Baseline Quality-of-Life of Caregivers of Patients With Heart Failure Prior to Advanced Therapies: Findings From the Sustaining Quality of Life of the Aged: Transplant or Mechanical Support (SUSTAIN-IT) Study.

BACKGROUND: We compared health-related quality of life (HRQOL), depressive symptoms, anxiety, and burden in caregivers of older patients with heart failure based on the intended therapy goal of the patient: awaiting heart transplantation (HT) with or without mechanical circulatory support (MCS) or prior to long-term MCS; and we identified factors associated with HRQOL. METHODS: Caregivers (n = 281) recruited from 13 HT and MCS programs in the United States completed measures of HRQOL (EQ-5D-3L), depressive symptoms (PHQ-8), anxiety (STAI-state), and burden (Oberst Caregiving Burden Scale). Analyses included ANOVA, Kruskal-Wallis tests, χ2 tests, and linear regression. RESULTS: The majority of caregivers were female, white spouses with ≤ 2 comorbidities, median [Q1,Q3] age = 62 [57.8, 67.0] years. Caregivers (HT with MCS = 87, HT without MCS = 98, long-term MCS = 96) reported similarly high baseline HRQOL (EQ-5D-3L visual analog scale median score = 90; P = 0.67 for all groups) and low levels of depressive symptoms. STAI-state median scores were higher in the long-term MCS group vs the HT groups with and without MCS, (38 vs 32 vs 31; P < 0.001), respectively. Burden (task: time spent/difficulty) differed significantly among groups. Caregiver factors (number of comorbidities, diabetes and higher anxiety levels) were significantly associated with worse caregiver HRQOL, R2 = 26%. CONCLUSIONS: Recognizing caregiver-specific factors, including comorbidities and anxiety, associated with the HRQOL of caregivers of these older patients with advanced HF may guide support strategies.

Report From the American Society of Transplantation Conference on Donor Heart Selection in Adult Cardiac Transplantation in the United States.

Cardiac transplantation remains the only definitive treatment for end-stage heart failure. Transplantation rates are limited by a shortage of donor hearts. This shortage is magnified because many hearts are discarded because of strict selection criteria and concern for regulatory reprimand for less-than-optimal posttransplant outcomes. There is no standardized approach to donor selection despite proposals to liberalize acceptance criteria. A donor heart selection conference was organized to facilitate discussion and generate ideas for future research. The event was attended by 66 participants from 41 centers with considerable experience in cardiac donor selection. There were state-of-the-art presentations on donor selection, with subsequent breakout sessions on standardizing the process and increasing utilization of donor hearts. Participants debated misconceptions and established agreement on donor and recipient risk factors for donor selection and identified the components necessary for a future donor risk score. Ideas for future initiatives include modification of regulatory practices to consider extended criteria donors when evaluating outcomes and prospective studies aimed at identifying the factors leading to nonacceptance of available donor hearts. With agreement on the most important donor and recipient risk factors, it is anticipated that a consistent approach to donor selection will improve rates of heart transplantation.

Splicing factors differentially expressed in psoriasis alter mRNA maturation of disease-associated EDA+ fibronectin.

The EDA+ fibronectin splicing variant is overexpressed in psoriatic non-lesional epidermis and sensitizes keratinocytes to mitogenic signals. However, regulation of its abundance is only partially understood. In our recent cDNA microarray experiment, we identified three SR-rich splicing factors-splicing factor, arginine/serine-rich 18 (SFRS18), peptidyl-prolyl cis-trans isomerase G (PPIG), and luc-7 like protein 3 (LUC7L3)-which might be implicated in the preactivated states of keratinocytes in psoriatic non-involved skin and could also contribute to the regulation of fibronectin mRNA maturation. In this study, we investigated the role of LUC7L3, PPIG, and SFRS18 in psoriasis and in the mRNA maturation process of fibronectin. Regarding tissue staining experiments, we were able to demonstrate a characteristic distribution of the splicing factors in healthy, psoriatic non-involved and involved epidermis. Moreover, the expression profiles of these SR-rich proteins were found to be very similar in synchronized keratinocytes. Contribution of splicing facwwtors to the EDA+ fibronectin formation was also confirmed: their siRNA silencing leads to altered fibronectin mRNA and protein expression patterns, suggesting the participation in the EDA domain inclusion. Our results indicate that LUC7L3, PPIG, and SFRS18 are not only implicated in EDA+ fibronectin formation, but also that they could possess multiple roles in psoriasis-associated molecular abnormalities.

Detection of a rare CDKN2A intronic mutation in a Hungarian melanoma-prone family and its role in splicing regulation.

BACKGROUND: The major locus for melanoma predisposition is the cell cycle regulatory CDKN2A gene on chromosome 9p21. However, the frequency of germline coding mutations of the CDKN2A gene is lower than expected in melanoma-prone families linked to chromosome 9p21. OBJECTIVES: To investigate whether the rare IVS1+37 G/C intronic mutation of the CDKN2A gene, recently identified in a Hungarian melanoma-prone family, influences mRNA splicing regulation. METHODS: CDKN2A minigenes containing the wild-type and the mutant intronic sequence were created and transfected into HeLa cells with the aim of studying the mRNA transcripts. RESULTS: The results revealed the emergence of a differential splicing pattern from the wild-type and the mutant minigene, suggesting that this mutation may alter the splicing of CDKN2A primary mRNA and therefore might have a pathogenetic role in familial melanoma. CONCLUSIONS: We believe that these results confirm the importance of the identification and characterization of CDKN2A intronic mutations with a view to improving our understanding of the pathogenesis, and explain why the frequency of germline coding mutations of the CDKN2A gene is lower than expected in melanoma-prone families linked to chromosome 9p21.

Long-term use of the CentriMag® Ventricular Assist System as a right ventricular assist device: a case report.

Right ventricular failure (RVF) following implantation of a left ventricular assist system (LVAS) is associated with high morbidity and mortality.( 1-4 ) Numerous centers have reported short-term use of the CentriMag (®) Ventricular Assist System (CVAS) (Levitronix LLC, Waltham, MA) for treatment of cardiogenic shock, decompensated heart failure and right ventricular failure (RVF) following LVAS implantation.( 5-9 ) The present report reviews the clinical course of a patient requiring long-term right ventricular support utilizing the CVAS, following a HeartMate (®) II LVAS (Thoratec Corp. Pleasanton, CA) implantation. Elevated cytotoxic antibody levels complicated the patient's treatment plan by precluding orthotropic heart transplantation. The CVAS operated for 304 days without mechanical difficulty until replaced with the HeartWare (®) Ventricular Assist System (HeartWare Inc. Miramar, FL).

A selective cytopheretic inhibitory device for use during cardiopulmonary bypass surgery.

BACKGROUND: Systemic inflammatory response syndrome (SIRS) can occur in association with cardiopulmonary bypass (CPB) surgery, resulting in multiple organ dysfunction (MOD). Activated neutrophils have been implicated as major inciting factors in this process. Neutrophil-depleting filters incorporated within the extracorporeal blood circuit during CPB have been developed and evaluated, with inconsistent clinical results. METHODS: A novel, biomimetic, selective cytopheretic device (SCD) was tested in vitro within a blood circuit to assess safety and interactions with blood components and further evaluated ex vivo in a bovine model of CPB surgery during ventricular assist device implantation. RESULTS: In vitro blood circuit studies demonstrated that the SCD reduces circulating neutrophils while maintaining low rates of hemolysis compared to current leukocyte-reduction filters. In the bovine CPB model, animals without SCD treatment (No SCD) demonstrated an increase in circulating white blood cell (WBC) and neutrophil counts, steadily increasing throughout CPB. SCD with only systemic heparin anticoagulation (SCD-H) acutely reduced neutrophils for the first 2 hrs of CPB, but followed with a greater than 6-fold increase in neutrophil counts. SCD treatment with regional citrate anticoagulation along the SCD circuit (SCD-C) reduced systemic neutrophil counts throughout 4 hrs of CPB despite lower amounts of eluted cells from the SCD. When analyzed for immature neutrophils, the control and SCD-H showed increasing counts at later time-points, not seen in the SCD-C group, suggesting a more complex mechanism of action than simple leukoreduction. CONCLUSIONS: These results suggest that SCD-C therapy may disrupt the systemic leukocyte response during CPB, leading to improved outcomes for CPB-mediated MOD.

Tissue-specific splicing pattern of fibronectin messenger RNA precursor during development and aging in rat.

Fibronectin isoforms are generated by the alternative splicing of a primary transcript derived from a single gene. In rat at least three regions of the molecule are involved: EIIIA, EIIIB, and V. This study investigated the splicing patterns of these regions during development and aging, by means of ribonuclease protection analysis. Between fetal and adult rat, the extent of inclusion of the EIIIA and/or EIIIB region in fibronectin mRNA varied according to the type of tissue analyzed; but the inclusion of the V region, and in particular the V25 alternative variant, was significantly higher in all fetal than in adult tissues. These data suggest a crucial role of the V25 variant, possibly related to its interaction with the alpha 4 beta 1 integrin receptor during development. On the other hand, during aging, the only significant change observed in the splicing pattern was a decrease in the EIIIA variant in brain. The high inclusion levels of the EIIIA and EIIIB regions in young adult brain suggest that these segments may play an important role in differentiated brain tissue. The decreasing levels of inclusion of the EIIIA segment in brain fibronectin mRNA during aging may be an age-related marker with functional consequences.

Sympathectomy alters bone architecture in adult growing rats.

Sympathetic nervous system (SNS) fibres and alpha- and beta-receptors are present in bone, indicating that the SNS may participate in bone metabolism. The importance of these observations is controversial because stimulation or inhibition of the SNS has had various effects upon both anabolic and catabolic activity in this tissue. In this study we evaluated the effects of pharmacological sympathectomy, using chronic treatment of maturing male rats with 40 mg of guanethidine/kg i.p., upon various parameters in bone. Double labelling with tetracycline injection was also performed 20 and 2 days before sacrifice. Bone mass, mineral content, density and histomorphometric characteristics in different skeletal regions were determined. Bone metabolic markers included urinary deoxypyridinoline and serum osteocalcin measurements. Guanethidine significantly reduced the accretion of lumbar vertebral bone and of mineral content and density, compared to controls. Femoral bone mineral content and density were also significantly reduced, compared to controls. Histomorphometric analyses indicated these effects were related to a reduction of cortical bone and mineral apposition rate at femoral diaphysials level. Both markers of bone metabolism were reduced in controls as they approached maturity. Guanethidine significantly decreased serum osteocalcin compared to controls, while urinary deoxypyridinoline was unchanged. These data indicate that guanethidine-induced sympathectomy caused a negative balance of bone metabolism, leading to decreased mass by regulating deposition rather than resorption during modeling and remodeling of bone.

Heart and lung transplantation in the United States, 1997-2006.

This article highlights trends in heart and lung transplantation between 1997 and 2006, drawing on data from the OPTN and SRTR. The total number of candidates actively awaiting heart transplantation declined by 45% over the last decade, dropping from 2414 patients in 1997 to 1327 patients in 2006. The overall death rates among patients awaiting heart transplantation declined over the same period. The distribution of recipients among the different status groups at the time of heart transplantation changed little between the inception of the new classification system in 1999 and 2005. Deaths in the first year after heart transplantation have steadily decreased. At the end of 2006, 2885 candidates were awaiting a lung transplant, up 10% from the 1997 count. The median time-to-transplant for listed patients decreased by 87% over the decade, dropping from 1053 days in 1997 to 132 days in 2006. Selection for listing and transplantation has shifted toward more urgent patients since the May 2005 implementation of a new lung allocation system based on survival benefit and urgency rather than waiting time. Only 31 heart-lung transplants were performed in 2006, down from a high of 62 in 1997.

Central ghrelin gastroprotection involves nitric oxide/prostaglandin cross-talk.

BACKGROUND AND PURPOSE: Ghrelin, a gut-brain peptide, is considered a gastroprotective factor in gastric mucosa. We investigated the role of prostaglandins (PG) and the possible interplay between PGs and nitric oxide (NO) in ghrelin gastroprotection against ethanol (EtOH)-induced gastric lesions. EXPERIMENTAL APPROACH: We examined the effects of (1) central ghrelin (4 mug per rat) injection on PGE(2) accumulation in normal or EtOH-lesioned gastric mucosa, (2) pretreatment with indomethacin (10 mg kg(-1), p.o.), a non-selective cyclooxygenase (COX) inhibitor, and with a selective COX-1, SC560 (5 mg kg(-1), p.o.) or COX-2 inhibitor, celecoxib (3.5 mg kg(-1), p.o.) on ghrelin gastroprotection against 50% EtOH (1 mL per rat)-induced gastric lesions, (3) the NO synthase inhibitor, L-NAME (70 mg kg(-1), s.c), on gastric PGE(2) content in ghrelin-treated rats and (4) central ghrelin on the expression of constitutive and inducible NOS and COX mRNA and on the localization of the immunoreactivity for COX-2 in the gastric mucosa exposed to EtOH. KEY RESULTS: Ghrelin increased PGE(2) in normal mucosa, whereas, it reversed the EtOH-induced PGE(2) surge. Ghrelin had no effect on mucosal COX-1 expression but reduced the EtOH-induced increase in COX-2 expression and immunoreactivity. Indomethacin and SC560, but not celecoxib, removed ghrelin gastroprotection. L-NAME prevented the PGE(2) surge induced by ghrelin and, like indomethacin, reduced EtOH-induced PGE(2) increase. Ghrelin enhanced eNOS expression and reduced iNOS mRNA. CONCLUSIONS AND IMPLICATIONS: This study shows that COX-1-derived PGs are mainly involved in ghrelin gastroprotection and that the constitutive-derived NO together with PGE(2) are involved in ghrelin gastroprotective activity.

Left ventricular systolic and diastolic dysfunction after infusion of tumor necrosis factor-alpha in conscious dogs.

We used a load-insensitive index of systolic left ventricular (LV) function and an analysis of diastolic pressure-dimension relationships to test the hypothesis that recombinant human (rh) tumor necrosis factor-alpha (TNF alpha) impairs LV function in dogs. Animals were studied 7-10 d after aseptic implantation of instrumentation to monitor cardiac output, external anterior-posterior LV diameter, and LV and pleural pressures. Data were analyzed from seven dogs that received active rhTNF alpha (100 micrograms/kg over 60 min) and from five dogs that received heat-inactivated rhTNF alpha. At 24 h after infusion of active rhTNF alpha, the slope of the LV end-diastolic dimension-stroke work relationship decreased significantly, indicating a decrement in LV systolic contractility. Simultaneously, LV unstressed dimension increased significantly, suggesting diastolic myocardial creep. The end-diastolic relationship between LV transmural pressure and normalized LV dimension (strain) was markedly displaced to the left, suggesting increased diastolic elastic stiffness. Despite these changes in LV performance, cardiac index was maintained by tachycardia. The abnormalities in LV function were resolved by 72 h. We conclude that rhTNF alpha reversibly impairs LV systolic and diastolic function in unanesthetized dogs. Because dysfunction occurs greater than 6 h after the infusion of rhTNF alpha and persists for 24-48 h, the mechanism underlying this phenomenon may involve secondary mediators or a change in myocardial gene expression.

Central nervous system site of action for the respiratory depressant effect of diacetylmorphine (heroin) in the cat.

The purpose of our study was to identify central nervous system sites involved in the respiratory depressant effect of drugs that stimulate opioid receptors. Diacetylmorphine (heroin) was administered into several cerebroventricular regions of chloralose-anesthetized cats, while monitoring pulmonary ventilation with a Fleisch pneumotachograph. Administration of heroin (17, 50, 150, and 450 micrograms) into the forebrain ventricles, which was restricted to these ventricles, resulted in no significant respiratory effects. In contrast, administration of heroin into either the fourth ventricle or the cisterna magna resulted in a significant (P less than 0.05) decrease in respiratory minute volume (VE). In the fourth ventricle this was because of a decrease in frequency (f) and in the cisterna magna, to a decrease in tidal volume (VT). Intravenous administration of heroin in the same dose-range produced a decrease in VE, which was primarily due to a decrease in f. Bilateral application of heroin (70 micrograms/side) to each of three ventral medullary surface sites (Mitchell's, Schlaefke's, and Loeschcke's areas) known to influence respiration elicited a decrease in VE only at Mitchell's area. This decrease was due to decreases in f and VT. The role of this site in the action of intravenously administered heroin was tested by topical application of naloxone to this area in animals with respiratory depression evoked by intravenous heroin. Bilateral application of naloxone (15 micrograms/side) to Mitchell's area restored breathing to normal. These results lead us to suggest that the site of heroin-induced respiratory depression is a specific area (Mitchell's area) on the ventral surface of the medulla.

Regulation of fibronectin EDA exon alternative splicing: possible role of RNA secondary structure for enhancer display.

The fibronectin primary transcript undergoes alternative splicing in three noncoordinated sites: the cassette-type EDA and EDB exons and the more complex IIICS region. We have shown previously that an 81-nucleotide region within the EDA exon is necessary for exon recognition and that this region contains at least two splicing-regulatory elements: a polypurinic enhancer (exonic splicing enhancer [ESE]) and a nearby silencer element (exonic splicing silencer [ESS]). Here, we have analyzed the function of both elements in different cell types. We have mapped the ESS to the nucleotide level, showing that a single base change is sufficient to abolish its function. Testing of the ESE and ESS elements in heterologous exons, individually or as part of the complete EDA regulatory region, showed that only the ESE element is active in different contexts. Functional studies coupled to secondary-structure enzymatic analysis of the EDA exon sequence variants suggest that the role of the ESS element may be exclusively to ensure the proper RNA conformation and raise the possibility that the display of the ESE element in a loop position may represent a significant feature of the exon splicing-regulatory region.

Results from a multicenter evaluation of the 4th generation Elecsys Troponin T assay.

BACKGROUND AND OBJECTIVE: Discrepancies between serum and heparin plasma samples have been described for many commercial troponin assays including the cardiac troponin T (cTnT) assay. Using the current 3rd generation Elecsys Troponin T immunoassay, heparin plasma cannot be recommended for the determination of cTnT due to systematic lower test results caused by a direct interference of the immunoassay by heparin. The purpose of the multicenter study was to evaluate the analytical performance of an improved 4th generation Elecsys Troponin T immunoassay with a special focus on the comparability of cTnT results determined in heparin plasma and serum. METHODS AND RESULTS: The multicenter evaluation was performed in 10 clinical laboratories according to a standardized protocol (Roche Diagnostics, Penzberg, Germany, Study No. B05P008). The Elecsys Troponin T immunoassay was performed on the Modular Analytics E170 and Elecsys 2010 systems. Intraassay imprecision (n = 21) and total imprecision (2 runs/d, 10 days, triplicate measurements) were evaluated using 2 commercial controls (Roche Diagnostics) and 6 different serum pools (cTnT: 0.0140 - 4.102 microg/L). Intraassay CVs ranged from 0.73 to 3.22%. Total imprecision CVs ranged from 3.61 to 35.45% (cTnT < 0.1 microg/L) and 1.82 to 9.09% (cTnT > 0.1 microg/L), respectively. The cut-off for myocardial necrosis was determined to be 0.03 microg/L using the 10% total imprecision CV criteria. Linearity was assessed by serial dilutions of 6 different serum samples using cTnT negative serum pools. Linearity was proven up to 21.3 microg/L (recoveries: 90% - 110%). Regression data of all comparison studies were calculated according to the method of Passing and Bablok. The method comparison between the 4th generation and the commercially available cTnT immunoassay showed highly similar results across the whole measuring range (0.01 - 25.0 microg/L): y = 1.024x -0.001, r = 0.998; n = 988. Using the commercially available cTnT reagent, the serum to heparin plasma comparison yielded a systematic bias to approximately 8% lower cTnT results in heparin plasma. However, suitable comparability was obtained using the 4th generation Elecsys cTnT assay. The regression analysis (serum vs. heparin plasma) across the studied measuring range (cTnT: 0.01 - 14 microg/L) yielded the following equation: y = 0.975x + 0.001; r = 0.986; n = 403. However, rare individual serum to matched heparin plasma samples still yielded poor comparability (deviation > 20%) using the 4th generation Elecsys Troponin T immunoassay. CONCLUSION: Our data confirm an excellent analytical performance of the improved troponin T immunoassay. Most importantly, no systematic bias between cTnT results determined in serum and heparin plasma was observed from data obtained in 7 evaluation sites. The performance of the 4th generation Elecsys Troponin T assay is therefore comparable to other commercially available troponin immunoassays. Further studies are necessary to investigate the cause of poor comparability of cTnT results in rare individual serum to matched heparin plasma samples.

Role of biochemical markers of bone remodeling in clinical practice.

Bone tissue is subject to remodeling throughout the lifetime of an individual. Through a continuous remodeling cycle, actuated via the so-called 'bone remodeling units', old bone is resorbed by osteoclasts with the formation of cavities that are subsequently filled by osteoblasts. Bone loss observed in old age and in women after menopause is due to an imbalance between bone resorption and formation. Biochemical markers provide a dynamic view of the remodeling process, which covers rate of turnover and pathogenesis, and should improve fracture risk prediction. Furthermore, they can be used to monitor the short-term effects of therapy, and indicate if an excessive slowing of the remodeling process is occurring. When searching for markers of bone remodeling, biochemists have focused mainly on skeletal molecules that can be dosed in plasma and/or urine, as indicators of osteoblast function (i.e. bone alkaline phosphatase, osteocalcin, procollagene I C- and N-terminal propeptides) or osteoclast function (i.e. pyridinium crosslinks, collagen I C- and N-terminal telopeptides). The clinical significance of any marker for bone remodeling depends on two fundamental characteristics: specificity and variability. If the objective is to monitor therapeutic efficacy, it seems most rational to use a resorption marker for drugs that act principally on osteoclast, such as estrogens or bisphosphonates, while for drugs that act principally on osteoblast, such as PTH-peptides a marker for bone formation would be more appropriate.

ATP release during cell swelling activates a Ca2+-dependent Cl- current by autocrine mechanism in mouse hippocampal microglia.

Microglia cells, resident immune cells of the brain, survey brain parenchyma by dynamically extending and retracting their processes. Cl- channels, activated in the cellular response to stretch/swelling, take part in several functions deeply connected with microglia physiology, including cell shape changes, proliferation, differentiation and migration. However, the molecular identity and functional properties of these Cl- channels are largely unknown. We investigated the properties of swelling-activated currents in microglial from acute hippocampal slices of Cx3cr1 +/GFP mice by whole-cell patch-clamp and imaging techniques. The exposure of cells to a mild hypotonic medium, caused an outward rectifying current, developing in 5-10 minutes and reverting upon stimulus washout. This current, required for microglia ability to extend processes towards a damage signal, was carried mainly by Cl- ions and dependent on intracellular Ca2+. Moreover, it involved swelling-induced ATP release. We identified a purine-dependent mechanism, likely constituting an amplification pathway of current activation: under hypotonic conditions, ATP release triggered the Ca2+-dependent activation of anionic channels by autocrine purine receptors stimulation. Our study on native microglia describes for the first time the functional properties of stretch/swelling-activated currents, representing a key element in microglia ability to monitor the brain parenchyma.

Evidence for a role of the GHS-R1a receptors in ghrelin inhibition of gastric acid secretion in the rat.

Ghrelin, the endogenous ligand of the GH secretagogue receptor (GHS-R) has been previously shown to inhibit gastric acid secretion in pylorus-ligated rats. Two isoforms of GHS-R have been identified: GHS-R(1a) and GHS-R(1b). The present study aimed: (i) to characterise the type of GHS-R involved in the central gastric inhibitory activity of ghrelin by using des-octanoyl ghrelin, and synthetic GHS-R(1a) agonist (EP1572) and antagonist (D-Lys(3)-GHRP-6) and (ii) to investigate the relationship between ghrelin and cortistatin (CST) in the control of gastric acid secretion by using the natural neuropeptide CST-14 and the synthetic octapeptide CST-8. The specific interactions of all the compounds with GHS-R(1a) were determined by comparing their ability to displace labelled ghrelin or somatostatin from its receptors on rat hypothalamic membranes or on rat cardiomyocyte, respectively. Intracerebroventricular administration of 0.01 and 1 nmol/rat des-octanoyl ghrelin did not affect gastric acid secretion in pylorus-ligated rats, whereas EP1572 either i.c.v. (0.01-1 nmol/rat) or i.p. (10 and 20 nmol/kg) inhibited acid gastric secretion. Preteatment with D-Lys(3)GHRP-6 (3 nmol/rat, i.c.v.) was able to remove the inhibitory action of ghrelin (0.01 nmol/rat, i.c.v.) on gastric acid volume and acid output, thus indicating that the type 1a GHS-R likely mediates the gastric inhibitory action of ghrelin. This is supported by binding data showing that D-Lys(3)GHRP-6, but not des-octanoyl ghrelin, binds to hypothalamic GHS-R. CST-14 (1 nmol/rat, i.c.v.) did not affect either basal or ghrelin inhibition of gastric acid secretion. CST-8 (1 nmol/rat, i.c.v.) was able to counteract the gastric ghrelin response. The observation that CST-14 binds both GHR-S and somatostatin receptors, whereas CST-8 specifically displaces only ghrelin binding, indicates that CST-8 behaves as a GHS-R(1a) antagonist.

Different skeletal regional response to continuous brain infusion of leptin in the rat.

This study was designed to evaluate whether or not continuous intracerebroventricular infusion of leptin (1.5 microg/rat/24 h, for 28 days) produced different regional response on the skeleton of growing rats. Leptin reduce the accretion of total femoral bone mineral content (BMC) and density (BMD). This effect was related to a reduction of metaphyseal femur as no changes were detected in the diaphysis. Despite the reduced accretion in the volumetric of both femur and tibia compared to controls, leptin had no significant effects on the lumbar vertebrae. Urine deoxypyrydinoline and serum osteocalcin remained more elevated in the leptin-treated group as compared to controls. The results demonstrate that long-term central infusion of leptin activates bone remodeling with a negative balance. Leptin induces distinct responses in the different structure of bone and in the axial and appendicular skeleton.

Difference in substrate specificity between human and mouse lysosomal acid lipase: low affinity for cholesteryl ester in mouse lysosomal acid lipase.

Lysosomal acid lipase (LAL) is essential for the intracellular degradation of cholesteryl esters (CE) and triacylglycerols (TG) that are delivered to lysosomes by low density lipoprotein (LDL) receptor mediated endocytosis. We have analysed the difference in the catalytic properties and substrate specificity of human and mouse LALs. LAL activities were measured in human and mouse fibroblasts and in HeLa cells transiently expressing wild-type or site-directed mutant LALs of the two species using the T7 vaccinia system. Cholesteryl esterase and triacylglycerol lipase activities were determined in cellular homogenates with a phospholipid/detergent vesicle assay, an assay frequently used to diagnose human LAL deficiency syndromes, and with LDL particles, a more physiological substrate. Characterisation of human and mouse LAL using these two assays demonstrated marked differences in their TG and CE hydrolysing activities. Compared to human LAL mouse LAL showed a much lower cholesteryl esterase activity in both assays used. The difference was more pronounced in the vesicle assay. The lower cholesteryl esterase activity of mouse LAL did not affect the LDL-CE degradation in intact fibroblasts. The analysis of site-directed mutants suggests a role of the non-conserved cysteine residue at position 240 in cholesteryl esterase activity in human LAL. Our results show a significant difference between human and mouse LAL in their specificity toward cholesteryl esters. The low cholesteryl esterase activity does not result in reduced LDL-cholesterol ester degradation in mouse fibroblasts in situ. In addition, this work emphasises the importance of the physical state of substrates in studies of the specificity and properties of lipolytic enzymes.