OBJECTIVE

To validate quantitative imaging techniques used to detect and measure steatosis with magnetic resonance (MR) imaging in an ob/ob mouse model of hepatic steatosis.

METHODS

The internal research animal and resource center approved this study. Twenty-eight male ob/ob mice in progressively increasing age groups underwent imaging and were subsequently sacrificed. Six ob/+ mice served as control animals. Fat fraction imaging was performed with a chemical shift-based water-fat separation method. The following three methods of conventional fat quantification were compared with imaging: lipid extraction and qualitative and quantitative histologic analysis. Fat fraction images were reconstructed with single- and multiple-peak spectral models of fat and with and without correction for T2* effects. Fat fraction measurements obtained with the different reconstruction methods were compared with the three methods of fat quantification, and linear regression analysis and two-sided and two-sample t tests were performed.

RESULTS

Lipid extraction and qualitative and quantitative histologic analysis were highly correlated with the results of fat fraction imaging (r(2) = 0.92, 0.87, 0.82, respectively). No significant differences were found between imaging measurements and lipid extraction (P = .06) or quantitative histologic (P = .07) measurements when multiple peaks of fat and T2* correction were included in image reconstruction. Reconstructions in which T2* correction, accurate spectral modeling, or both were excluded yielded lower agreement when compared with the results yielded by other techniques. Imaging measurements correlated particularly well with histologic grades in mice with low fat fractions (intercept, -1.0% +/-1.2 [standard deviation]).

CONCLUSIONS

MR imaging can be used to accurately quantify fat in vivo in an animal model of hepatic steatosis and may serve as a quantitative biomarker of hepatic steatosis.

The Escherichia coli protein YjeQ represents a protein family whose members are broadly conserved in bacteria and have been shown to be indispensable to the growth of E. coli and Bacillus subtilis [Arigoni, F., et al. (1998) Nat. Biotechnol. 16, 851]. Proteins of the YjeQ family contain all sequence motifs typical of the vast class of P-loop-containing GTPases, but show a circular permutation, with a G4-G1-G3 pattern of motifs as opposed to the regular G1-G3-G4 pattern seen in most GTPases. All YjeQ family proteins display a unique domain architecture, which includes a predicted N-terminal OB-fold RNA-binding domain, the central permuted GTPase module, and a zinc knuckle-like C-terminal cysteine cluster. This domain architecture suggests a possible role for YjeQ as a regulator of translation. YjeQ was overexpressed, purified to homogeneity, and shown to contain 0.6 equiv of GDP. Steady state kinetic analyses indicated slow GTP hydrolysis, with a k(cat) of 9.4 h(-)(1) and a K(m) for GTP of 120 microM (k(cat)/K(m) = 21.7 M(-)(1) s(-)(1)). YjeQ also hydrolyzed other nucleoside triphosphates and deoxynucleotide triphosphates such as ATP, ITP, and CTP with specificity constants (k(cat)/K(m)) ranging from 0.2 to 1.0 M(-)(1) s(-)(1). Pre-steady state kinetic analysis of YjeQ revealed a burst of nucleotide hydrolysis for GTP described by a first-order rate constant of 100 s(-)(1) as compared to a burst rate of 0.2 s(-)(1) for ATP. In addition, a variant in the G1 motif of YjeQ (S221A) was substantially impaired for GTP hydrolysis (0.3 s(-)(1)) with a less significant impact on the steady state rate (1.8 h(-)(1)). In summary, E. coli YjeQ is an unusual, circularly permuted P-loop-containing GTPase, which catalyzes GTP hydrolysis at a rate 45 000 times greater than that of turnover.

Human DNA ligase III has essential functions in nuclear and mitochondrial DNA replication and repair and contains a PARP-like zinc finger (ZnF) that increases the extent of DNA nick joining and intermolecular DNA ligation, yet the bases for ligase III specificity and structural variation among human ligases are not understood. Here combined crystal structure and small-angle X-ray scattering results reveal dynamic switching between two nick-binding components of ligase III: the ZnF-DNA binding domain (DBD) forms a crescent-shaped surface used for DNA end recognition which switches to a ring formed by the nucleotidyl transferase (NTase) and OB-fold (OBD) domains for catalysis. Structural and mutational analyses indicate that high flexibility and distinct DNA binding domain features in ligase III assist both nick sensing and the transition from nick sensing by the ZnF to nick joining by the catalytic core. The collective results support a "jackknife model" in which the ZnF loads ligase III onto nicked DNA and conformational changes deliver DNA into the active site. This work has implications for the biological specificity of DNA ligases and functions of PARP-like zinc fingers.

OBJECTIVE

Interstitial cells of Cajal (ICC) express the receptor tyrosine kinase, KIT, the receptor for stem cell factor. In the gastrointestinal (GI) tract, ICC are pacemaker cells that generate spontaneous electrical slow waves, and mediate inputs from motor neurons. Absence or loss of ICC are associated with GI motility disorders, including those consequent of diabetes. Studies of ICC have been hampered by the low density of these cells and difficulties in recognizing these cells in cell dispersions.

METHODS

Kit(+/copGFP) mice harboring a copepod super green fluorescent protein (copGFP) complementary DNA, inserted at the Kit locus, were generated. copGFP(+) ICC from GI muscles were analyzed using confocal microscopy and flow cytometry. copGFP(+) ICC from the jejunum were purified by a fluorescence-activated cell sorter and validated by cell-specific markers. Kit(+/copGFP) mice were crossbred with diabetic Lep(+/ob) mice to generate compound Kit(+/copGFP);Lep(ob/ob) mutant mice. copGFP(+) ICC from compound transgenic mice were analyzed by confocal microscopy.

RESULTS

copGFP in Kit(+/copGFP) mice colocalized with KIT immunofluorescence and thus was predominantly found in ICC. In other smooth muscles, mast cells were also labeled, but these cells were relatively rare in the murine GI tract. copGFP(+) cells from jejunal muscles were Kit(+) and free of contaminating cell-specific markers. Kit(+/copGFP);Lep(ob/ob) mice displayed ICC networks that were dramatically disrupted during the development of diabetes.

CONCLUSIONS

Kit(+/copGFP) mice offer a powerful new model to study the function and genetic regulation of ICC phenotypes. Isolation of ICC from animal models will help determine the causes and responses of ICC to therapeutic agents.

Neuropeptide Y (NPY), a 36 amino acid neuromodulator that is secreted by neurons throughout the peripheral and central nervous system, has been implicated in the control of many physiological processes. We have begun to examine its role in regulation of appetite, behavior, and excitotoxicity by examining mice that are unable to produce NPY as a consequence of gene inactivation. These mutant mice are remarkably normal when reared under standard vivarium conditions. Despite considerable evidence that NPY plays a central role in stimulating appetite, NPY-deficient mice eat normally, grow normally, and refeed after a fast normally. Furthermore, all of their endocrine responses to fasting are normal. The response of NPY-null mice to diet-induced obesity, chemically induced obesity (monosodium glutamate and gold thioglucose), and genetic-based obesity (lethal yellow agouti, Ay; uncoupling protein-diphtheria toxin transgenics, UCP-DT) were all normal. However, NPY deficiency does partially ameliorate the obesity and all of the adverse endocrine effects of leptin deficiency in ob/ob mice. NPY-null mice as well as mice deficient in both NPY and leptin are more sensitive to leptin, suggesting that NPY may normally have a tonic inhibitory action on leptin-mediated satiety signals. NPY-null mice display the normal voracious feeding response to injected NPY. Thus, the only condition where we have observed a role for NPY in body-weight regulation is in the context of complete leptin deficiency--where absence of NPY is beneficial. The activity and general behavior of NPY-null mice are normal. They appear to have normal spatial and contextual learning ability; however, they manifest more anxiety under some conditions. NPY-null mice occasionally display spontaneous, seizure-like events. They also are less able to terminate seizures induced by GABA receptor antagonists or glutamate receptor agonists. These observations are consistent with previous data suggesting that NPY plays an important role in dampening excitotoxicity.

The intercellular and intracellular distribution of the movement protein (MP) of the Ob tobamovirus was examined in infected leaf tissues using an infectious clone of Ob in which the MP gene was translationally fused to the gene encoding the green fluorescent protein (GFP) of Aequorea victoria. In leaves of Nicotiana tabacum and N. benthamiana, the modified virus caused fluorescent infection sites that were visible as expanding rings. Microscopy of epidermal cells revealed subcellular patterns of accumulation of the MP:GFP fusion protein which differed depending upon the radial position of the cells within the fluorescent ring. Punctate, highly localized fluorescence was associated with cell walls of all of the epidermal cells within the infection site, and apparently represents association of the fusion protein with plasmodesmata; furthermore, fluorescence was retained in cell walls purified from infected leaves. Within the brightest region of the fluorescent ring, the MP:GFP was observed in irregularly shaped inclusions in the cortical regions of infected cells. Fluorescent filamentous structures presumed to represent association of MP:GFP with microtubules were observed, but were distributed differently within the infection sites on the two hosts. Within cells containing filaments, a number of fluorescent bodies, some apparently streaming in cytoplasmic strands, were also observed. The significance of these observations is discussed in relation to MP accumulation, targeting to plasmodesmata, and degradation.

BACKGROUND

Deinococcus radiodurans R1 is one of the most radiation-resistant organisms known and is able to repair an unusually large amount of DNA damage without induced mutation. Single-stranded DNA-binding (SSB) protein is an essential protein in all organisms and is involved in DNA replication, recombination and repair. The published genomic sequence from Deinococcus radiodurans includes a putative single-stranded DNA-binding protein gene (ssb; DR0100) requiring a translational frameshift for synthesis of a complete SSB protein. The apparently tripartite gene has inspired considerable speculation in the literature about potentially novel frameshifting or RNA editing mechanisms. Immediately upstream of the ssb gene is another gene (DR0099) given an ssb-like annotation, but left unexplored.

RESULTS

A segment of the Deinococcus radiodurans strain R1 genome encompassing the ssb gene has been re-sequenced, and two errors involving omitted guanine nucleotides have been documented. The corrected sequence incorporates both of the open reading frames designated DR0099 and DR0100 into one contiguous ssb open reading frame (ORF). The corrected gene requires no translational frameshifts and contains two predicted oligonucleotide/oligosaccharide-binding (OB) folds. The protein has been purified and its sequence is closely related to the Thermus thermophilus and Thermus aquaticus SSB proteins. Like the Thermus SSB proteins, the SSBDr functions as a homodimer. The Deinococcus radiodurans SSB homodimer stimulates Deinococcus radiodurans RecA protein and Escherichia coli RecA protein-promoted DNA three-strand exchange reactions with at least the same efficiency as the Escherichia coli SSB homotetramer.

CONCLUSIONS

The correct Deinococcus radiodurans ssb gene is a contiguous open reading frame that codes for the largest bacterial SSB monomer identified to date. The Deinococcus radiodurans SSB protein includes two OB folds per monomer and functions as a homodimer. The Deinococcus radiodurans SSB protein efficiently stimulates Deinococcus radiodurans RecA and also Escherichia coli RecA protein-promoted DNA strand exchange reactions. The identification and purification of Deinococcus radiodurans SSB protein not only allows for greater understanding of the SSB protein family but provides an essential yet previously missing player in the current efforts to understand the extraordinary DNA repair capacity of Deinococcus radiodurans.

Despite intensive research, the mechanism of the rapid retardation in the rates of cellobiohydrolase (CBH) catalyzed cellulose hydrolysis is still not clear. Interpretation of the hydrolysis data has been complicated by the inability to measure the catalytic constants for CBH-s acting on cellulose. We developed a method for measuring the observed catalytic constant (k(obs)) for CBH catalyzed cellulose hydrolysis. It relies on in situ measurement of the concentration of CBH with the active site occupied by the cellulose chain. For that we followed the specific inhibition of the hydrolysis of para-nitrophenyl-beta-D-lactoside by cellulose. The method was applied to CBH-s TrCel7A from Trichoderma reesei and PcCel7D from Phanerochaete chrysosporium and their isolated catalytic domains. Bacterial microcrystalline cellulose, Avicel, amorphous cellulose, and lignocellulose were used as substrates. A rapid decrease of k(obs) in time was observed on all substrates. The k(obs) values for PcCel7D were about 1.5 times higher than those for TrCel7A. In case of both TrCel7A and PcCel7D, the k(obs) values for catalytic domains were similar to those for intact enzymes. A model where CBH action is limited by the average length of obstacle-free way on cellulose chain is proposed. Once formed, productive CBH-cellulose complex proceeds with a constant rate determined by the true catalytic constant. After encountering an obstacle CBH will "get stuck" and the rate of further cellulose hydrolysis will be governed by the dissociation rate constant (k(off)), which is low for processive CBH-s.

BACKGROUND

Sepsis is a potentially deadly disease that often is caused by gram-positive bacteria, in particular Staphylococcus aureus (S. aureus). As there are few effective therapies for sepsis, increased basic knowledge about factors predisposing is needed.

RESULTS

The purpose of this study was to study the effect of Western diet on mortality induced by intravenous S. aureus inoculation and the immune functions before and after bacterial inoculation. Here we show that C57Bl/6 mice on high-fat diet (HFD) for 8 weeks, like genetically obese Ob/Ob mice on low-fat diet (LFD), have increased mortality during S. aureus-induced sepsis compared with LFD-fed C57Bl/6 controls. Bacterial load in the kidneys 5-7 days after inoculation was increased 10-fold in HFD-fed compared with LFD-fed mice. At that time, HFD-fed mice had increased serum levels and fat mRNA expression of the immune suppressing cytokines interleukin-1 receptor antagonist (IL-1Ra) and IL-10 compared with LFD-fed mice. In addition, HFD-fed mice had increased serum levels of the pro-inflammatory IL-1beta. Also, HFD-fed mice with and without infection had increased levels of macrophages in fat. The proportion and function of phagocytosing granulocytes, and the production of reactive oxygen species (ROS) by peritoneal lavage cells were decreased in HFD-fed compared with LFD-fed mice.

CONCLUSIONS

Our findings imply that chronic HFD disturb several innate immune functions in mice, and impairs the ability to clear S. aureus and survive sepsis.

The gene 2.5 protein (gp2.5) of bacteriophage T7 is a single-stranded DNA (ssDNA) binding protein that has essential roles in DNA replication and recombination. In addition to binding DNA, gp2.5 physically interacts with T7 DNA polymerase and T7 primase-helicase during replication to coordinate events at the replication fork. We have determined a 1.9-A crystal structure of gp2.5 and show that it has a conserved OB-fold (oligosaccharide/oligonucleotide binding fold) that is well adapted for interactions with ssDNA. Superposition of the OB-folds of gp2.5 and other ssDNA binding proteins reveals a conserved patch of aromatic residues that stack against the bases of ssDNA in the other crystal structures, suggesting that gp2.5 binds to ssDNA in a similar manner. An acidic C-terminal extension of the gp2.5 protein, which is required for dimer formation and for interactions with the T7 DNA polymerase and the primase-helicase, appears to be flexible and may act as a switch that modulates the DNA binding affinity of gp2.5.

The adult subventricular zone (SVZ) supports neural stem cell self-renewal and differentiation and continually gives rise to new neurons throughout adult life. The mechanisms orienting the migration of neuroblasts from the SVZ to the olfactory bulb (OB) via the rostral migratory stream (RMS) have been extensively studied, but factors controlling neuroblast exit from the SVZ remain poorly explored. The morphogen Sonic Hedgehog (Shh) displays proliferative and survival activities toward neural stem cells and is an axonal chemoattractant implicated in guidance of commissural axons during development. We identify here the presence of Shh protein in SVZ extracts and in the cerebrospinal fluid of adult mice, and we demonstrate that migrating neuroblasts in the SVZ and RMS express the Shh receptor Patched. We show that Shh displays a chemoattractive activity in vitro on SVZ-derived neuronal progenitors, an effect blocked by Cur61414, a Smoothened antagonist. Interestingly, Shh-expressing cells grafted above the RMS of adult mice exert a chemoattractive activity on migrating neuroblasts in vivo, thus inducing their accumulation and deviation from their normal migratory pathway. Furthermore, the adenoviral transfer of Shh into the lateral ventricle or the blocking of Shh present in the SVZ of adult mice using its physiological antagonist Hedgehog interacting protein or neutralizing Shh antibodies provides in vivo evidence that Shh can retain SVZ-derived neuroblasts. The ability to modulate the number of neuroblasts leaving the SVZ and reaching the OB through the chemoattractive activity of Shh suggests a novel degree of plasticity in cell migration of this adult stem cell niche.

Leptin exerts important effects on the regulation of food intake and energy expenditure by acting in the brain. Leptin action is mediated by the interaction with a receptor that is alternatively spliced, resulting in at least five different isoforms. The long form (OB-Rb) has a long intracellular domain that is essential for intracellular signal transduction. The specific aim of this study was to further investigate the role that the brain may play in the pathogenesis of obesity in humans. We studied the expression of OB-R mRNA (both short or common and long isoforms) in the brains of obese, lean and diabetic subjects, by in situ hybridization, semiquantitative RT-PCR and Northern blots analysis. We used two alternative probes: one that recognizes all known splice variants (OB-Ra) and a second that recognizes only the long form, OB-Rb. Several brain regions, including hypothalamus, cerebellum, neocortex, entorrhinal cortex, amygdala, and rostral medulla, were evaluated. In situ hybridization studies revealed that both OB-Ra and OB-Rb mRNAs are widely distributed in the human brain. The specific hybridization signal with both probes was detected exclusively in the cytoplasm of the cell body, dendrites and proximal axons of neurons. Hypothalamic nuclei, Purkinje cells and dentate nuclei of the cerebellum, inferior olivary and cranial nerves nuclei in the medulla, amygdala and neurons from both neocortex and entorrhinal cortex demonstrated positive signals. The hybridization signal obtained in ependyma was lower than that in neurons and no specific hybridization was detected in glial cells. No significant differences were identified among the regions or among the three groups studied. These results match those previously obtained by us [Couce et al.: Neuroendocrinology 1997;66:145] in which the distribution of the OB-R protein in the human brain was first described. RT-PCR indicated that the OB-Rb was highly expressed in the hypothalamus and cerebellum. No significant differences of OB-Ra or OB-Rb mRNA expression were identified in lean or obese individuals in these two cerebral regions. The levels of OB-Rb were significantly higher in cerebellum compared to hypothalamus in lean and obese individuals. The original hypothesis that OB-Rb is present only in the hypothalamus needs to be reconsidered. This OB-Rb isoform seems to be widely expressed in the human brain with highest levels in the cerebellum. Obesity and hyperleptinemia appears not to be associated with down-regulation of the OB-Rb in the human brain.

OBJECTIVE

To investigate the effect of television food advertising on children's food intake, specifically whether childhood obesity is related to a greater susceptibility to food promotion.

METHODS

The study was a within-subject, counterbalanced design. The children were tested on two occasions separated by two weeks. One condition involved the children viewing food advertisements followed by a cartoon, in the other condition the children viewed non-food adverts followed by the same cartoon. Following the cartoon, their food intake and choice was assessed in a standard paradigm.

METHODS

The study was conducted in Liverpool, UK.

METHODS

Fifty-nine children (32 male, 27 female) aged 9-11 years were recruited from a UK school to participate in the study. Thirty-three children were normal-weight (NW), 15 overweight (OW) and 11 obese (OB).

RESULTS

Exposure to food adverts produced substantial and significant increases in energy intake in all children (P < 0.001). The increase in intake was largest in the obese children (P = 0.04). All children increased their consumption of high-fat and/or sweet energy-dense snacks in response to the adverts (P < 0.001). In the food advert condition, total intake and the intake of these specific snack items correlated with the children's modified age- and gender-specific body mass index score.

CONCLUSIONS

These data suggest that obese and overweight children are indeed more responsive to food promotion, which specifically stimulates the intake of energy-dense snacks.

Pancreatic β cells adapt to compensate for increased metabolic demand during insulin resistance. Although the microRNA pathway has an essential role in β cell proliferation, the extent of its contribution is unclear. Here, we report that miR-184 is silenced in the pancreatic islets of insulin-resistant mouse models and type 2 diabetic human subjects. Reduction of miR-184 promotes the expression of its target Argonaute2 (Ago2), a component of the microRNA-induced silencing complex. Moreover, restoration of miR-184 in leptin-deficient ob/ob mice decreased Ago2 and prevented compensatory β cell expansion. Loss of Ago2 during insulin resistance blocked β cell growth and relieved the regulation of miR-375-targeted genes, including the growth suppressor Cadm1. Lastly, administration of a ketogenic diet to ob/ob mice rescued insulin sensitivity and miR-184 expression and restored Ago2 and β cell mass. This study identifies the targeting of Ago2 by miR-184 as an essential component of the compensatory response to regulate proliferation according to insulin sensitivity.

Leptin is detected in the sera, and leptin receptors are expressed in the cerebrum of mouse embryos, suggesting that leptin plays a role in cerebral development. Compared with the wild type, leptin-deficient (ob/ob) mice had fewer cells at embryonic day (E) 16 and E18 and had fewer 5-bromo-2'-deoxyuridine(+) cells at E14 and E16 in the neuroepithelium. Intracerebroventricular leptin injection in E14 ob/ob embryos increased the number of neuroepithelium cells at E16. In cultured neurosphere cells, leptin treatment increased Hes1 mRNA expression and maintained neural progenitors. Astrocyte differentiation was induced by low-dose (0.1 microg/ml) but not high-dose (1 microg/ml) leptin. High-dose leptin decreased Id mRNA and increased Ngn1 mRNA in neurosphere cells. The neuropeptide Y mRNA level in the cortical plate was lower in ob/ob than the wild type at E16 and E18. These results suggest that leptin maintains neural progenitors and is related to glial and neuronal development in embryos.

OBJECTIVE

To characterize equine bone marrow (BM)-derived mesenchymal stem cell (MSC) growth characteristics and frequency as well as their adipogenic and osteogenic differentiation potential.

METHODS

In vitro experimental study.

METHODS

Foals (n=3, age range, 17-51 days) and young horses (n=5, age range, 9 months to 5 years).

METHODS

Equine MSCs were harvested and isolated from sternal BM aspirates and grown up to passage 10 to determine cell-doubling (CD) characteristics. Limit dilution assays were performed on primary and passaged MSCs to determine the frequency of colony-forming units with a fibroblastic phenotype (CFU-F), and the frequency of MSC differentiation into adipocytes (CFU-Ad) and osteoblasts (CFU-Ob).

RESULTS

Initial MSC isolates had a lag phase with a significantly longer CD time (DT=4.9+/-1.6 days) compared with the average DT (1.4+/-0.22 days) of subsequent MSC passages. Approximately 1 in 4224+/-3265 of the total nucleated BM cells displayed fibroblast colony-forming activity. Primary MSCs differentiated in response to adipogenic and osteogenic inductive conditions and maintained their differentiation potential during subsequent passages.

CONCLUSIONS

The frequency, in vitro growth rate, and adipogenic and osteogenic differentiation potential of foals and young adult horses are similar to those documented for BM MSCs of other mammalian species.

CONCLUSIONS

The results have direct relevance to the use of BM as a potential source of adult stem cells for tissue engineering applications in equine veterinary medicine.

OBJECTIVE

To examine the association between breakfast skipping and type of breakfast consumed with overweight/obesity, abdominal obesity, other cardiometabolic risk factors and the metabolic syndrome (MetS).

METHODS

Cross-sectional. Three breakfast groups were identified, breakfast skippers (BS), ready-to-eat-cereal (RTEC) consumers and other breakfast (OB) consumers, using a 24 h dietary recall. Risk factors were compared between the breakfast groups using covariate-adjusted statistical procedures.

METHODS

The 1999–2006 National Health and Nutrition Examination Survey, USA.

METHODS

Young adults (20–39 years of age).

RESULTS

Among these young adults (n 5316), 23.8% were BS, 16.5% were RTEC consumers and 59.7% were OB consumers. Relative to the BS, the RTEC consumers were 31%, 39%, 37%, 28%, 23%, 40% and 42% less likely to be overweight/obese or have abdominal obesity, elevated blood pressure, elevated serum total cholesterol, elevated serum LDL-cholesterol, reduced serum HDL-cholesterol or elevated serum insulin, respectively. Relative to the OB consumers, the BS were 1.24, 1.26 and 1.44 times more likely to have elevated serum total cholesterol, elevated serum LDL-cholesterol or reduced serum HDL-cholesterol, respectively. Relative to the OB consumers, the RTEC consumers were 22%, 31% and 24% less likely to be overweight/ obese or have abdominal obesity or elevated blood pressure, respectively. No difference was seen in the prevalence of the MetS by breakfast skipping or type of breakfast consumed.

CONCLUSIONS

Results suggest that consumption of breakfast, especially that included an RTEC, was associated with an improved cardiometabolic risk profile in U.S. young adults. Additional studies are needed to determine the nature of these relationships.

1. We have determined the binding characteristics of [(125)I]-(Pyr(1))Apelin-13, a putative ligand for the APJ orphan receptor in human cardiovascular and rat tissue and investigated the functional properties of (Pyr(1))Apelin-13 in human saphenous vein. 2. The binding of [(125)I]-(Pyr(1))Apelin-13 to sections of human heart tissue was time dependent and rapid at 23 degrees C. Data were fitted to a single site model with an association rate constant (k(obs)) of 0.115 min(-1). [(125)I]-(Pyr(1))Apelin-13 also dissociated from a single site with a dissociation rate constant of 0.0105 min(-1). 3. In saturation binding experiments [(125)I]-(Pyr(1))Apelin-13 bound to human left ventricle with a K(D) value of 0.35+/-0.08 nM, B(max) of 4.3+/-0.9 fmol mg(-1) protein with a Hill slope of 0.97+/-0.04 and to the right atria with a K(D) of 0.33+/-0.09 nM, B(max) of 3.1+/-0.6 fmol mg(-1) protein and a Hill slope of 0.93+/-0.05. 4. [(125)I]-(Pyr(1))Apelin-13 binding sites were localized using autoradiography to human cardiovascular tissue, including coronary artery, aorta and saphenous vein grafts. In rat tissue a high density of receptors were localized to the molecular layer of the rat cerebellum, rat lung, rat heart and low levels in the rat kidney cortex. 2. (Pyr(1))Apelin-13 potently contracted human saphenous vein with a pD(2) value of 8.4+/-0.2 (n=8). The maximum response elicited by the peptide was 22.6+/-6% of 100 mM KCl. 6. We provide the first evidence of APJ receptor expression, relative densities and functional properties of (Pyr(1))Apelin-13 in human cardiovascular tissue.

OBJECTIVE

To provide an initial abuse liability assessment of an electronic cigarette (EC) in current tobacco cigarette smokers.

METHODS

The first of four within-subject sessions was an EC sampling session that involved six, 10-puff bouts (30 seconds inter-puff interval), each bout separated by 30 minutes. In the remaining three sessions participants made choices between 10 EC puffs and varying amounts of money, 10 EC puffs and a varying number of own brand cigarette (OB) puffs, or 10 OB puffs and varying amounts of money using the multiple-choice procedure (MCP). The MCP was completed six times at 30-minute intervals, and one choice was reinforced randomly at each trial.

METHODS

Clinical laboratory.

METHODS

Twenty current tobacco cigarette smokers.

METHODS

Sampling session outcome measures included plasma nicotine, cardiovascular response and subjective effects. Choice session outcome was the cross-over value on the MCP.

RESULTS

EC use resulted in significant nicotine delivery, tobacco abstinence symptom suppression and increased product acceptability ratings. On the MCP, participants chose to receive 10 EC puffs over an average of $1.06 or three OB puffs and chose 10 OB puffs over an average of $1.50 (P < 0.003).

CONCLUSIONS

Electronic cigarettes can deliver clinically significant amounts of nicotine and reduce cigarette abstinence symptoms and appear to have lower potential for abuse relative to traditional tobacco cigarettes, at least under certain laboratory conditions.

Cytoplasmic Ca2+ concentration ([Ca2+]i) and insulin secretion were monitored in single ob/ob mouse pancreatic islets stimulated by glucose. After culture in 5.5 mM of the sugar, islets responded to 11 mM glucose with pulsatile insulin secretion synchronized with oscillations of [Ca2+]i (0.3-0.5/min). Most islets also showed superimposed regular rapid [Ca2+]i oscillations and insulin transients of similar frequency. Whereas the amplitude of the slow insulin pulses increased in 20 mM glucose, the [Ca2+]i oscillations were replaced by a sustained increase. After culture in the absence of sugar, there was little rise of [Ca2+]i during exposure to 11 mM glucose and only a slight secretory response, which, however, was pulsatile. The slow secretory pulses in the presence of 11 mM glucose were augmented after culture in 11 or 20 mM glucose despite a sustained elevation of [Ca2+]i. Although pulsatile insulin release was not always associated with [Ca2+]i oscillations, the data indicate that the slow and fast [Ca2+]i oscillations do correspond to pulsatile insulin secretion.

Diabetic kidney disease (DKD) remains the most common cause of end-stage kidney disease despite multifactorial intervention. We demonstrated that increased cholesterol in association with downregulation of ATP-binding cassette transporter ABCA1 occurs in normal human podocytes exposed to the sera of patients with type 1 diabetes and albuminuria (DKD(+)) when compared with diabetic patients with normoalbuminuria (DKD(-)) and similar duration of diabetes and lipid profile. Glomerular downregulation of ABCA1 was confirmed in biopsies from patients with early DKD (n = 70) when compared with normal living donors (n = 32). Induction of cholesterol efflux with cyclodextrin (CD) but not inhibition of cholesterol synthesis with simvastatin prevented podocyte injury observed in vitro after exposure to patient sera. Subcutaneous administration of CD to diabetic BTBR (black and tan, brachiuric) ob/ob mice was safe and reduced albuminuria, mesangial expansion, kidney weight, and cortical cholesterol content. This was followed by an improvement of fasting insulin, blood glucose, body weight, and glucose tolerance in vivo and improved glucose-stimulated insulin release in human islets in vitro. Our data suggest that impaired reverse cholesterol transport characterizes clinical and experimental DKD and negatively influences podocyte function. Treatment with CD is safe and effective in preserving podocyte function in vitro and in vivo and may improve the metabolic control of diabetes.

The behavioral and neuroendocrine effects of the adipose tissue-derived circulating protein, leptin, appear to be mediated by the hypothalamus. We have investigated whether the leptin receptor gene is expressed in hindbrain regions known to be involved in the processing of satiety and energetic signals of peripheral origin. In the mouse, gene expression was detected in the nucleus of the solitary tract, lateral parabrachial nucleus, and medullary reticular nucleus and diffusely elsewhere by in situ hybridization. Receptor messenger RNA in these neuronal areas consisted largely, if not exclusively, of the long splice variant, Ob-Rb. Presumed short receptor splice variants were abundantly expressed in the leptomeninges and the choroid plexus of the fourth ventricle. Similar levels of leptin receptor gene expression were present in the hindbrain of lean and obese (ob/ob) mice. The leptin receptor gene was expressed comparatively weakly in the nucleus of the solitary tract of the rat and was not detectable in the lateral parabrachial nucleus. However, by contrast with the mouse, a high level of receptor gene expression was observed in the cerebellum of the rat. A number of rodent hindbrain sites expressing the leptin receptor gene are activated by circulating leptin and may form a monitoring/signaling pathway to complement more direct hypothalamic interactions.

OBJECTIVE

Morbid obesity may be accompanied by diabetes and painful diabetic neuropathy, a poorly understood condition that is manifested by mechanical or thermal allodynia and hyperalgesia. Recent studies have highlighted the importance of T-type calcium channels (T-channels) in peripheral nociception; therefore, our goal was to examine the function of these channels in the pathophysiology and development of painful diabetic neuropathy.

METHODS

In vivo testing of mechanical and thermal sensation, morphometric peripheral nerve studies, and electrophysiological and biochemical measurements were used to characterize the role of T-channels and the development of painful diabetic neuropathy in leptin-deficient (ob/ob) mice.

RESULTS

We found that ob/ob mice developed significant mechanical and thermal hypersensitivity early in life that coincided with hyperglycemia and was readily reversed with insulin therapy. These disturbances were accompanied by significant biophysical and biochemical modulation of T-channels in dorsal root ganglion neurons as measured by a large increase in the amplitude of T-currents and the expression of mRNA. The most prevalent subtype, alpha1H (Ca(v)3.2), was most strongly affected. Moreover, (3beta,5alpha,17beta)-17-hydroxyestrane-3-carbonitrile (ECN), a novel neuroactive steroid and selective T-channel antagonist, provided dose-dependent alleviation of neuropathic thermal and mechanical hypersensitivity in diabetic ob/ob mice.

CONCLUSIONS

Our results indicate that pharmacological antagonism of T-channels is potentially an important novel therapeutic approach for the management of painful diabetic neuropathy.

Olfactory sensory neuron (OSN) axons coalesce to form the olfactory nerve (ON) and then grow from the olfactory epithelium to the olfactory bulb (OB), enter the olfactory nerve layer (ONL), reorganize extensively, and innervate specific glomeruli. Within the ON and ONL a population of glial cells, the olfactory ensheathing cells (OECs), surround OSN axon fascicles. To better understand the relationship between OECs and axon fascicles in the ONL of the adult mouse, we used confocal microscopy and antibodies to the low affinity nerve growth factor receptor p75 (p75), glial fibrillary acidic protein (GFAP), neuropeptide Y (NPY), and S-100 to identify glia. Antibodies to olfactory marker protein (OMP) and neuronal cell adhesion molecule (NCAM) were used to identify OSN axons. Electron microscopy characterized the ONL ultrastructure. We found that glial processes were not uniformly distributed in the ONL of the mouse. The p75(+) OEC processes were restricted to the ON and the outer ONL sublamina, and oriented parallel to the plane of the OB layers. In the inner ONL NPY(+) OEC-like processes were seen. GFAP(+) processes were restricted to the inner ONL sublamina, the ONL/GL boundary, and the GL, where they delineated loosely aggregated axon fascicles that entered the glomeruli obliquely. S-100(+) processes and somata were distributed throughout the ONL; the outer and inner ONL were equivalent in their S-100 staining. Ultrastructural studies showed that, although OECs could be identified in both the outer and inner ONL, in the latter, their relationship to bundles of OEC axons appeared less orderly than seen in the outer ONL. Our data demonstrate a differential organization of the ONL that could subserve distinct functions; axon extension may occur predominantly in the outermost ONL, whereas glomerular targeting occurs in the inner sublamina of the ONL.