3H-glucose. Data were analyzed with a modified minimal model segregating suppression of EGP by insulin using a non-linear mixed-effects population approach. FSIGT provided estimates of SI, glucose effectiveness SG, and distribution volume. EHC provided estimates of SI, SG, glycolysis, and suprabasal insulin concentration for 50% EGP inhibition. Obesity appears to affect glucose distribution but not utilization at basal insulin, and reduces SI estimated by FSIGT and EHC. Differences in SI between FSIGT and EHC depend on different descriptions of EGP inhibition by insulin. Finally, glucose disposal at basal insulin appears to occur entirely through glycolysis, whereas significant amounts of glucose are sequestrated from oxidation during EHC.

Expression and purification of feline thyrotropin (fTSH): immunological detection and bioactivity of heterodimeric and yoked glycoproteins.

The goal of this study was to express and purify recombinant feline TSH as a possible immunoassay standard or pharmaceutical agent. Previously cloned feline common glycoprotein alpha (CGA) and beta subunits were ligated into the mammalian expression vector pEAK10. The feline CGA-FLAG and beta subunits were cloned separately into the pEAK10 expression vector, and transiently co-transfected into PEAK cells. Similarly, previously cloned and sequenced yoked (single chain) fTSH (yfTSH) and the CGA-FLAG sequences were ligated into the same vector, and stable cell lines selected by puromycin resistance. Expression levels of at least 1 microg/ml were achieved for both heterodimeric and yoked fTSH forms. The glycoproteins were purified in one step using anti-FLAG immunoaffinity column chromatography to high purity. The molecular weights of feline CGA-FLAG subunit, beta subunit and yfTSH were 20.4, 17, and 45 kDa, respectively. Both heterodimeric and yoked glycoproteins were recognized with approximately 40% detection by both a commercial canine TSH immunoassay and an in-house canine TSH ELISA. The yoked glycoprotein exhibited parallelism with the heterodimeric form in the in-house ELISA, supporting their possible use as immunoassay standards. In bioactivity assays, the heterodimeric and yoked forms of fTSH were 12.5 and 3.4% as potent as pituitary source bovine TSH at displacing (125)I-bTSH and 45 and 24% as potent in stimulating adenylate cyclase activity in human TSH receptor-expressing JP09 cells. However, in addition to reduced receptor binding affinity, the recombinant glycohormones produced a reduced maximal effect at maximal concentration (E(max)) suggesting the possibility of the recombinant glycohormone constructs acting as partial agonists at the human TSH receptor.

Activity and tissue-specific expression of lipases and tumor-necrosis factor alpha in lean and obese cats.

Post-heparin plasma activity of lipoprotein lipase (LPL) and hepatic lipase (HL), and fat and muscle activity of LPL were measured in neutered lean and obese cats. Lipoprotein lipase, hormone-sensitive lipase (HSL), and tumor necrosis factor a (TNF) mRNA were measured in muscle and fat tissue with real-time PCR using primers for feline LPL, HSL, and TNF. Lipoprotein lipase plasma and fat activity and fat mRNA levels were significantly lower (50, 80, and 50%, respectively) in obese cats than lean cats, whereas the muscle/fat ratio of LPL was significantly higher in obese compared to lean cats. The activity of HL was not different between the groups. Hormone-sensitive lipase mRNA levels were significantly higher in obese than lean cats. The level of fat TNF also was significantly higher in obese cats than in lean cats, whereas the level in muscle was not different. The lower LPL activity and mRNA expression in fat and the higher LPL and HSL mRNA expression in muscle in obese cats compared to lean cats expectedly favor a redistribution of fatty acids from fat to muscle tissue where they can be deposited or used for energy in times of need. Tumor necrosis factor alpha may regulate this repartitioning process through suppression of adipocyte LPL.

Glucose inhibits the high-affinity (Ca2+ + Mg2+)-ATPase in the plasma membrane of a glucose-responsive insulinoma.

(Ca2+ + Mg2+)-ATPase enzyme activity of a purified plasma membrane preparation from a glucose responsive rat insulinoma, was characterized as Ca2(+)-dependent dephosphorylation of [gamma-32P]ATP. A high-affinity enzyme with a Km(ATP) ranging from 20 to 30 microM and a Km(Ca2+) of 1 microM was identified. Glucose inhibited this high-affinity enzyme in a dose-dependent manner, with no significant inhibition at a concentration between 0 and 5 mM, 50% inhibition at 13.3 mM and 94.5% inhibition at 30 mM. The inhibitory effect of glucose was immediate and rapidly reversible. The effect was stereospecific for the alpha-anomer. These findings support the concept that glucose acts directly at the beta-cell plasma membrane and is involved in the maintenance of elevated intracellular free calcium concentrations associated with insulin release by directly or indirectly inhibiting energy-dependent calcium efflux. Glyceraldehyde (20 mM) increased enzyme activity 3-fold, while other metabolic fuels had no effect. This suggests that inhibition of the enzyme is not an obligatory requirement for insulin release. Calmodulin stimulated the enzyme activity in calmodulin-depleted but not in undepleted membranes. Trifluoperazine (30-100 microM) inhibited (Ca2+ + Mg2+)-ATPase in a dose-dependent manner (14-61% activity) and the activity was also inhibited by vanadate (0.1-1.0 mM) and NaCl (150 mM).