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Current thalassemia gene therapy protocols require the collection of hematopoietic stem/progenitor cells (HSPCs), in vitro culture, lentivirus vector transduction, and retransplantation into myeloablated patients. Because of cost and technical complexity, it is unlikely that such protocols will be applicable in developing countries, where the greatest demand for a [beta]-thalassemia therapy lies. We have developed a simple in vivo HSPC gene therapy approach that involves HSPC mobilization and an intravenous injection of integrating HDAd5/35++ vectors. Transduced HSPCs homed back to the bone marrow, where they persisted long-term. HDAd5/35++ vectors for in vivo gene therapy of thalassemia had a unique capsid that targeted primitive HSPCs through human CD46, a relatively safe SB100X transposase-based integration machinery, a micro-LCR-driven [gamma]-globin gene, and an MGMT(P140K) system that allowed for increasing the therapeutic effect by short-term treatment with low-dose [O.sup.6]-benzylguanine plus bis-chloroethylnitrosourea. We showed in 'healthy' human CD46-transgenic mice and in a mouse model of thalassemia intermedia that our in vivo approach resulted in stable [gamma]-globin expression in the majority of circulating red blood cells. The high marking frequency was maintained in secondary recipients. In the thalassemia model, a near-complete phenotypic correction was achieved. The treatment was well tolerated. This cost-efficient and 'portable' approach could permit a broader clinical application of thalassemia gene therapy., Introduction Thalassemia is one of the most common inherited diseases in humans worldwide (1), resulting from absent ([beta]O/[beta]O) or deficient ([beta]+/[beta]+) [beta]-globin chain synthesis. Approximately 60,000 children are born annually [...]

Long-term consumption of high-protein (HP) diets at 35% of energy is postulated to negatively influence bone health. Previous studies have not comprehensively examined the biochemical, physical, and biomechanical properties of bone required to arrive at this conclusion. Our objective in this study was to examine the Iong-term effect of a HP diet on bone metabolism, mass, and strength in rats. Adult female Sprague-Dawley rats (n = 80) were randomized to receive for 4, 8, 12, or 17 mo a normal-protein (NP) control diet (15% of energy) ora HP diet (35% of energy). Diets were balanced for calcium because the protein sources were rich in calcium. At each time point, measurements included weight, body composition, and bone mass using dual-energy X-ray absorptiometry, mechanical strength at the mid-diaphysis of femur and tibia, microarchitecture of femurs using microcomputerized tomography and serum osteocalcin, carboxy-terminal crosslinks of type I collagen (CTX), insulin-like growth factor-1 (IGF-1), leptin, and adiponectin. Effects of diet, time, and their interaction were tested using factorial ANOVA. The HP diet resulted in Iower body weight, total body, and abdominal fat and higher lean mass. Serum leptin and adiponectin were greater in HP-fed than in NP-fed rats, but IGF-1 did not differ between the groups. Whereas the HP diet resulted in higher relative bone mineral content (g/kg)in the femur, tibia, and vertebrae, serum osteocalcin and CTX and bone internal architecture and biomechanical strength were unaffected. In conclusion, HP diets at 35% of energy Iower body fat content without hindering the mechanical and weight-bearing properties of bone. J. Nutr. 139: 2099-2105, 2009. doi: 10.3945/jn.109.106377

BACKGROUND: Postnatal lead exposure in children and animals produces alterations in the visual system primarily characterized by decreases in the rod-mediated (scotopic) electroretinogram (ERG) amplitude (subnormality). In contrast, low-level gestational Pb exposure (GLE) increases the amplitude of scotopic ERGs in children (supernormality). OBJECTIVES: The goal of this study was to establish a rat model of human equivalent GLE and to determine dose-response effects on scotopic ERGs and on retinal morphology, biochemistry, and dopamine metabolism in adult offspring. METHODS: We exposed female Long-Evans hooded rats to water containing 0, 27 (low), 55 (moderate), or 109 (high) ppm of Pb beginning 2 weeks before mating, throughout gestation, and until postnatal day (PND) 10. We measured maternal and litter indices, blood Pb concentrations (BPb), retinal Pb concentrations, zinc concentrations, and body weights. On PND90, we performed the retinal experiments. RESULTS: Peak BPb concentrations were < 1, 12, 24, and 46 [micro]g/dL in control, low-, moderate- and high-level GLE groups, respectively, at PNDs 0-10. ERG supernormality and an increased rod photoreceptor and rod bipolar cell neurogenesis occurred with low- and moderate-level GLE. In contrast, high-level GLE produced ERG subnormality, rod cell loss, and decreased retinal Zn levels. GLE produced dose-dependent decreases in dopamine and its utilization. CONCLUSIONS: Low- and moderate-level GLE produced persistent scotopic ERG supernormality due to an increased neurogenesis of cells in the rod signaling pathway and/or decreased dopamine utilization, whereas high-level GLE produced rod-selective toxicity characterized by ERG subnormality. The ERG is a differential and noninvasive biomarker of GLE. The inverted U-shaped dose-response curves reveal the sensitivity and vulnerability of the developing retina to GLE. KEY WORDS: bipolar cells, development, dopamine, electroretinograms, gestation, lead, neurogenesis, rod photoreceptors, scotopic, zinc. Environ Health Perspect 116:618-625 (2008). doi:10.1289/ehp.11268 available via http://dx.doi.org/ [Online 22 February 2008], The adverse effects of low-level developmental lead exposure [blood lead (BPb) [less than or equal to] 10 [micro]g/dL] on cognitive, auditory, and visual-motor function are well documented (Canfield et al. [...]

The National Toxicology Program (NTP) is currently reviewing its research portfolio as part of its efforts to implement the NTP Roadmap to achieve the NTP Vision for the 21st century. This review includes a recent workshop, "Hormonally Induced Reproductive Tumors--Relevance of Rodent Bioassays," held 22-24 May 2006, that was organized to determine the adequacy and relevance to human disease outcome of rodent models currently used in the 2-year bioassay for four types of hormonally induced reproductive tumors (ovary, mammary gland, prostate, and testis). In brief, none of the workshop&apos;s breakout groups felt the currently used models are sufficient. For some types of tumors such as prostate, no adequate animal models exist, and for others such as ovary, the predominant tumors in humans are of different cellular origins than those induced by chemicals in rodents. This inadequacy of current models also applies to the testis, although our more complete understanding of the responses of Leydig cells to hormonal changes in rats may prove predictive for effects in humans other than cancer. All breakout groups recommended that the NTP consider modifying its testing protocols (i.e., age at exposure, additional end points, etc.) and/or using alternative models (i.e., genetically engineered models, in vitro systems, etc.) to improve sensitivity. In this article we briefly review the workshop&apos;s outcome and outline some next steps forward in pursuing the workshop&apos;s recommendations. Breakout group reports and additional information on the workshop, including participants, presentations, public comments and background materials, are posted on the NTP website. Key words: animal models, breast, endocrine, hormone, mammary gland, National Toxicology Program, ovary, prostate, reproductive tumors, species differences, testis. Environ Health Perspect 115:1351-1356 (2007). doi:10.1289/ehp.10135 available via http://dx.doi.org/ [Online 5 July 2007], The National Toxicology Program (NTP) hosted the workshop "Hormonally Induced Reproductive Tumors--Relevance of Rodent Bioassays" on 22-24 May 2006 in Raleigh, North Carolina, to discuss the adequacy of rodent models [...]

BACKGROUND: Quantum dots (QDs) are autofluorescent semiconductor nanocrystals that can be used for in vivo biomedical imaging. However, we know little about their in vivo disposition and health consequences. OBJECTIVES: We assessed the tissue disposition and pharmacokinetics of QD705 in mice. METHODS: We determined quantitatively the blood and tissue kinetics of QD705 in mice after single intravenous (iv) injection at the dose of 40 pmol for up to 28 days. Inductively coupled plasma- mass spectrometry (ICP-MS) measurement of cadmium was the primary method of quantification of QD705. Fluorescence light microscopy revealed the localization of QD705 in tissues. RESULTS: Plasma half-life of QD705 in mice was short (18.5 hr), but ICP-MS analyses revealed QD705 persisted and even continued to increase in the spleen, liver, and kidney 28 days after an iv dose. Considerable time-dependent redistribution from body mass to liver and kidney was apparent between 1 and 28 days postdosing. The recoveries at both time points were near 100%; all QD705s reside in the body. Neither fecal nor urinary excretion of QD705 was detected appreciably in 28 days postdosing. Fluorescence microscopy demonstrated deposition of QD705 in the liver, spleen, and kidneys. CONCLUSION: Judging from the continued increase in the liver (29-42% of the administered dose), kidney (1.5-9.2%), and spleen (4.8-5.2%) between 1 and 28 days without any appreciable excretion, QD705 has a very long half-life, potentially weeks or even months, in the body and its health consequences deserve serious consideration. KEY WORDS: ICP-MS, ckinetics, nanoparticles, quantum dot 705, redistribution, mice. Environ Health Perspect 115:1339-1343 (2007). doi:10.1289/ehp.10290 available via http://dx.doi.org/ [Online 14 June 2007], Quantum dots (QDs) are semiconductor nanocrystals approximately 2-100 nm in diameter. Structurally, they have a metalloid core and a cap or shell (Dabbousi et al. 1997). Organic coatings can be [...]

NOD mice with knockout of both native insulin genes and a mutated proinsulin transgene, alanine at position B16 in preproinsulin (B16:A-dKO mice), do not develop diabetes. Transplantation of NOD islets, [...]

Over the last three decades, experiments in several mammalian species have shown that the febrile response to bacterial endotoxin is attenuated late in pregnancy. More recent evidence has established that the expression of nitric oxide synthase (NOS) enzymes is increased in the brain late in pregnancy. The current study investigated the possible role of brain nitric oxide in mediating the phenomenon of fever suppression. Core body temperature ([T.sub.b]) of near-term pregnant rats (day 19 and 20) was measured following inhibition of brain NOS and intraperitoneal injection of LPS (50 [micro]g/kg); they were compared with both day 15 pregnant and virgin animals. Intracerebroventricular injection with an inhibitor of NOS, [N.sup.G]-monomethyl-L-arginine citrate (L-NMMA; 280 [micro]g), in near-term pregnant rats restored the febrile response to LPS. As expected, near-term dams that received intracerebroventricular vehicle + IP LPS did not increase [T.sub.b], in contrast to the 1.0 [+ or -] 0.2[degrees]C rise in [T.sub.b] in dams treated with ICV L-NMMA + IP LPS (P < 0.01). In virgin females and day 15 pregnant controls receiving this treatment, the increases in [T.sub.b] were 1.5 [+ or -] 0.3[degrees]C and 1.6 [+ or -] 0.4[degrees]C, respectively. Thus, blockade of brain NOS restored the febrile response to LPS in near-term dams; at 5 h postinjection, [T.sub.b] was 60-70% of that observed in virgins and day 15 pregnant animals. Intracerebroventricular L-NMMA alone did not induce a significant change in [T.sub.b] in any group. These results suggest that the mechanism underlying the suppression of the febrile response in near-term pregnancy is mediated by nitric oxide signaling in the brain. nitric oxide synthase; [N.sup.G]-monomethyl-L-arginine citrate; lipopolysaccharide; core body temperature; thermoregulation doi: 10.1152/ajpregu.00032.2007

The signaling molecule nitric oxide (NO), first described as endothelium-derived relaxing factor (EDRF), acts as physiological activator of NO-sensitive guanylyl cyclase (NO-GC) in the cardio-vascular, gastrointestinal, and nervous systems. Besides NO-GC, other NO targets have been proposed; however, their particular contribution still remains unclear. Here, we generated mice deficient for [[beta].sub.1] subunit of NO-GC, which resulted in complete loss of the enzyme. GC-KO mice have a life span of 3-4 weeks but then die because of intestinal dysmotility; however, they can be rescued by feeding them a fiber-free diet. Apparently, NO-GC is absolutely vital for the maintenance of normal peristalsis of the gut. GC-KO mice show a pronounced increase in blood pressure, underlining the importance of NO in the regulation of smooth muscle tone in vivo. The lack of an NO effect on aortic relaxation and platelet aggregation confirms NO-GC as the only NO target regulating these two functions, excluding cGMP-independent mechanisms. Our knockout model completely disrupts the NO/cGMP signaling cascade and provides evidence for the unique role of NO-GC as NO receptor. cardiovascular | knockout mice | cGMP | platelet aggregation | smooth muscle relaxation

Proper functioning of the human circadian timing system is crucial to physical and mental health. Much of what we know about this system is based on experimental protocols that induce the desynchronization of behavioral and physiological rhythms within individual subjects, but the neural (or extraneural) substrates for such desynchronization are unknown. We have developed an animal model of human internal desynchrony in which rats are exposed to artificially short (22-h) light--dark cycles. Under these conditions, locomotor activity, sleep--wake, and slow-wave sleep (SWS) exhibit two rhythms within individual animals, one entrained to the 22-h light--dark cycle and the other free-running with a period >24 h ([tau]>24 h). Whereas core body temperature showed two rhythms as well, further analysis indicates this variable oscillates more according to the [tau]>24 h rhythm than to the 22-h rhythm, and that this oscillation is due to an activity-independent circadian regulation. Paradoxical sleep (PS), on the other hand, shows only one free-running rhythm. Our results show that, similarly to humans, (i) circadian rhythms can be internally dissociated in a controlled and predictable manner in the rat and (ii) the circadian rhythms of sleep--wake and SWS can be desynchronized from the rhythms of PS and core body temperature within individual animals. This model now allows for a deeper understanding of the human timekeeping mechanism, for testing potential therapies for circadian dysrhythmias, and for studying the biology of PS and SWS states in a neurologically intact model. suprachiasmatic

Noncoplanar polychlorinated biphenyls (PCBs) are widely dispersed in human environment and tissues. Here, an exemplar noncoplanar PCB was fed to rat dams during gestation and throughout three subsequent nursing weeks. Although the hearing sensitivity and brainstem auditory responses of pups were normal, exposure resulted in the abnormal development of the primary auditory cortex (A1). A1 was irregularly shaped and marked by internal nonresponsive zones, its topographic organization was grossly abnormal or reversed in about half of the exposed pups, the balance of neuronal inhibition to excitation for A1 neurons was disturbed, and the critical period plasticity that underlies normal postnatal auditory system development was significantly altered. These findings demonstrate that developmental exposure to this class of environmental contaminant alters cortical development. It is proposed that exposure to noncoplanar PCBs may contribute to common developmental disorders, especially in populations with heritable imbalances in neurotransmitter systems that regulate the ratio of inhibition and excitation in the brain. We conclude that the health implications associated with exposure to noncoplanar PCBs in human populations merit a more careful examination. autism | brain development | cortical maps | environment | sensory systems

BACKGROUND: Inhalation of diesel exhaust particles (DEPs) is characterized by lung injury and inflammation, with significant increases in the numbers of polymorphonuclear leukocytes and alveolar macrophages. This influx of cellular [...]

BACKGROUND: Ground corncob animal bedding and corn food products contain substances that disrupt endocrine function in rats. The disruptors were identified as isomeric mixtures of tetra-hydrofurandiols (THF-diols; 9,12-oxy-10,13-dihydroxyoctadecanoic acid and [...]

Objective: Electroconvulsive therapy (ECT) is a widely used and effective treatment for mood disorders and appears to have positive effects on the motor symptoms of Parkinson's disease (PD), improving motor [...]

Hyperosmotic intravenous infusions of NaCl are more potent for inducing drinking and vasopressin (AVP) secretion than equally osmotic solutions of glucose or urea. The fact that all three solutes increased cerebrospinal fluid osmolality and sodium concentration led the investigators to conclude that critical sodium receptors or osmoreceptors for stimulating drinking and AVP secretion were outside the blood-brain barrier (BBB) in the circumventricular organs (CVOs). We tested an obvious prediction of this hypothesis: that all three solutes should increase c-Fos-like immunoreactivity (Fos-ir) inside the BBB, but that only NaCl should increase Fos-ir in the CVOs. We gave intravenous infusions of 3.0 Osm/l NaCl, glucose, or urea to rats for 11 or 22 min at 0.14 ml/min and perfused the rats for assay of Fos-ir at 90 min. Controls received isotonic NaCl at the same volume. Drinking latency was measured, but water was then removed. Drinking consistently occurred with short latency during hyperosmotic NaCl infusions only. Fos-ir in the forebrain CVOs, the subfornical organ, and organum vasculosum laminae terminalis was consistently elevated only by hyperosmotic NaCl. However, all three hyperosmotic solutes potently stimulated Fos-ir in the supraoptic and paraventricular nuclei of the hypothalamus inside the BBB. Hyperosmotic NaCl greatly elevated Fos-ir in the area postrema, but even glucose and urea caused moderate elevations that may be related to volume expansion rather than osmolality. The data provide strong support for the conclusion that the osmoreceptors controlling drinking are located in the CVOs. area postrema; c-Fos; drinking; hypernatremia; organum vasculosum laminae terminalis; osmoreceptors; paraventricular nucleus of the hypothalamus; subfornical organ; supraoptic nucleus

The passive and active length-tension relationships of isolated rat mesenteric lymphatics (-150 [micro]m ID), and adjacent small arteries (~240 [micro]m) and veins (-275 [mciro]m) were compared under isometric conditions using a wire myograph. About 60% of the lymphatic vessels developed spontaneous contractions in physiological saline solution at nominal preload. To maximally activate smooth muscle, 145 mM [K.sup.+] + 5 X [10.sup.-5] M norepinephrine was used for arteries, and 145 mM [K.sup.+] + 1 x [10.sup.-6] M substance P was used for lymphatics and veins. In response, arteries exhibited monotonic force development to a plateau level, whereas lymphatics and veins showed biphasic force development, consisting of a transient force peak followed by partial relaxation to a plateau over ~5 min. The passive and the active length-tension curves were similar in shape among all three vessels. However, the maximal active tension of arteries (3.4 [+ or -] 0.42 mN/mm) was significantly greater than peak active tension (0.59 [+ or -] 0.04 mN/mm) or plateau tension (0.20 [+ or -] 0.04 mN/mm) in small veins and greater than peak active tension (0.34 [+ or -] 0.02 mN/mm) or plateau tension (0.21 [+ or -] 0.02 mN/mm) in lymphatics. Maximal active medial wall stress was similar between lymphatics and veins but was approximately fivefold higher in small arteries. For lymphatics, the pressure calculated from the optimal preload was significantly higher than that found previously in isobaric studies of isolated lymphatics, suggesting the capacity to operate at higher than normal pressures for increased responsiveness. Our results represent the first mechanical comparisons of arterial, venous, and lymphatic vessels in the same vasculature. vascular mechanics; wall stress; strain; isometric; preload; contraction

The brain and testis retain selenium better than other tissues during selenium deficiency. Studies of mice with selenoprotein P (Sepp1) dereted (Sepp[1.sup.-/-] mice) showed that brain and testis selenium revels are largely dependent on Sepp1. Therefore, we examined tissue selenium in mice fed varying amounts of selenium and in Sepp[1.sup.-/-] mice to characterize better the role(s) of Sepp1. Mice were fed a selenium-deficient diet for 8 wk supplemented with selenium as selenite from none to 0.25 mg/kg diet and tissue selenium was measured. Brain and testis maintained their selenium better than did liver, kidney, and muscle when dietary selenium was limiting but testis selenium fell sharply in the group fed the deficient diet. Brain retained its selenium well, even in the group fed the deficient diet. After intravenous injection of [sup.75]Se-Sepp1 into Sepp[1.sup.-/-] and Sepp[1.sup.+/+] mice, qualitative differences between brain and testis [sup.75]Se uptake were noted, further suggesting differences in their uptake of selenium from Sepp1. Finally, selenium was measured in brain regions of Sepp[1.sup.-/-] and Sepp[1.sup.+/+] mice fed the diet supplemented with 1 mg selenium/kg and Sepp[1.sup.+/+] mice fed the deficient diet. Deletion of Sepp1 and selenium deficiency each lowered selenium a similar amount in cortex, midbrain, brainstem, and cerebellum. Selenium in the hippocampus was lowered by deletion of Sepp1 but not by selenium deficiency. These results suggest that Sepp1 is more important for maintaining selenium in the hippocampus than in other brain regions. They also confirm the position of the brain at the apex of the organ selenium hierarchy.

We examined whether antigen-specific immune responses are lower in mice with protein energy malnutrition (PEM mice) compared with nourished (control) mice. The mechanisms underlying reduced antigen-specific immune responses of PEM mice were evaluated through analysis of the functional capacities of antigen-presenting dendritic cells (DC). PEM mice were produced by subjecting male C57BL/6 mice for 52 wk to a daily food intake equivalent to 70% of the mean amount consumed by the control mice that consumed food ad libitum. PEM mice and control mice were immunized with hepatitis B vaccine containing hepatitis B surface antigen (H BsAg) at 52 wk and hu moral and cellular immune responses to H BsAg were evaluated at 58 wk. Lymphoproliferative assays were performed to assess the functional capacities of lymphocytes and DC. After 52 wk of food restriction, PEM mice had a 49% lower body weight than controls, almost no subcutaneous fat, severe muscle wasting, and atrophied spleen. All control mice developed antibodies to HBsAg (anti-HBs) in the sera and HBsAg-specific lymphocytes in the spleen as a result of immunization with the hepatitis B vaccine. PEM mice, however, were almost unresponsive to immunization with the hepatitis B vaccine. In PEM mice, the numbers of spleen DC, the T lymphocyte stimulatory capacities of DC, and their production of IL-12p70 and I FN-[gamma], was less than those of control mice (P < 0.05). We suggest that chronic undernutrition disrupts antigen- specific immune responses and that this disruption can be attributed at least in part to reduced frequencies and impaired functions of DC.

Neural prostheses for restoring lost functions can benefit from selective activation of nerves with limited number and density of electrodes. Here, we show by simulations and animal experiments that multlpoint simultaneous stimulation with a surface electrode array can selectively activate nerves in a bundle at a desired location in between the array or at a desired depth, which are referred to as lateral or depth-wise gating stimulation, respectively. The stimulation broadly generates action potentials with cathodic source electrodes, and simultaneously blocks unnecessary propagatian with downstream anodie gate electrodes. In general, stimulation with a small diameter electrode can affect a nearly hemispherical region, while a large electrode is effective at a more vertically compressed region, i.e., a surface of nerve bundle. The gating stimulation takes advantage of the size effects by utilizing an asymmetrical electrode array. The array of the lateral gating stimulation is designed to have four electrodes; a pair of large source electrodes and a pair of small gate electrodes. The depth-wise gating stimulation array consists of two electrodes; a large gate and small source electrodes. The simulation first demonstrated that appropriate combination of currents at the source and gate electrodes can change recruitment patterns of nerves with lateral or depth-wise selectivity as desired. We then applied the lateral gating stimulation an the rat spinal cords and obtained a preliminary support for the feasibility. Index Terms--Extracellular stimulation, functional electrical stimulation (FES), neural prosthesis, , rat, spinal cord.

The stochastic state point process filter (SSPPF) and steepest descent point process filter (SDPPF) are adaptive filter algorithms for state estimation from point process observations that have been used to track neural receptive field plasticity and to decode the representations of biological signals in ensemble neural spiking activity. The SSPPF and SDPPF are constructed using, respectively, Gaussian and steepest descent approximations to the standard Bayes and Chapman-Kolmogorov (BCK) system of filter equations. To extend these approaches for constructing point process adaptive filters, we develop sequential Monte Carlo (SMC) approximations to the BCK equations in which the SSPPF and SDPPF serve as the proposal densities. We term the two new SMC point process filters SMC-PPFs and SMC-PPFD, respectively. We illustrate the new filter algorithms by decoding the wind stimulus magnitude from simulated neural spiking activity in the cricket cercal system. The SMC-PPFs and SMC-PPFD provide more accurate state estimates at low number of particles than a conventional bootstrap SMC filter algorithm in which the state transition probability density is the proposal density. We also use the SMC-PPFs algorithm to track the temporal evolution of a spatial receptive field of a rat hippocampal neuron recorded while the animal foraged in an open environment. Our results suggest an approach for constructing point process adaptive filters using SMC methods. Index Terms--Adaptive filtering, hidden Markov models, point processes, sequential Monte Carlo, state estimation.

Abstract Acute administration of the psychotomimetic phencyclidine (PCP) can mimic some features of schizophrenia, while a repeated treatment regimen of PCP may provide a more effective way to model in [...]

We rescued the embryonic lethality of global PPAR[gamma] knockout by breeding Mox2-Cre (MORE) mice with floxed PPAR[gamma] mice to inactivate PPAR[gamma] in the embryo but not in trophoblasts and created [...]

Neutropenia and neutrophil dysfunction are common in many diseases, although their etiology is often unclear. Previous views held that there was a single ER enzyme, glucose-6-phosphatase-[alpha] (G6Pase-[alpha]), whose activity--limited to [...]

In humans, inactivating mutations in the gene of the thyroid hormone transporter monocarboxylate transporter 8 (MCT8; SLC16A2) lead to severe forms of psychomotor retardation combined with imbalanced thyroid hormone serum [...]

Adult mice carrying a null mutation of the prostanoid receptor EP3R (EP3[R.sup.-/-] mice) exhibit increased frequency of feeding during the light cycle of the day and develop an obese phenotype under a normal fat diet fed ad libitum. EP3[R.sub.-/-] mice show increased motor activity, which is not sufficient to offset the increased feeding leading to increased body weight. Altered 'nocturnal' activity and feeding behavior is present from a very early age and does not seem to require age-dependent factors for the development of obesity. Obesity in EP3[R.sub.-/-] mice is characterized by elevated leptin and insulin levels and >20% higher body weight compared with WT littermates. Abdominal and subcutaneous fat and increased liver weight account for the weight increase in EP3[R.sub.-/-] mice. These observations expand the roles of prostaglandin [E.sub.2] signaling in metabolic regulation beyond the reported stimulation of leptin release from adipose tissue to involve actions mediated by EP3R in the regulation of sleep architecture and feeding behavior. The findings add to the growing literature on links between inflammatory signaling and obesity. body weight | insulin | leptin | prostanoid receptor | signaling

Adult stem cells represent unique populations of undifferentiated cells with self-renewal capacity. In many tissues, stem cells divide less often than their progeny. It has been widely speculated, but largely untested, that their undifferentiated and quiescent state may make stem cells more efficient as donors for cloning by nuclear transfer (NT). Here, we report the use of nuclei from hair follicle stem cells and other skin keratinocytes as NT donors. When keratinocyte stem cells (KSCs) were used as NT donors, 19 liveborn mice were obtained, 9 of which survived to adulthood. Embryonic keratinocytes and cumulus cells also gave rise to cloned mice. Although cloning efficiencies were similar ( adult stemcells | embryos | cloning | keratinocyte | nuclear transfer

Time-delay circuitries in which a transcription factor processes independent input parameters can modulate NF-[kappa]B activation, manage quorum-sensing cross-talk, and control the circadian clock. We have constructed a synthetic mammalian gene network that processes four different input signals to control either immediate or time-delayed transcription of specific target genes. BirA-mediated ligation of biotin to a biotinylation signal-containing VP16 transactivation domain triggers heterodimerization of chimeric VP16 to a streptavidin-linked tetracycline repressor (TetR). At increasing biotin concentrations up to 20 nM, TetR-specific promoters are gradually activated (off to on, input signal 1), are maximally induced at concentrations between 20 nM and 10 [micro]M, and are adjustably shut off at biotin levels exceeding 10 [micro]M (on to off, input signal 2). These specific expression characteristics with a discrete biotin concentration window emulate a biotin-triggered bandpass filter. Removal of biotin from the culture environment (input signal 3) results in time-delayed transgene expression until the intracellular biotinylated VP16 pool is degraded. Because the TetR component of the chimeric transactivator retains its tetracycline responsiveness, addition of this antibiotic (input signal 4) overrides biotin control and immediately shuts off target gene expression. Biotin-responsive immediate, bandpass filter, and time-delay transcription characteristics were predicted by a computational model and have been validated in standard cultivation settings or biopharmaceutical manufacturing scenarios using trangenic CHO-K1 cell derivatives and have been confirmed in mice. Synthetic gene circuitries provide insight into structure-function correlations of native signaling networks and foster advances in gene therapy and biopharmaceutical manufacturing. biological circuit | biopharmaceutical manufacturing | gene switch | synthetic biology | biotin

CASK is an evolutionarily conserved multidomain protein composed of an N-terminal [Ca.sup.2+]/calmodulin-kinase domain, central PDZ and SH3 domains, and a C-terminal guanylate kinase domain. Many potential activities for CASK have been suggested, including functions in scaffolding the synapse, in organizing ion channels, and in regulating neuronal gene transcription. To better define the physiological importance of CASK, we have now analyzed CASK 'knockdown' mice in which CASK expression was suppressed by [approximately equal to] 70%, and CASK knockout (KO) mice, in which CASK expression was abolished. CASK knockdown mice are viable but smaller than WT mice, whereas CASK KO mice die at first day after birth. CASK KO mice exhibit no major developmental abnormalities apart from a partially penetrant cleft palate syndrome. In CASK-deficient neurons, the levels of the CASK-interacting proteins Mints, Veli/ Mals, and neurexins are decreased, whereas the level of neuroligin 1 (which binds to neurexins that in turn bind to CASK) is increased. Neurons lacking CASK display overall normal electrical properties and form ultrastructurally normal synapses. However, glutamatergic spontaneous synaptic release events are increased, and GABAergic synaptic release events are decreased in CASK-deficient neurons. In contrast to spontaneous neurotransmitter release, evoked release exhibited no major changes. Our data suggest that CASK, the only member of the membrane-associated guanylate kinase protein family that contains a [Ca.sup.2+]/calmodulin-dependent kinase domain, is required for mouse survival and performs a selectively essential function without being in itself required for core activities of neurons, such as membrane excitability, [Ca.sup.2+]-triggered presynaptic release, or postsynaptic receptor functions. CaM kinase | MAGUK | neurexin | neurotransmitter release | synapse

Fragile X syndrome is a common heritable form of mental retardation in humans. Recent neuroanatomical studies indicate an apparent immature appearance of neurons in fragile X syndrome patients and fragile X mental retardation protein (FMRP)-knockout mice, an animal model of this condition. In this work, we investigated possible alterations in synaptic plasticity in the neocortex of FMRP-knockout mice. Extracellular field potentials were recorded from the deep-layer visual neocortex. Long-term potentiation (LTP) was severely attenuated in brain slices from knockout mice relative to that observed in slices from wild-type mice. Considering that neocortical LTP can involve both NMDA receptor-dependent and -independent mechanisms, we attempted to distinguish the nature of LTP attenuated in the knockout condition. In slices from wild-type mice, LTP was partially attenuated by the NMDA receptor antagonist 3-[([+ or -])-2-carboxypiperazin-4-yl]-propyl-1-phosphate (CPP); however, the general metabotropic glutamate receptor (mGluR) antagonist [alpha]-methyl-4-carboxyphenylglycine (MCPG) strongly attenuated LTP, resulting in a response indistinguishable from that observed in slices from knockout mice. The selective mGluRs antagonist 2-methyl-6-(phenylethynyl)-pyridine (MPEP) attenuated LTP to a similar degree as did MCPG in wild-type slices, but MPEP did not alter the reduced potentiation in knockout slices. Our results suggest that LTP in layer V visual neocortex depends primarily on mGlu[R.sub.5] activation. Our data also indicate that mGlu[R.sub.5]mediated synaptic plasticity is absent in the neocortex of FMRP-knockout mice. Such an alteration may contribute to the cognitive and learning deficits exhibited in these mice as well as in fragile X syndrome. fragile X mental retardation protein | long-term potentiation | synaptic plasticity | visual cortex

There is emerging evidence for the existence of secretory pathways for superoxide dismutase (SOD1) mutants linked to amyotrophic lateral sclerosis (ALS) and for neurotoxicity of extracellular mutant SOD1. This evidence led us to test immunization protocols aiming to reduce the burden of extracellular SOD1 mutants in nervous tissue of mice models of ALS, by using bacterially purified recombinant SOD1 mutant protein as an immunogen. First, a vaccination was tested on a G37R SOD1 mouse strain with late-onset disease exhibiting levels of mutant SOD1 protein at 4-fold higher than normal SOD1 levels. Repeated injections of adjuvant/ SOD1 mutant with a final booster injection before symptoms at 6 months of age were effective in delaying disease onset and extending the life span of G37R SOD1 mice by >4 weeks. Western blot analysis with a monoclonal antibody specific to mutant SOD1 forms provided evidence of clearance of SOD1 species in the spinal cord of vaccinated G37R SOD1 mice. In contrast, this vaccination approach failed to confer significant protection in G93A SOD1 mice with extreme overexpression of mutant SOD1. Nonetheless, a passive immunization through intraventricular infusion of purified anti-human SOD1 antibody with osmotic minipump succeeded in alleviating disease symptoms and prolonging the life span of G93A SOD1 mice. From these results, we propose that immunization strategies should be considered as potential avenues for treatment of familial ALS caused by SOD1 mutations. vaccination | immunotherapy | antibody

Type I diabetes in both humans and nonobese diabetic (NOD) mice results from autoreactive T cell destruction of insulin-producing cells. Cure of type 1 diabetes may require both reversal of autoimmunity and regeneration of [beta] cells. Induction of chimerism via allogeneic hematopoietic cell transplantation has been shown to reestablish tolerance in both prediabetic and diabetic NOD mice. However, it is unclear whether this therapy augments [beta] cell regeneration. Furthermore, this procedure usually requires total body irradiation conditioning of recipients. The toxicity of total body irradiation conditioning and potential for graft-versus-host disease (GVHD) limit the application of allogeneic hematopoietic cell transplantation for treating type 1 diabetes. Here we report that injection of donor bone marrow and [CD4.sup.+] T cell-depleted spleen cells induced chimerism without causing GVHD in overtly diabetic NOD mice conditioned with anti-CD3/CD8 and that induction of chimerism in new-onset diabetic NOD mice led to elimination of insulitis, regeneration of host [beta] cells, and reversal of hyperglycemia. Therefore, this radiation-free GVHD preventive approach for induction of chimerism may represent a viable means for reversing type 1 diabetes. hematopoietic cell transplantation | type 1 diabetes | anti-CD3-conditioning | reversal of diabetes | reversal of autoimmunity

Mast cells are immune sentinels that participate in the defense against bacteria and parasites. Resident within the joint, mast cells become activated in human rheumatoid arthritis and are implicated in the pathogenesis of experimental murine synovitis. However, their arthritogenic role remains undefined. Using a model of autoantibody-induced arthritis, we show that mast cells contribute to the initiation of inflammation within the joint by elaboration of IL-1. Mast cells become activated to produce this cytokine via the IgG immune complex receptor Fc[gamma]/RIII. Interestingly, mast cells become dispensable for the perpetuation of arthritis after delivery of IL-1, highlighting the contribution of this lineage to arthritis induction. These findings illuminate a mechanism by which mast cells can participate in the pathogenesis of autoimmune inflammatory arthritis and provide insights of potential relevance to human rheumatoid arthritis. cytokine | mouse model | synovitis

Radiotracer kinetic modeling in small animals with Positron Emission Tomography (PET) requires the determination of the blood tracer concentration as a function of time. A continuous blood counting system was designed to measure the input function from rats and mice in real time. The system consists of a flow-through [beta] counter made of silicon PIN photodiodes and a [micro]I syringe pump. The latter draws blood continuously from an implanted venous or arterial catheter, at a user selected rate. The direct/3 detection by photodiodes minimizes the shield footprint next to the animal and reduces the counter sensitivity to ambient gamma radiation. The device is entirely remote controlled for sampling protocol selection, tuning, and real time monitoring of measured parameters. It can be hooked to a computer or fully integrated with the LabPET[TM] scanner for blood counting during dynamic PET imaging experiments. The counter sensitivity to the most popular PET radioisotopes ([sup.18]F,[sup.84]Cu,[sup.13]N, [sup.11]C) ranges from 7.1 to 46.8 Bq/[micro]. Its linearity is better than 98% up to 46 kBq/[micro]l for [sup.18]F, and a 1.4 s dispersion constant was measured at a rate of 250 [micro]1/min with rat whole blood in a PE10 catheter. Due to its optimized mechanical design and compact shielding, the counter sensitivity to radioactive background is only 5 counts per second (cps) for a 37 MBq [sup.18]F source 10 cm away from the detector. Accurate time-activity curves have been obtained from rats and mice in dynamic PET imaging studies. Index Terms--Beta [beta] detector, blood sampling, pharmacokinetic modeling, small animal PET.

A deficiency in essential fatty acid metabolism has been reported in diabetes. Nutritional supplementations with (n-6) or (n-3) PUFA have differential efficiency on parameters of diabetic neuropathy, including nerve conduction velocity (NCV) and nerve blood flow (NBF). The aim of this study was to compare the neuroprotective effects of [gamma]-linolenic acid (GLA)-lipoic acid (LA) conjugate (GLA-LA) and docosahexaenoic acid (DHA)-enriched phospholipids (PL) supplementations on NCV and NBF. Streptozotocin-induced diabetic (D) and control (C) rats were supplemented for 8 wk with either DHA-enriched PL at a dose of 30 mg * [kg.sup.-1] * [d.sup.-1] (DDHA and CDHA) or with corn oil enriched with GLA-LA at a dose of 30 mg * [kg.sup.-1] * [d.sup.-1] (DGLA and CGLA). Moreover, a C and D group received no supplementation. After 8 wk, NCV (-30%) and NBF (-50%) were lower in the D group than in the C group. Supplementation with GLA-LA totally prevented the decrease in NCV and NBF in the DGLA group, in which values did not differ from group C. Supplementation with DHA only partially prevented the decrease in NCV in the DDHA group, in which value was different from groups C and D and did not affect NBF. We conclude that at the low doses used, supplementation with GLA-LA is more effective than supplementation with DHA in preventing experimental diabetic neuropathy. The difference could be due in part to an antioxidant protective effect of LA on GLA.

Glutathione (GSH) concentration affects cell proliferation and apoptosis in intestinal and other cell lines in vitro. However, in vivo data on gut mucosal GSH redox status and cell turnover are limited. We investigated the effect of altered GSH redox status on the ileal mucosa in a rat model of short bowel syndrome following massive small bowel resection (SBR). Rats underwent 80% mid-jejunoileal resection (RX) or small bowel transection (TX; as operative controls), with administration of either saline or D, L-buthionine-sulfoximine (BSO), a specific inhibitor of cellular GSH synthesis. Ileal mucosal redox, morphology, and indices of cell proliferation and apoptosis were determined at different days after surgery. Ileal GSH redox status was assessed by GSH and GSH disulfide (GSSG) concentrations and the redox potential of GSH/GSSG ([E.sub.h]). Ileal lipid peroxidation [free malondialdehyde (MDA)] was measured as an index of lipid peroxidation. BSO markedly decreased ileal mucosal GSH, oxidized GSH/GSSG [E.sub.h], and increased MDA content without inducing morphological damage as assessed by light or electron microscopy. As expected, SBR stimulated adaptive growth of ileal villus height and total mucosal height at 7 d after surgery, but this response was unaffected by BSO treatment despite a modest increase in crypt cell apoptosis. Ileal cell proliferation (crypt cell bromodeoxyuridine incorporation) increased at 2 d after SBR but was unaffected by BSO. Collectively, our in vivo data show that marked depletion of ileal GSH and oxidation of the GSH redox pool does not alter indices of ileal epithelial proliferation or SBR-induced ileal mucosal adaptive growth.

Sleep is one of the most noticeable and widespread phenomena occurring in multicellular animals. Nevertheless, no consensus for a theory of its origins has emerged. In particular, no explicit, quantitative theory exists that elucidates or distinguishes between the myriad hypotheses proposed for sleep. Here, we develop a general, quantitative theory for mammalian sleep that relates many of its fundamental parameters to metabolic rate and body size. Several mechanisms suggested for the function of sleep can be placed in this framework, e.g., cellular repair of damage caused by metabolic processes as well as cortical reorganization to process sensory input. Our theory leads to predictions for sleep time, sleep cycle time, and rapid eye movement time as functions of body and brain mass, and it explains, for example, why mice sleep [approximately equal to] 14 hours per day relative to the 3.5 hours per day that elephants sleep. Data for 96 species of mammals, spanning six orders of magnitude in body size, are consistent with these predictions and provide strong evidence that time scales for sleep are set by the brain's, not the whole-body, metabolic rate. allometric scaling | brain | cellular repair | metabolic rate | sleep times

Notch-mediated induction of Nodal at the vertebrate node is a critical step in initiating left-right (LR) asymmetry. In mice and zebrafish we show that Baf60c, a subunit of the Swi/Snf-like BAF chromatin remodeling complex, is essential for establishment of LR asymmetry. Baf60c knockdown mouse embryos fail to activate Nodal at the node and also have abnormal node morphology with mixing of crown and pit cells. In cell culture, Baf60c is required for Notch-dependent transcriptional activation and functions to stabilize interactions between activated Notch and its DNA-binding partner, RBP-J. Brg1 is also required for these processes, suggesting that BAF complexes are key components of nuclear Notch signaling. We propose a critical role for Baf60c in Notch-dependent transcription and LR asymmetry. chromatin | Swi/Snf | node

Myelomeningocele (MMC) is the most common cause of neurogenic bladder dysfunction (NBD). We recently developed a novel retinoic acid (RA)-induced MMC model in fetal rats. The objective of this study was to use this model to assess functional and structural characteristics of the detrusor muscle in MMC-associated NBD. Time-dated pregnant Sprague-Dawley rats were gavage fed 60 mg/kg RA dissolved in olive oil or olive oil alone [embryonic day 10 (E10)]. Bladder specimens from olive oil-exposed fetuses (OIL; n = 71), MMC (n = 79), and RA-exposed-no MMC (RA, n = 62) were randomly assigned for functional and histopathological evaluation and protein analysis. Contractility responses to field and agonist-mediated stimulation (KCl and bethanecol) were analyzed. The expression patterns of [alpha]-smooth muscle actin, myosin, desmin, vimentin, and collagen III and I were analyzed by immunohistochemistry and Western blotting. Spatial and temporal distribution of nerve fibers within the detrusor muscle was monitored by neurotubulin-[beta]-III throughout gestation. Neither OIL, MMC, nor RA detrusor responded to field stimulation. MMC bladder strips showed a significant decrease in contractility after KCl and bethanechol stimulation compared with OIL and RA bladders. Bladder detrusor morphology and expression patterns of smooth muscle markers were similar between groups. Detrusor muscles in OIL and RA fetuses were densely innervated, possessing abundant intramural ganglia and nerve trunks that branch to supply smooth muscle bundles. In MMC bladders, neurotubulin-[beta]-III-positive nerve fibers were markedly decreased with advancing gestational age and were almost completely absent at term (E22). We conclude that the biomechanical properties of fetal rat MMC bladders are analogous to that seen in humans with MMC-associated NBD. Decreased nerve density indicates loss of peripheral neural innervation throughout gestation. The early observation of decreased innervation and decreased contractility in the absence of morphologic abnormalities in muscle structure or extracellular matrix supports a pathophysiological hypothesis that denervation is the primary insult preceding the observed alterations in bladder muscle structure and function. all-trans retinoic acid; contractility

Proximal tubules were isolated from control and acidotic rats by collagenase digestion and Percoll density gradient centrifugation. Western blot analysis indicated that the tubules were ~95% pure. The samples were analyzed by two-dimensional difference gel electrophoresis (DIGE) and DeCyder software was used to quantify the temporal changes in proteins that exhibit enhanced or reduced expression. The mass-to-charge ratios and the amino acid sequences of the recovered tryptic peptides were determined by MALDI-TOF/TOF mass spectrometry and the proteins were identified using Mascot software. This analysis confirmed the well-characterized adaptive responses in glutaminase (GA), glutamate dehydrogenase (GDH), and phosphoenolpyruvate carboxykinase (PEPCK). This approach also identified 17 previously unrecognized proteins that are increased with ratios of 1.5 to 5.6 and 16 proteins that are decreased with ratios of 0.67 to 0.03 when tubules from 7-day acidotic vs. control rats were compared. Some of these changes were confirmed by Western blot analysis. Temporal studies identified proteins that were induced either with rapid kinetics similar to PEPCK or with more gradual profiles similar to GA and GDH. All of the mRNAs that encode the latter proteins contain an AU sequence that is homologous to the pH response element found in GA mRNA. Thus selective mRNA stabilization may be a predominant mechanism by which protein expression is increased in response to acidosis. difference gel electrophoresis; mass spectrometry; phosphoenolpyruvate carboxykinase; glutaminase; mRNA stabilization

Brain/kidney (B/K) protein is a novel double C2-like-domain protein that is highly expressed in rat brain and kidney, but its cellular localization and functional role in the kidney are still undetermined. We examined the cellular localization of B/K protein in the rat kidney under normal and ischemic conditions. Ischemia-reperfusion (I/R) injury was induced by clamping both renal arteries for 45 rain, and animals were killed at 1 and 6 h and 1, 2, 3, 5, 7, 14, and 28 days after the reperfusion. Kidney tissues were processed for immunohistochemistry and immunoblot analyses using rabbit anti-B/K polyclonal antibodies. In control kidneys, B/K protein was expressed primarily in distal tubules including the thick ascending limb, distal convoluted and connecting tubules, and collecting duct. Notably, B/K protein was also expressed in the straight portion (S3 segment), but not in the S 1 or S2, of proximal tubules, and podocytes of the glomerulus. In rat kidneys with I/R injury, expression of B/K protein was differentially regulated according to the anatomic location. In distal tubules, overall expression of B/K protein was markedly decreased. On the other hand, I/R injury significantly increased B/K protein expression in the S3 segment of the outer medulla as well as in the rat proximal tubular epithelial cell line NRK-52E in vitro. Furthermore, B/K protein was strongly expressed in many exfoliated cells in the lumen and urine. These findings suggest that B/K protein is closely associated with cell death in proximal tubules, which are vulnerable to I/R injury in the kidney. brain/kidney protein; cell death

By crossing mice with expression of Cre recombinase under control of the endogenous renin promoter (Sequeira Lopez ML, Pentz ES, Nomasa T, Smithies O, Gomez RA. Dev Cell 6: 719-728, 2004) with mice in which exon 1 of the Gnas gene was flanked by loxP sites (Chen M, Gavrilova O, Liu J, Xie T, Deng C, Nguyen AT, Nackers LM, Lorenzo J, Shen L, Weinstein LS. Proc Natl Acad Sci USA), we generated animals with preferential and nearly complete excision of Gs[alpha] in juxtaglomemlar granular (JG) cells. Compared with wild-type animals, mice with conditional Gs[alpha] deficiency had markedly reduced basal levels of renin expression and very low plasma renin concentrations. Furthermore, the acute release responses to furosemide, hydralazine, and isoproterenol were virtually abolished. Consistent with a state of primary renin depletion, Gs[alpha]-deficient mice had reduced arterial blood pressure, reduced levels of aldosterone, and a low glomemlar filtration rate. Renin content and renin secretion of JG cells in primary culture were drastically reduced, and the stimulatory response to the addition of [PGE.sub.2] or isoproterenol was eliminated. Unexpectedly, Gs[alpha] recombination was also observed in the renal medulla, and this was associated with a vasopressin-resistant concentrating defect. Our study shows that Cre recombinase under control of the renin promoter can be used for the excision of floxed targets from JG cells. We conclude that Gs[alpha]-mediated signal transduction is essential and nonredundant in the control of renin synthesis and release. juxtaglomerular granular cell culture; furosemide; hydralazine; aldosterone; cyclooxygenase 2; urine concentration

Gravidas with obesity and diabetes ('diabesity') may transmit this syndrome to their children through genetic and nongenetic mechanisms. Here, we used the [Lepr.sup.db/+] diabese mouse to examine the magnitude of these transmission modes, focusing on adipose tissue (AT). We compared the following six groups: wild-type (+/+) offspring from +/+ or db/+ dams (different early life environment) and db/+ offspring from db/+ dams, fed a standard or high-fat diet. Weight gain (0-8 wk) was higher in +/+ offspring from db/+ vs. +/+ mothers, and even higher in db/+ vs. +/+ offspring from db/+ mothers. In addition, we observed a stepwise increase in AT and adipocyte size in +/+ from +/+ mice, +/+ from db/+ mice, and db/+ mice at 8 wk. Differences in weight and adiposity between +/+ offspring from db/+ vs. +/+ dams were more pronounced in males than in females. Leptin and apelin mRNA levels in white and brown AT were higher in +/+ offspring from db/+ vs. +/+ dams; however, leptin, apelin, and tumor necrosis factor-or expression were boosted more robustly in db/+ offspring. The high-fat diet amplified AT differences between db/+ vs. +/+ offspring from db/+ dams, but not between +/+ offspring from db/+ vs. +/+ dams. Moreover, db/+ but not +/+ offspring from db/+ mothers were insulin-resistant and hyperinsulinemic after a glucose challenge. In conclusion, the genetic transmission of the diabesity phenotype clearly prevailed, but the early-life diabesity environment had discernible effects on postnatal weight gain as well as on adipocyte size and adipokine expression at a postpubertal age. apelin; leptin

HIV infection is associated with abnormal lipid metabolism, body fat redistribution, and altered energy expenditure. The pathogenesis of these complex abnormalities is unclear. Viral protein R (Vpr), an HIV-1 accessory protein, can regulate gene transcription mediated by the glucocorticoid receptor and peroxisome proliferator-activated receptor-[gamma] and affect mitochondrial function in vitro. To test the hypothesis that expression of Vpr in liver and adipocytes can alter lipid metabolism in vivo, we engineered mice to express Vpr under control of the phosphoenolpyruvate carboxykinase promoter in a tissue-specific and inducible manner and investigated the effects of dietary fat, indinavir, and dexamethasone on energy metabolism and body composition. The transgenic mice expressed Vpr mRNA in white and brown adipose tissues and liver and immunoaffinity capillary electrophoresis revealed that they had free Vpr protein in the plasma. Compared with wild-type (WT) animals, Vpr mice had lower plasma triglyceride levels after 6 wk (P < 0.05) but not after 10 wk of a high-fat diet and lower plasma cholesterol levels after 10 wk of high-fat diet (P < 0.05). Treatment with dexamethasone obviated group differences, whereas indinavir had no significant independent effect on lipids. In the fasted state, Vpr mice had a higher respiratory quotient than WT mice (P < 0.05). These data provide the first in vivo evidence that HIV-1 Vpr expressed at low levels in adipose tissues and liver can 1) circulate in the blood, 2) regulate lipid and fatty acid metabolism, and 3) alter fuel selection for oxidation in the fasted state. human immunodeficiency virus lipodystrophy; glucose; triglycerides; cholesterol; energy expenditure; fat oxidation

Abnormal autonomic nerve traffic has been associated with a number of peripheral neuropathies and cardiovascular disorders prompting the development of genetically altered mice to study the genetic and molecular components of these diseases. Autonomic function in mice can be assessed by directly recording sympathetic nerve activity. However, murine sympathetic spikes are typically detected using a manually adjusted voltage threshold and no unsupervised detection methods have been developed for the mouse. Therefore, we tested the performance of several unsupervised spike detection algorithms on simulated murine renal sympathetic nerve recordings, including an automated amplitude discriminator and wavelet-based detection methods which used both the discrete wavelet transform (DWT) and the stationary wavelet transform (SWT) and several wavelet threshold rules. The parameters of the wavelet methods were optimized by comparing basal sympathetic activity to postmortem recordings and recordings made during pharmacological suppression and enhancement of sympathetic activity. In general, SWT methods were found to outperform amplitude discriminators and DWT methods with similar wavelet coefficient thresholding algorithms when presented with simulations with varied mean spike rates and signal-to-noise ratios. A SWT method which estimates the noise level using a 'noise-only' wavelet scale and then selectively thresholds scales containing the physiologically important signal information was found to have the most robust spike detection. The proposed noise-level estimation method was also successfully validated during pharmacological interventions. Index Terms--Denoising, mice, spike detection, sympathetic nerve activity, wavelet.

Experiments were performed to test the postulate that exercise training (ExT) improves the blunted renal excretory response to acute volume expansion (VE), in part, by normalizing the neural component of the volume reflex typically observed in chronic heart failure (HF). Diuretic and natriuretic responses to acute VE were examined in sedentary and ExT groups of rats with either HF or sham-operated controls. Experiments were performed in anesthetized (Inactin) rats 6 wk after coronary ligation surgery. Histological data indicated that there was a 34.9 [+ or -] 3.0% outer and 42.5 [+ or -] 3.2% inner infarct of the myocardium in the HF group. Sham rats had no observable damage to the myocardium. In sedentary rats with HF, VE produced a blunted diuresis (46% of sham) and natriuresis (35% of sham) compared with sham-operated control rats. However, acute VE-induced diuresis and natriuresis in ExT rats with HF were comparable to sham rats and significantly higher than sedentary HF rats. Renal denervation abolished the salutary effects of ExT on renal excretory response to acute VE in HF. Since glomerular filtration rates were not significantly different between the groups, renal hemodynamic changes may not account for the blunted renal responses in rats with HF. Additional experiments confirmed that renal sympathetic nerve activity responses to acute VE were blunted in sedentary HF rats; however, ExT normalized the renal sympathoinhibition in HF rats. These results confirm an impairment of neurally mediated excretory responses to acute VE in rats with HF. ExT restored the blunted excretory responses as well as the renal sympathoinhibitory response to acute VE in HF rats. Thus the beneficial effects of ExT on cardiovascular regulation in HF may be partly due to improvement of the neural component of volume reflex. sympathetic nerve activity; renal function; myocardial infarction

The adult proximal tubule is a low-resistance epithelium where there are high rates of both active transcellular and passive paracellular NaCl transport. We have previously demonstrated that the neonatal rabbit and rat proximal tubule have substantively different passive paracellular transport properties than the adult proximal tubule, which results in a maturational change in the paracellular passive flux of ions. Neonatal proximal tubules have a higher [P.sub.Na]/[P.sub.cl] ratio and lower chloride and bicarbonate permeabilities than adult proximal tubules. Claudins are a large family of proteins which are the gate keepers of the paracellular pathway, and claudin isoform expression determines the permeability characteristics of the paracellular pathway. Previous studies have shown that claudins 1, 2, 3, 4, 5, 7, 8, 10, 11, 12, 15, and 16 are expressed in the adult mouse kidney. To determine whether there are developmental claudin isoforms, we compared the claudin isoforms present in the neonatal and adult kidney using RT-PCR to detect mRNA of claudin isoforms. Claudin 6, claudin 9, and claudin 13 were either not expressed or barely detectable in the adult mouse kidney using traditional PCR, but were expressed in the neonatal mouse kidney. Using real-time RT-PCR, we were able to detect a low level of claudin 6 mRNA expression in the adult kidney compared with the neonate, but claudin 9 and claudin 13 were only detected in the neonatal kidney. There was the same maturational decrease in these claudin proteins with Western blot analysis. Immunohistochemistry showed high levels of expression of claudin 6 in neonatal proximal tubules, thick ascending limb, distal convoluted tubules, and collecting ducts in a paracellular distribution but there was no expression of claudin 6 in the adult kidney. Using real-time RT-PCR claudin 6 and 9 mRNA were present in 1-day-old proximal convoluted tubules and were virtually undetectable in proximal convoluted tubules from adults. Claudin 13 was not detectable in neonatal or adult proximal convoluted tubules. In summary, we have identified developmentally expressed claudin isoforms, claudin 6, claudin 9, and claudin 13. These paracellular proteins may play a role in the maturational changes in paracellular permeability. renal tubule; renal development; paracellular pathway; neonatal kidney