Development of a real-time PCR assay for rapid detection and quantification of Photobacterium damselae subsp. piscicida in fish tissues.

The availability of a rapid and accurate method for the diagnosis of Photobacterium damselae subsp. piscicida (Phdp), able to discriminate its strictly correlated subsp. damselae (Phdd), formally known as Vibrio damsela, is essential for managing fish pasteurellosis outbreaks in farmed fish. A single-step, high-sensitivity real-time PCR assay for simultaneous detection and quantification of P. damselae was designed targeting partial of the sequence of the bamB gene and tested for specificity and sensitivity on laboratory-generated samples as well as on experimentally infected seabream tissue samples. With a limit of detection (LOD) of one copy in pure bacterial DNA, the sensitivity was higher than all methods previously reported. Validation in target and non-target bacterial species proved the assay was able to discriminate Phdd-Phdp subspecies from diverse hosts/geographical origins and between non-target species. In addition, two SNPs in the target amplicon region determine two distinctive qPCR dissociation curves distinguishing between Phdp-Phdd. This is the first time that a molecular method for P. damselae diagnosis combines detection, quantification and subspecies identification in one step. The assay holds the potential to improve the knowledge of infection dynamics and the development of better strategies to control an important fish disease.

Histopathological Findings Associated With Gastroesophageal Reflux Disease and Aspiration After Lung Transplantation: Initial Brazilian Single-Center Experience.

BACKGROUND: Gastro-esophageal reflux disease (GERD) and broncho-aspiration (BA) are known to increase the risk for chronic lung allograft dysfunction (CLAD). However, specific lung injury mechanisms are not clearly known. The objective of the study was to describe histopathological findings in surveillance lung transbronchial biopsies that can be correlated with episodes of BA in the lung allograft. METHODS: This retrospective analysis of surveillance transbronchial biopsies was performed in lung transplant recipients, with available data of broncho-alveolar fluid (cultures and cytology), lung function parameters, and esophageal functional tests. RESULTS: Were analyzed 11 patients, divided into 3 groups: (1) GERD group: 4 patients with GERD and CLAD diagnosis; (2) control group: 2 patients without GERD or CLAD; and (3) BA group: 5 patients with foreign material in lung biopsies. A histopathological pattern of neutrophilic bronchitis (NB) was present in 4 of 4 cases in the GERD group and in 1 of 5 cases in the BA group in 2 or more biopsy samples; culture samples were all negative; the 5 NB-positive patients developed CLAD and died (3/5) or needed re-transplantation (2/5). The other 3 patients in the BA group had GERD without NB or CLAD. Both patients in the control group had transient NB in biopsies with positive cultures but remained free of CLAD. CONCLUSIONS: Surveillance transbronchial biopsies may provide useful information other than the evaluation of acute cellular rejection and can help to identify high-risk patients for allograft dysfunction related to gastro-esophageal reflux.

Posterior reversible encephalopathy syndrome in lung transplantation: 5 case reports.

Posterior reversible encephalopathy syndrome (PRES) is a cliniconeuroradiologic entity characterized by typical neurologic symptoms with characteristic cerebral image alterations. It has been reported in solid organ transplantations, especially related to the use of calcineurin inhibitors. The incidence of PRES in lung transplantation is unknown and probably under-reported in the literature. Here we describe 5 cases of PRES after bilateral lung transplantation. One of the reported cases was the first in the literature in which the neurologic onset precluded the introduction of calcineurin inhibitor. Therefore, although calcineurin inhibitors are known to play an important role in the development of PRES in the setting of lung transplantation, other causes seems to be involved in the physiopathology of this syndrome.

Fungal infection by Mucorales order in lung transplantation: 4 case reports.

Mucorales is a fungus that causes systemic, highly lethal infections in immunocompromised patients. The overall mortality of pulmonary mucormycosis can reach 95%. This work is a review of medical records of 200 lung transplant recipients between the years of 2003 and 2013, in order to identify the prevalence of Mucorales in the Lung Transplantation service of Heart Institute (InCor), Hospital das Clínicas da Universidade de São Paulo, Brazil, by culture results from bronchoalveolar lavage and necropsy findings. We report 4 cases found at this analyses: 3 in patients with cystic fibrosis and 1 in a patient with bronchiectasis due to Kartagener syndrome. There were 2 unfavorable outcomes related to the presence of Mucorales, 1 by reduction of immunosuppression, another by invasive infection. Another patient died from renal and septic complications from another etiology. One patient was diagnosed at autopsy just 5 days after lung transplantation, with the Mucor inside the pulmonary vein with a precise, well-defined involvement only of donor's segment, leading to previous colonization hypothesis. There are few case reports of Mucorales infection in lung transplantation in the literature. Surveillance for the presence of Mucor can lead to timely fungal treatment and reduce morbidity and mortality in the immunocompromised patients, especially lung transplant recipients.

Tolerability of nausea and vomiting and associations with weight loss in a randomized trial of liraglutide in obese, non-diabetic adults.

BACKGROUND: Liraglutide 3.0 mg, with diet and exercise, produced substantial weight loss over 1 year that was sustained over 2 years in obese non-diabetic adults. Nausea was the most frequent side effect. OBJECTIVE: To evaluate routinely collected data on nausea and vomiting among individuals on liraglutide and their influence on tolerability and body weight. DESIGN: A randomized, placebo-controlled, double-blind 20-week study with an 84-week extension (sponsor unblinded at 20 weeks, open-label after 1 year) in eight European countries (Clinicaltrials.gov: NCT00422058). SUBJECTS: After commencing a 500-kcal/day deficit diet plus exercise, 564 participants (18-65 years, body mass index (BMI) 30-40 kg m(-2)) were randomly assigned (after a 2-week run-in period) to once-daily subcutaneous liraglutide (1.2, 1.8, 2.4 or 3.0 mg), placebo or open-label orlistat (120 mg × 3 per day). After 1 year, participants on liraglutide/placebo switched to liraglutide 2.4 mg, and subsequently, to liraglutide 3.0 mg (based on 20-week and 1-year results, respectively). RESULTS: The intention-to-treat population comprised 561 participants (n=90-98 per arm, age 45.9±10.3 years, BMI 34.8±2.7 kg m(-2) (mean±s.d.)). In year 1, more participants reported ⩾1 episode of nausea/vomiting on treatment with liraglutide 1.2-3.0 mg (17-38%) than with placebo or orlistat (both 4%, P⩽0.001). Most episodes occurred during dose escalation (weeks 1-6), with 'mild' or 'moderate' symptoms. Among participants on liraglutide 3.0 mg, 48% reported some nausea and 13% some vomiting, with considerable variation between countries, but only 4 out of 93 (4%) reported withdrawals. The mean 1-year weight loss on treatment with liraglutide 3.0 mg from randomization was 9.2 kg for participants reporting nausea/vomiting episodes, versus 6.3 kg for those with none (a treatment difference of 2.9 kg (95% confidence interval 0.5-5.3); P=0.02). Both weight losses were significantly greater than the respective weight losses for participants on placebo (P<0.001) or orlistat (P<0.05). Quality-of-life scores at 20 weeks improved similarly with or without nausea/vomiting on treatment with liraglutide 3.0 mg. CONCLUSION: Transient nausea and vomiting on treatment with liraglutide 3.0 mg was associated with greater weight loss, although symptoms appeared tolerable and did not attenuate quality-of-life improvements. Improved data collection methods on nausea are warranted.

Safety, tolerability and sustained weight loss over 2 years with the once-daily human GLP-1 analog, liraglutide.

OBJECTIVE: Having demonstrated short-term weight loss with liraglutide in this group of obese adults, we now evaluate safety/tolerability (primary outcome) and long-term efficacy for sustaining weight loss (secondary outcome) over 2 years. DESIGN: A randomized, double-blind, placebo-controlled 20-week study with 2-year extension (sponsor unblinded at 20 weeks, participants/investigators at 1 year) in 19 European clinical research centers. SUBJECTS: A total of 564 adults (n=90-98 per group; body mass index 30-40 kg m(-2)) enrolled, 398 entered the extension and 268 completed the 2-year trial. Participants received diet (500 kcal deficit per day) and exercise counseling during 2-week run-in, before being randomly assigned (with a telephone or web-based system) to once-daily subcutaneous liraglutide (1.2, 1.8, 2.4 or 3.0 mg, n=90-95), placebo (n=98) or open-label orlistat (120 mg × 3, n=95). After 1 year, liraglutide/placebo recipients switched to liraglutide 2.4 mg, then 3.0 mg (based on 20-week and 1-year results, respectively). The trial ran from January 2007-April 2009 and is registered with Clinicaltrials.gov, number NCT00480909. RESULTS: From randomization to year 1, liraglutide 3.0 mg recipients lost 5.8 kg (95% confidence interval 3.7-8.0) more weight than those on placebo and 3.8 kg (1.6-6.0) more than those on orlistat (P0.0001; intention-to-treat, last-observation-carried-forward). At year 2, participants on liraglutide 2.4/3.0 mg for the full 2 years (pooled group, n=184) lost 3.0 kg (1.3-4.7) more weight than those on orlistat (n=95; P<0.001). Completers on liraglutide 2.4/3.0 mg (n=92) maintained a 2-year weight loss of 7.8 kg from screening. With liraglutide 3.0 mg, 20-week body fat decreased by 15.4% and lean tissue by 2.0%. The most frequent drug-related side effects were mild to moderate, transient nausea and vomiting. With liraglutide 2.4/3.0 mg, the 2-year prevalence of prediabetes and metabolic syndrome decreased by 52 and 59%, with improvements in blood pressure and lipids. CONCLUSION: Liraglutide is well tolerated, sustains weight loss over 2 years and improves cardiovascular risk factors.

Impact of cytomegalovirus infection in lung transplant patients under universal prophylaxis: single-center experience in Brazil.

INTRODUCTION: Cytomegalovirus (CMV) infection, a common complication in lung transplant (LT) patients, is associated with worse outcomes. Therefore, prophylaxis and surveillance with preemptive treatment is recommended. OBJECTIVES: Describe the epidemiology and impact on mortality of CMV infection in LT patients receiving CMV prophylaxis. METHODS: Single-center retrospective cohort of LT recipients from August 2003 to March 2008. We excluded patients with survival or follow-up shorter than 30 days. We reviewed medical charts and all CMV pp65 antigen results. RESULTS: Forty-seven patients met the inclusion criteria and 19 (40%) developed a CMV event: eight CMV infections, seven CMV syndromes, and 15 CMV diseases. The mean number of CMV events for each patient was 1.68 +/- 0.88. Twelve patients developed CMV events during prophylaxis (5/12 had CMV serology D+/R-). Forty-six of the 47 patients had at least one episode of acute rejection (mean 2.23 +/- 1.1). Median follow-up was 22 months (range = 3-50). There were seven deaths. Upon univariate analysis, CMV events were related to greater mortality (P = .04), especially if the patient experienced more than two events (P = .013) and if the first event occurred during the first 3 months after LT (P = .003). Nevertheless, a marginally significant relationship between CMV event during the first 3 months after LT and mortality was observed in the multivariate analysis (hazards ratio: 7.46; 95% confidence interval: 0.98-56.63; P = .052). Patients with CMV events more than 3 months post-LT showed the same survival as those who remained CMV-free. CONCLUSION: Prophylaxis and preemptive treatment are safe and effective; however, the patients who develop CMV events during prophylaxis experience a worse prognosis.

Extracellular PBEF/NAMPT/visfatin activates pro-inflammatory signalling in human vascular smooth muscle cells through nicotinamide phosphoribosyltransferase activity.

AIMS/HYPOTHESIS: Extracellular pre-B cell colony-enhancing factor/nicotinamide phosphoribosyltransferase/visfatin (ePBEF/NAMPT/visfatin) is an adipocytokine, whose circulating levels are enhanced in metabolic disorders, such as diabetes mellitus and obesity. Here, we explored the ability of ePBEF/NAMPT/visfatin to promote vascular inflammation, as a condition closely related to atherothrombotic diseases. We specifically studied the ability of PBEF/NAMPT/visfatin to directly activate pathways leading to inducible nitric oxide synthase (iNOS) induction in cultured human aortic smooth muscle cells, as well as the mechanisms involved. METHODS: iNOS levels and extracellular signal-regulated kinase (ERK) 1/2 activity were determined by western blotting. Nuclear factor (NF)-kappaB activity was assessed by electrophoretic mobility shift assay. RESULTS: ePBEF/NAMPT/visfatin (10-250 ng/ml) induced iNOS in a concentration-dependent manner. At a submaximal concentration (100 ng/ml), ePBEF/NAMPT/visfatin time-dependently enhanced iNOS levels up to 18 h after stimulation. Over this time period, ePBEF/NAMPT/visfatin elicited a sustained activation of NF-kappaB and triggered a biphasic ERK 1/2 activation. By using the respective ERK 1/2 and NF-kappaB inhibitors, PD98059 and pyrrolidine dithiocarbamate, we established that iNOS induction by ePBEF/NAMPT/visfatin required the consecutive upstream activation of ERK 1/2 and NF-kappaB. The pro-inflammatory action of ePBEF/NAMPT/visfatin was not prevented by insulin receptor blockade. However, exogenous nicotinamide mononucleotide, the product of NAMPT activity, mimicked NF-kappaB activation and iNOS induction by ePBEF/NAMPT/visfatin, while the NAMPT inhibitor APO866 prevented the effects of ePBEF/NAMPT/visfatin on iNOS and NF-kappaB. CONCLUSIONS/INTERPRETATION: Through its intrinsic NAMPT activity, ePBEF/NAMPT/visfatin appears to be a direct contributor to vascular inflammation, a key feature of atherothrombotic diseases linked to metabolic disorders.

Bacterial and fungal pneumonias after lung transplantation.

OBJECTIVE: The aim of this study was to evaluate the epidemiology of bacterial and fungal pneumonia in lung transplant (LT) recipients and to assess donor-to-host transmission of these microorganisms. MATERIALS AND METHODS: We retrospectively studied all positive cultures from bronchoalveolar lavage (BAL) of 49 lung transplant recipients and their donors from August 2003 to April 2007. RESULTS: There were 108 episodes of pneumonia during a medium follow-up of 412 days (range, 1-1328 days). The most frequent microorganisms were: Pseudomonas aeruginosa (n = 36; 33.3%), Staphylococcus aureus (n = 29; 26.8%), and Aspergillus spp. (n = 18; 16%). Other fungal infections were due to Fusarium spp., Cryptococcus neoformans, and Paracoccidioides brasiliensis. Of the 31 donors with positive BAL, 15 had S. aureus. There were 21 pretransplant colonized recipients (43%) and 16 of them had suppurative underlying lung disease. P. aeruginosa was the most frequent colonizing organism (59% of pretransplant positive cultures). There were 11 episodes of bacteremia and lungs were the source in 5 cases. Sixteen deaths occurred and 6 (37.5%) were due to infection. Statistical analyses showed association between pretransplant colonizing microorganisms from suppurative lung disease patients and pneumonias after lung transplantation (RR = 4.76; P = .04; 95% CI = 1.02-22.10). No other analyzed factor was significant. CONCLUSIONS: Bacterial and fungal infections are frequent and contribute to higher mortality in lung transplant recipients. P. aeruginosa is the most frequent agent of respiratory infections. This study did not observe any impact of donor lung organisms on pneumonia after lung transplantation. Nevertheless, we demonstrated an association between pretransplant colonizing microorganisms and early pneumonias in suppurative lung transplant recipients.

Relationship of serum leptin concentration with age, gender, and biomedical parameters in healthy, non-obese subjects.

It is known that the circulating levels of leptin, the adipocyte hormone implicated in the control of energy balance, are correlated with fat body mass (FBM), although the influences of other physiological conditions are not fully understood. We investigated the relationships of serum leptin concentration with age, gender, and 36 hormone-metabolic parameters in a sample of a well defined healthy population (n=246; age range 20-93 years), and in subgroups of lean individuals according to their body mass index (BMI), within similar age range and gender distribution. Only insulin secretion (positively) and testosteronemia (negatively, in males) show direct correlations. The other relationships are not significant but throughout collaborating variables, such as serum lipids, especially through FBM, lean body mass (LBM) through insulin secretion, and gender through FBM. In males, LBM correlates with insulin secretions, s-IGF-1 and with s-testosterone. The relationship between insulin secretion and LBM persists up to advanced age. From the present study it may be concluded that the positive relationship of leptin with insulin secretion and the negative one with testosterone, indicate direct implications of leptin in insulin signaling, as well as in male sexual development. Finally, the fact that the amount of secreted insulin depends on LBM and the latter on testosterone and IGF-1, indicates the importance of muscle mass in the control of insulin secretion.

L-calcium channel blockade induced by diltiazem inhibits proliferation, migration and F-actin membrane rearrangements in human vascular smooth muscle cells stimulated with insulin and IGF-1.

During the atheroma plaque formation, smooth muscle cells (SMC) have to change their differentiated phenotype in order to proliferate, migrate and synthesize collagen. These phenotypic changes are stimulated by insulin and IGF-1, and we have studied the effect of L-type calcium channel blockade produced by diltiazem on such changes. Mitotic activity was measured using bromodeoxyuridine DNA incorporation, the migration capability as chemotaxis index in a Boyden chamber, and cytoskeleton changes related to SMC movement in immunofluorescence studies. Diltiazem (10(-7)-10(-6) M) reduced insulin-induced mitotic activity in cultured human vascular SMC more effectively than in IGF-1-induced mitotic activity, but at 10(-5) M, the inhibitory effects were similar. Diltiazem also showed a clear inhibition of migration ability, both under basal conditions (p < 0.05) and after addition of insulin (p = 0.0001) and IGF-1 (p < 0.0001). Finally, diltiazem inhibited membrane ruffling induced both by insulin and IGF-1 in a similar manner, and similar results were obtained with SMC from rat aorta. We conclude that substances blocking the L-type calcium channels such as diltiazem, could inhibit those processes which in vivo lead SMC to form the atheroma plaque.

Role of prevention in the contention of the obesity epidemic.

Obesity has become one of the major health burdens of the westernized world with an increasing number of people affected at any age. Although genetic factors explain around 40% of individual susceptibility to the disease, obesity may and should be controlled through interventions on the individual behaviour and on the social environment. Very promising to this aim is the role of prevention. Several levels of action have been established as well as different types of interventions according to the targeted population. Of special importance is the contention of childhood obesity with home and school as privileged settings for intervention. Despite a general acknowledging of the urgency for effective preventive measures at social, economic and political level to contrast the increasing prevalence of obesity, no clear nationwide policies have yet been established, and the educational and public health measures adopted so far lack that coordination and integration that the magnitude of the situation requires.

When the ICU patient refuses vital supports.

The aim of this paper is to discuss the problem of caring for ICU patients who request forgoing of intensive supports; in particular, evaluating their competence and building effective relationships among the patients themselves, their relatives and the health care team. The histories of 2 adult competent ICU patients 2 asked that vital intervention be forgone are presented, together with the discussion of problems posed by such a request and of possible solutions which respect patients' autonomy and bystanders needs. These patients were hospitalized in an Italian general 14 bed ICU. The patients' request were weighted against their prognosis and their view of life, in order to evaluate their reliability. Also the refusal of either treatment or outcome was evaluated, in order to effectively safeguard the patients' interests. The request of one patient was satisfied and he was allowed to die, after all the involved people had understood and accepted the final decision. The second patient, who was somehow forced to undergo intensive treatment, is alive and satisfied with having been cured. In Italy too, autonomy is an increasingly applied principle in end-of-life decisions in ICUs. It is usually tempered by a consideration about the patients' best interest as perceived by involved bystanders.

Principles, protocols and patients: the practical management of a limit in ICU.

The definition and management of a limit is a common but sometimes extremely complex problem in the Intensive Care Unit (ICU). Guidelines and consensus documents have been published in order to help clinicians. Yet, many controversial issues are brought into question. Legal rules are sometimes vague and derive more from the interpretation of various and unrelated principles (which vary from country to country) than from a clear ad hoc law. In this sense, the practical management of a limit in ICU is usually run by a dual normativity: an external one, which derives from the cultural, moral and legal values of the society, and an internal one, which depends on the particular clinical and human situation, namely the values of everybody involved in (the patient, his/her relatives, the health staff) and the relationships among these people. The considerable freedom left for the decision by an open communication is a great and favourable potential which must be used in the interest of the patient's and of his/her family.

Bioethical issues related to continuous renal replacement therapy in intensive care patients.

OBJECTIVE: To examine the ethical approach of intensivists and nephrologists to continuous renal replacement therapy (CRRT). DESIGN: A questionnaire. SETTING: The First International Course on Critical Care Nephrology. PARTICIPANTS: The participants in the course (around 500). RESULTS: Most participants think that establishing ethical criteria for managing CRRT is a medical task, as clinicians have adequate criteria for defining futility. However, many responders would grant the request of starting futile CRRT or would maintain it if requested by the family. Only 55% believe that informed consent is necessary for initiating CRRT; one out of four would start or maintain unwanted life-saving CRRT. In case of lack of equipment, the majority would select the patients, excluding the worst one or on a "first-come, first-served" basis. Withholding and withdrawing are regarded differently by most responders. Again, most think that every vital support should be withdrawn when futile, but practical and psychological aspects still influence the final decision. Responders think that ethics critical care committees can help in the management of ethical problems in ICU. CONCLUSIONS: Our results show that several ethical questions are still unsolved and that practical and psychological aspects of the treatment process can be stronger than bioethical principles.

Adipocyte differentiation factor (ADF): a protein secreted by mature fat cells that induces preadipocyte differentiation in culture.

A factor that is released into the culture medium of mature adipocytes and promotes the differentiation (adipogenic conversion) of preadipocytes has been partially characterized. The factor acts in a dose-dependent manner on preadipocytes to produce up to a four-fold increase in triacylglycerol (triglyceride) content and a nine-fold increase in glycerol-3-phosphate dehydrogenase (GPDH) activity, a marker of the late phase of differentiation of preadipocytes. The material appears to be a protein, since it has a molecular weight (Superose-12 gel exclusion chromatography) of about 53 kDa, an isoelectric point (pl) of 4.7-4.9, and is inactivated by the proteases papain and chymotrypsin and extremes of pH (2 and 12). Considerations of molecular weight, isoelectric point, stability to specific proteases, and especially to the action of chemical agents [the adipogenic activity is not affected by either an oxidizing (KIO4) or a reducing agent (DTT)], lead to the conclusion that the differentiation factor is distinct from known cytokines. The authors suggest that the protein be designated adipocyte differentiation factor (ADF). ADF in vivo may act as a cytokine paracrine agent to regulate the differentiation of preadipocytes.

Increase in insulin secretion with age: its clinical importance in evaluating abnormal secretions focused on diabetes type II and obesity.

The urinary C-peptide excretion was measured in a healthy standardized population sample of 160 subjects from 20 to 90 years of age, homogeneously distributed by age and sex. Urinary C-peptide excretion corresponded to 7% of the total amount released. The daily C-peptide excretion was 61.23 +/- 2.2 (S.E.) microg in the whole sample which corresponds to 41.9 +/- 1.5 IU of insulin secreted/day (I(CP)d), without sex differences. There is an increase of the I(CP)d value from the young to the healthy middle-aged person, but when the results were corrected for standard amounts of excreted creatinine (1 g) and urea (22 g) the age-dependent increase is to be observed during the whole adult life span. Assuming that cross-sectionally observed data are representative of the individual changes, it is concluded that age alone increases insulin secretion. The results which may be useful as reference values for clinical application were as follows: (A) in 5 diabetes type II patients in which the I(CP)d value was measured several times a week, the intraindividual variation coefficient was 10.9 +/- 7.2%;(B) in a sample of 47 type II diabetic patients of both sexes, between 51 and 70 years of age, a clear correlation was found between I(CP)d and the results of the glucagon stimulation test, mainly regarding the relationship between I(CP)d and the planimetrically measured area under the curve (r = 0.7, P < 0.0001); (C) in 7 obese non-diabetic individuals of similar ages the influence of the hypocaloric diet on the I(CP)d value was more evident than the use of C-peptide blood determinations before or after glucagon. Finally, the I(CP)d values of type II diabetes patients with insulin requirement (n = 27) were significantly lower than in the healthy control group (31.1 +/- 24.0 vs. 45.0 +/- 20.4), while diabetic patients without insulin requirement showed significantly higher values (73.0 +/- 33.0) (n = 27). These clinical studies primarily focused on the physiology of human ageing justify the measurement of C-peptide urinary excretion for evaluating daily insulin secretion in patients with type II diabetes.

RFLP analysis of human chromosome 11 region q13 in multiple symmetric lipomatosis and multiple endocrine neoplasia type 1-associated lipomas.

Six lipomas from patients affected by Multiple Symmetric Lipomatosis (MSL) and by Multiple Endocrine Neoplasia Type 1 (MEN 1) were analyzed for loss of heterozygosity on chromosome 11 region q12-13 using four RFLPs. Allelic loss for the D11S146 locus was found only in one visceral MEN 1-associated lipoma. Lipomas that exhibited a lack of allelic lesions were analyzed for an eventual abnormal amount or a defective function of the Gs protein by studying the Gs alpha subunit gene, codons 201 and 207, by PCR and TGGE techniques. All the samples were negative for activating mutations.

Catecholamine-sensitive lipolysis in the rat: different loci for effect of age on the lipolytic cascade in epididymal vs perirenal fat cells.

The biochemical locus of the decrease of lipolytic responsiveness to catecholamines in the aging rat has not heretofore been completely identified. Although increased sensitivity to the inhibitory action of adenosine is the likely explanation for the decrease during maturation, the nature of the age effect during senescence has been unclear. In order to determine whether the proximal or distal portion of the lipolytic pathway is involved, we have studied the lipolytic effect of the distally acting cyclic AMP analogue, 8-(4-chlorophenylthioadenosine)3'5'-monophosphate (cyclic) (Cl-cAMP) on rat fat cells from both the epididymal and perirenal fat pads of mature (6 mo) and senescent (24 mo) Fischer 344 rats. Using an adenosine (N6-1-2-phenylisopropyl-adenosine; PIA) regulated system, the lipolytic response to epinephrine (glycerol release) was measured simultaneously with that to Cl-cAMP. The effects of age on lipolysis are greatly influenced by the anatomic site of origin of the fat cells. The epididymal cells of the old rats showed no decreased responsiveness to either epinephrine or Cl-cAMP. However, the perirenal cells of the old rats showed a grossly impaired maximal response to both epinephrine (60% decrease relative to young; p < .005) and Cl-cAMP (42% and 58% decrease in 2 sets of experiments; p < .05 and .04, respectively). Although decreased lipolytic response to epinephrine in epididymal cells was not seen in these studies, this has been clearly shown in earlier work, suggesting that diminished response to epinephrine is demonstrable only when the system is not already maximally inhibited by PIA.(ABSTRACT TRUNCATED AT 250 WORDS)