Population Selection and Sequencing of Caenorhabditis elegans Wild Isolates Identifies a Region on Chromosome III Affecting Starvation Resistance.

To understand the genetic basis of complex traits, it is important to be able to efficiently phenotype many genetically distinct individuals. In the nematode Caenorhabditis elegans, individuals have been isolated from diverse populations around the globe and whole-genome sequenced. As a result, hundreds of wild strains with known genome sequences can be used for genome-wide association studies (GWAS). However, phenotypic analysis of these strains can be laborious, particularly for quantitative traits requiring multiple measurements per strain. Starvation resistance is likely a fitness-proximal trait for nematodes, and it is related to metabolic disease risk in humans. However, natural variation in C. elegans starvation resistance has not been systematically characterized, and precise measurement of the trait is time-intensive. Here, we developed a population-selection-and-sequencing-based approach to phenotype starvation resistance in a pool of 96 wild strains. We used restriction site-associated DNA sequencing (RAD-seq) to infer the frequency of each strain among survivors in a mixed culture over time during starvation. We used manual starvation survival assays to validate the trait data, confirming that strains that increased in frequency over time are starvation-resistant relative to strains that decreased in frequency. Further, we found that variation in starvation resistance is significantly associated with variation at a region on chromosome III. Using a near-isogenic line (NIL), we showed the importance of this genomic interval for starvation resistance. This study demonstrates the feasibility of using population selection and sequencing in an animal model for phenotypic analysis of quantitative traits, documents natural variation of starvation resistance in C. elegans, and identifies a genomic region that contributes to such variation.

Insulin/IGF Signaling and Vitellogenin Provisioning Mediate Intergenerational Adaptation to Nutrient Stress.

The roundworm C. elegans reversibly arrests larval development during starvation [1], but extended early-life starvation reduces reproductive success [2, 3]. Maternal dietary restriction (DR) buffers progeny from starvation as young larvae, preserving reproductive success [4]. However, the developmental basis of reduced fertility following early-life starvation is unknown, and it is unclear how maternal diet modifies developmental physiology in progeny. We show here that extended starvation in first-stage (L1) larvae followed by unrestricted feeding results in a variety of developmental abnormalities in the reproductive system, including proliferative germ-cell tumors and uterine masses that express neuronal and epidermal cell fate markers. We found that maternal DR and reduced maternal insulin/insulin-like growth factor (IGF) signaling (IIS) increase oocyte provisioning of vitellogenin lipoprotein, reducing penetrance of starvation-induced abnormalities in progeny, including tumors. Furthermore, we show that maternal DR and reduced maternal IIS reduce IIS in progeny. daf-16/FoxO and skn-1/Nrf, transcriptional effectors of IIS, are required in progeny for maternal DR and increased vitellogenin provisioning to suppress starvation-induced abnormalities. daf-16/FoxO activity in somatic tissues is sufficient to suppress starvation-induced abnormalities, suggesting cell-nonautonomous regulation of reproductive system development. This work reveals that early-life starvation compromises reproductive development and that vitellogenin-mediated intergenerational insulin/IGF-to-insulin/IGF signaling mediates adaptation to nutrient availability.

Developmental fidelity is imposed by genetically separable RalGEF activities that mediate opposing signals.

The six C. elegans vulval precursor cells (VPCs) are induced to form the 3°-3°-2°-1°-2°-3° pattern of cell fates with high fidelity. In response to EGF signal, the LET-60/Ras-LIN-45/Raf-MEK-2/MEK-MPK-1/ERK canonical MAP kinase cascade is necessary to induce 1° fate and synthesis of DSL ligands for the lateral Notch signal. In turn, LIN-12/Notch receptor is necessary to induce neighboring cells to become 2°. We previously showed that, in response to graded EGF signal, the modulatory LET-60/Ras-RGL-1/RalGEF-RAL-1/Ral signal promotes 2° fate in support of LIN-12. In this study, we identify two key differences between RGL-1 and RAL-1. First, deletion of RGL-1 confers no overt developmental defects, while previous studies showed RAL-1 to be essential for viability and fertility. From this observation, we hypothesize that the essential functions of RAL-1 are independent of upstream activation. Second, RGL-1 plays opposing and genetically separable roles in VPC fate patterning. RGL-1 promotes 2° fate via canonical GEF-dependent activation of RAL-1. Conversely, RGL-1 promotes 1° fate via a non-canonical GEF-independent activity. Our genetic epistasis experiments are consistent with RGL-1 functioning in the modulatory 1°-promoting AGE-1/PI3-Kinase-PDK-1-AKT-1 cascade. Additionally, animals lacking RGL-1 experience 15-fold higher rates of VPC patterning errors compared to the wild type. Yet VPC patterning in RGL-1 deletion mutants is not more sensitive to environmental perturbations. We propose that RGL-1 functions to orchestrate opposing 1°- and 2°-promoting modulatory cascades to decrease developmental stochasticity. We speculate that such switches are broadly conserved but mostly masked by paralog redundancy or essential functions.

Pervasive Positive and Negative Feedback Regulation of Insulin-Like Signaling in Caenorhabditis elegans.

The Caenorhabditis elegans insulin-like signaling network supports homeostasis and developmental plasticity. The genome encodes 40 insulin-like peptides and one known receptor. Feedback regulation has been reported, but the extent of feedback and its effect on signaling dynamics in response to changes in nutrient availability has not been determined. We measured messenger RNA expression for each insulin-like peptide, the receptor daf-2, components of the PI3K pathway, and its transcriptional effectors daf-16/FoxO and skn-1/Nrf at high temporal resolution during transition from a starved, quiescent state to a fed, growing state in wild type and mutants affecting daf-2/InsR and daf-16/FoxO. We also analyzed the effect of temperature on insulin-like gene expression. We found that most PI3K pathway components and insulin-like peptides are affected by signaling activity, revealing pervasive positive and negative feedback regulation at intra- and intercellular levels. Reporter gene analysis demonstrated that the daf-2/InsR agonist daf-28 positively regulates its own transcription and that the putative agonist ins-6 cross-regulates DAF-28 protein expression through feedback. Our results show that positive and negative feedback regulation of insulin-like signaling is widespread, giving rise to an organismal FoxO-to-FoxO signaling network that supports homeostasis during fluctuations in nutrient availability.

Neurohormonal signaling via a sulfotransferase antagonizes insulin-like signaling to regulate a Caenorhabditis elegans stress response.

Insulin and insulin-like signaling regulates a broad spectrum of growth and metabolic responses to a variety of internal and environmental stimuli. For example, the inhibition of insulin-like signaling in C. elegans mediates its response to both osmotic stress and starvation. We report that in response to osmotic stress the cytosolic sulfotransferase SSU-1 antagonizes insulin-like signaling and promotes developmental arrest. Both SSU-1 and the DAF-16 FOXO transcription factor, which is activated when insulin signaling is low, are needed to drive specific responses to reduced insulin-like signaling. We demonstrate that SSU-1 functions in a single pair of sensory neurons to control intercellular signaling via the nuclear hormone receptor NHR-1 and promote both the specific transcriptional response to osmotic stress and altered lysophosphatidylcholine metabolism. Our results show the requirement of a sulfotransferase-nuclear hormone receptor neurohormonal signaling pathway for some but not all consequences of reduced insulin-like signaling.

Food perception without ingestion leads to metabolic changes and irreversible developmental arrest in C. elegans.

BACKGROUND: Developmental physiology is very sensitive to nutrient availability. For instance, in the nematode Caenorhabditis elegans, newly hatched L1-stage larvae require food to initiate postembryonic development. In addition, larvae arrested in the dauer diapause, a non-feeding state of developmental arrest that occurs during the L3 stage, initiate recovery when exposed to food. Despite the essential role of food in C. elegans development, the contribution of food perception versus ingestion on physiology has not been delineated. RESULTS: We used a pharmacological approach to uncouple the effects of food (bacteria) perception and ingestion in C. elegans. Perception was not sufficient to promote postembryonic development in L1-stage larvae. However, L1 larvae exposed to food without ingestion failed to develop upon return to normal culture conditions, instead displaying an irreversible arrest phenotype. Inhibition of gene expression during perception rescued subsequent development, demonstrating that the response to perception without feeding is deleterious. Perception altered DAF-16/FOXO subcellular localization, reflecting activation of insulin/IGF signaling (IIS). The insulin-like peptide daf-28 was specifically required, suggesting perception in chemosensory neurons, where it is expressed, regulates peptide synthesis and possibly secretion. However, genetic manipulation of IIS did not modify the irreversible arrest phenotype caused by food perception, revealing that wild-type function of the IIS pathway is not required to produce this phenotype and that other pathways affected by perception of food in the absence of its ingestion are likely to be involved. Gene expression and Nile red staining showed that food perception could alter lipid metabolism and storage. We found that starved larvae sense environmental polypeptides, with similar molecular and developmental effects as perception of bacteria. Environmental polypeptides also promoted recovery from dauer diapause, suggesting that perception of polypeptides plays an important role in the life history of free-living nematodes. CONCLUSIONS: We conclude that actual ingestion of food is required to initiate postembryonic development in C. elegans. We also conclude that polypeptides are perceived as a food-associated cue in this and likely other animals, initiating a signaling and gene regulatory cascade that alters metabolism in anticipation of feeding and development, but that this response is detrimental if feeding does not occur.

Ral Signals through a MAP4 Kinase-p38 MAP Kinase Cascade in C. elegans Cell Fate Patterning.

C. elegans vulval precursor cell (VPC) fates are patterned by an epidermal growth factor (EGF) gradient. High-dose EGF induces 1° VPC fate, and lower dose EGF contributes to 2° fate in support of LIN-12/Notch. We previously showed that the EGF 2°-promoting signal is mediated by LET-60/Ras switching effectors, from the canonical Raf-MEK-ERK mitogen-activated protein (MAP) kinase cascade that promotes 1° fate to the non-canonical RalGEF-Ral that promotes 2° fate. Of oncogenic Ras effectors, RalGEF-Ral is by far the least well understood. We use genetic analysis to identify an effector cascade downstream of C. elegans RAL-1/Ral, starting with an established Ral binding partner, Exo84 of the exocyst complex. Additionally, RAL-1 signals through GCK-2, a citron-N-terminal-homology-domain-containing MAP4 kinase, and PMK-1/p38 MAP kinase cascade to promote 2° fate. Our study delineates a Ral-dependent developmental signaling cascade in vivo, thus providing the mechanism by which lower EGF dose is transduced.

daf-16/FoxO promotes gluconeogenesis and trehalose synthesis during starvation to support survival.

daf-16/FoxO is required to survive starvation in Caenorhabditis elegans, but how daf-16IFoxO promotes starvation resistance is unclear. We show that daf-16/FoxO restructures carbohydrate metabolism by driving carbon flux through the glyoxylate shunt and gluconeogenesis and into synthesis of trehalose, a disaccharide of glucose. Trehalose is a well-known stress protectant, capable of preserving membrane organization and protein structure during abiotic stress. Metabolomic, genetic, and pharmacological analyses confirm increased trehalose synthesis and further show that trehalose not only supports survival as a stress protectant but also serves as a glycolytic input. Furthermore, we provide evidence that metabolic cycling between trehalose and glucose is necessary for this dual function of trehalose. This work demonstrates that daf-16/FoxO promotes starvation resistance by shifting carbon metabolism to drive trehalose synthesis, which in turn supports survival by providing an energy source and acting as a stress protectant.

Insulin-like signalling to the maternal germline controls progeny response to osmotic stress.

In 1893 August Weismann proposed that information about the environment could not pass from somatic cells to germ cells, a hypothesis now known as the Weismann barrier. However, recent studies have indicated that parental exposure to environmental stress can modify progeny physiology and that parental stress can contribute to progeny disorders. The mechanisms regulating these phenomena are poorly understood. We report that the nematode Caenorhabditis elegans can protect itself from osmotic stress by entering a state of arrested development and can protect its progeny from osmotic stress by increasing the expression of the glycerol biosynthetic enzyme GPDH-2 in progeny. Both of these protective mechanisms are regulated by insulin-like signalling: insulin-like signalling to the intestine regulates developmental arrest, while insulin-like signalling to the maternal germline regulates glycerol metabolism in progeny. Thus, there is a heritable link between insulin-like signalling to the maternal germline and progeny metabolism and gene expression. We speculate that analogous modulation of insulin-like signalling to the germline is responsible for effects of the maternal environment on human diseases that involve insulin signalling, such as obesity and type-2 diabetes.

L1 arrest, daf-16/FoxO and nonautonomous control of post-embryonic development.

Post-embryonic development is governed by nutrient availability. L1 arrest, dauer formation and aging illustrate how starvation, anticipation of starvation and caloric restriction have profound influence on C. elegans development, respectively. Insulin-like signaling through the Forkhead box O transcription factor daf-16/FoxO regulates each of these processes. We recently reported that ins-4, ins-6 and daf-28 promote L1 development from the intestine and chemosensory neurons, similar to their role in dauer development. daf-16 functions cell-nonautonomously in regulation of L1 arrest, dauer development and aging. Discrepancies in daf-16 sites of action have been reported in each context, but the consensus implicates epidermis, intestine and nervous system. We suggest technical limitations of the experimental approach responsible for discrepant results. Steroid hormone signaling through daf-12/NHR is known to function downstream of daf-16 in control of dauer development, but signaling pathways mediating cell-nonautonomous effects of daf-16 in aging and L1 arrest had not been identified. We recently showed that daf-16 promotes L1 arrest by inhibiting daf-12/NHR and dbl-1/TGF-ß Sma/Mab signaling, two pathways that promote L1 development in fed larvae. We will review these results on L1 arrest and speculate on why there are so many signals and signaling centers regulating post-embryonic development.

Relationship Between Measures of Adiposity, Arterial Inflammation, and Subsequent Cardiovascular Events.

BACKGROUND: The objective of this study was to evaluate how different measures of adiposity are related to both arterial inflammation and the risk of subsequent cardiovascular events. METHODS AND RESULTS: We included individuals who underwent (18)F-fluorodeoxyglucose positron emission tomography/computed tomography imaging for oncological evaluation. Subcutaneous adipose tissue (SAT) volume, visceral adipose tissue (VAT) volume, and VAT/SAT ratio were determined. Additionally, body mass index, metabolic syndrome, and aortic (18)F-fluorodeoxyglucose uptake (a measure of arterial inflammation) were determined. Subsequent development of cardiovascular disease (CVD) events was adjudicated. The analysis included 415 patients with a median age of 55 (P25-P75: 45-65) and a median body mass index of 26.4 (P25-P75: 23.4-30.9) kg/m(2). VAT and SAT volume were significantly higher in obese individuals. VAT volume (r=0.290; P<0.001) and VAT/SAT ratio (r=0.208; P<0.001) were positively correlated with arterial inflammation. Thirty-two subjects experienced a CVD event during a median follow-up of 4 years. Cox proportional hazard models showed that VAT volume and VAT/SAT ratio were associated with CVD events (hazard ratio [95% confidence interval]: 1.15 [1.06-1.25]; P<0.001; 3.60 [1.88-6.92]; P<0.001, respectively). Body mass index, metabolic syndrome, and SAT were not predictive of CVD events. CONCLUSIONS: Measures of visceral fat are positively related to arterial inflammation and are independent predictors of subsequent CVD events. Individuals with higher measures of visceral fat as well as elevated arterial inflammation are at highest risk for subsequent CVD events. The findings suggest that arterial inflammation may explain some of the CVD risk associated with adiposity.

dbl-1/TGF-ß and daf-12/NHR Signaling Mediate Cell-Nonautonomous Effects of daf-16/FOXO on Starvation-Induced Developmental Arrest.

Nutrient availability has profound influence on development. In the nematode C. elegans, nutrient availability governs post-embryonic development. L1-stage larvae remain in a state of developmental arrest after hatching until they feed. This "L1 arrest" (or "L1 diapause") is associated with increased stress resistance, supporting starvation survival. Loss of the transcription factor daf-16/FOXO, an effector of insulin/IGF signaling, results in arrest-defective and starvation-sensitive phenotypes. We show that daf-16/FOXO regulates L1 arrest cell-nonautonomously, suggesting that insulin/IGF signaling regulates at least one additional signaling pathway. We used mRNA-seq to identify candidate signaling molecules affected by daf-16/FOXO during L1 arrest. dbl-1/TGF-ß, a ligand for the Sma/Mab pathway, daf-12/NHR and daf-36/oxygenase, an upstream component of the daf-12 steroid hormone signaling pathway, were up-regulated during L1 arrest in a daf-16/FOXO mutant. Using genetic epistasis analysis, we show that dbl-1/TGF-ß and daf-12/NHR steroid hormone signaling pathways are required for the daf-16/FOXO arrest-defective phenotype, suggesting that daf-16/FOXO represses dbl-1/TGF-ß, daf-12/NHR and daf-36/oxygenase. The dbl-1/TGF-ß and daf-12/NHR pathways have not previously been shown to affect L1 development, but we found that disruption of these pathways delayed L1 development in fed larvae, consistent with these pathways promoting development in starved daf-16/FOXO mutants. Though the dbl-1/TGF-ß and daf-12/NHR pathways are epistatic to daf-16/FOXO for the arrest-defective phenotype, disruption of these pathways does not suppress starvation sensitivity of daf-16/FOXO mutants. This observation uncouples starvation survival from developmental arrest, indicating that DAF-16/FOXO targets distinct effectors for each phenotype and revealing that inappropriate development during starvation does not cause the early demise of daf-16/FOXO mutants. Overall, this study shows that daf-16/FOXO promotes developmental arrest cell-nonautonomously by repressing pathways that promote larval development.

Ral and Rheb GTPase activating proteins integrate mTOR and GTPase signaling in aging, autophagy, and tumor cell invasion.

Diverse environmental cues converge on and are integrated by the mTOR signaling network to control cellular growth and homeostasis. The mammalian Tsc1-Tsc2 GTPase activating protein (GAP) heterodimer is a critical negative regulator of Rheb and mTOR activation. The RalGAPα-RalGAPß heterodimer shares sequence and structural similarity with Tsc1-Tsc2. Unexpectedly, we observed that C. elegans expresses orthologs for the Rheb and RalA/B GTPases and for RalGAPα/ß, but not Tsc1/2. This prompted our investigation to determine whether RalGAPs additionally modulate mTOR signaling. We determined that C. elegans RalGAP loss decreased lifespan, consistent with a Tsc-like function. Additionally, RalGAP suppression in mammalian cells caused RalB-selective activation and Sec5- and exocyst-dependent engagement of mTORC1 and suppression of autophagy. Unexpectedly, we also found that Tsc1-Tsc2 loss activated RalA/B independently of Rheb-mTOR signaling. Finally, RalGAP suppression caused mTORC1-dependent pancreatic tumor cell invasion. Our findings identify an unexpected crosstalk and integration of the Ral and mTOR signaling networks.

Arterial inflammation in bronchial asthma.

BACKGROUND: Bronchial asthma is a chronic inflammatory condition associated with increased cardiovascular (CV) events. Here, we assess arterial inflammation, using 18F-fluorodeoxyglucose positron emission tomography/computed tomography imaging (FDG-PET/CT), in patients with bronchial asthma and low to intermediate Framingham risk scores (FRS). METHODS: A total of 102 patients underwent FDG-PET/CT imaging for clinical indications. Thirty-four patients (mean age 54.9 ± 16.1) with mild asthma and no known atherosclerotic disease were compared to 2 non-asthmatic groups. The first control group (n = 34) were matched by age, gender, and FRS. The second control group (n = 34) had clinical atherosclerosis and were matched by gender. Thereafter, arterial FDG uptake on PET images was determined, while blinded to patient identifiers. RESULTS: Target-to-background-ratio (TBR) in the aorta was higher in asthmatics vs non-asthmatic FRS-matched controls (1.96 ± 0.26 vs 1.76 ± 0.20; P < .001). The aortic TBR remained elevated in asthmatics vs non-asthmatic controls after adjusting traditional CV risk factors (P < .001). An inverse correlation was observed between FDG uptake and lung function, FEV1 (P = .02) and peak flow (P = .03). CONCLUSIONS: Bronchial asthma is associated with increased arterial inflammation beyond that estimated by current risk stratification tools. Further studies are required to evaluate whether attenuation of systemic inflammation will decrease CV events.

Phenolic acids suppress adipocyte lipolysis via activation of the nicotinic acid receptor GPR109A (HM74a/PUMA-G).

Phenolic acids are found in abundance throughout the plant kingdom. Consumption of wine or other rich sources of phenolic acids, such as the "Mediterranean diet," has been associated with a lower risk of cardiovascular disease. The underlying mechanism(s), however, has remained unclear. Here, we show that many phenolic acids, including those from the hydroxybenzoic and hydroxycinnamic acid classes, can bind and activate GPR109A (HM74a/PUMA-G), the receptor for the antidyslipidemic agent nicotinic acid. In keeping with this activity, treatment with a number of phenolic acids, including cinnamic acid, reduces lipolysis in cultured human adipocytes and in fat pats isolated from wild-type mice but not from mice deficient of GPR109A. Oral administration of cinnamic acid significantly reduces plasma levels of FFA in the wild type but not in mice deficient of GPR109A. Activation of GPR109A by phenolic acids may thus contribute to a cardiovascular benefit of these plant-derived products.

MMP-2 mediates ethanol-induced invasion of mammary epithelial cells over-expressing ErbB2.

Ethanol is a tumor promoter and may enhance the metastasis of breast cancer. We have previously demonstrated that over-expression of ErbB2 promoted ethanol-mediated invasion of mammary epithelial cells and breast cancer cells. However, the underlying cellular/molecular mechanisms remain unknown. By gelatin zymography, we showed that over-expression of ErbB2 increased the production of matrix metalloproteinase-2 (MMP-2) and MMP-9 in human mammary epithelial cells (HB2). Transient or stable transfection of ErbB2 cDNA to HB2 cells upregulated the transcripts and the activity of the MMP-2/-9 gene promoter; the upregulation of MMP-2/-9 expression was mediated by p38 mitogen-activated protein kinase (p38 MAPK) and phosphatidylinositol 3-kinase (PI3K). Although ethanol, at physiologically relevant concentrations (100-400 mg/dl), did not affect the production of MMP-2/-9, it activated MMP-2 in HB2 cells over-expressing ErbB2 (HB2(ErbB2)), but not HB2 cells; it enhanced the cleavage of proform MMP-2 (72 kDa) to an active form (62 kDa). The activation was dependent on c-jun N-terminal kinases (JNKs) and reactive oxygen species (ROS). On the other hand, ethanol affected neither the expression nor the activation of MMP-9. Selective inhibitors of MMP-2 (SB-3CT and OA-Hy) and antioxidants significantly inhibited ethanol-stimulated invasion of HB2(ErbB2) cells. Furthermore, knocking down MMP-2 by small interference RNA also induced a partial blockage on ethanol-promoted invasion of HB2(ErbB2) cells. Thus, ethanol-stimulated invasion of cells over-expressing ErbB2 was mediated, at least partially, by MMP-2 activation.

Detection of biological threat agents by real-time PCR: comparison of assay performance on the R.A.P.I.D., the LightCycler, and the Smart Cycler platforms.

BACKGROUND: Rapid detection of biological threat agents is critical for timely therapeutic administration. Fluorogenic PCR provides a rapid, sensitive, and specific tool for molecular identification of these agents. We compared the performance of assays for 7 biological threat agents on the Idaho Technology, Inc. R.A.P.I.D., the Roche LightCycler, and the Cepheid Smart Cycler. METHODS: Real-time PCR primers and dual-labeled fluorogenic probes were designed to detect Bacillus anthracis, Brucella species, Clostridium botulinum, Coxiella burnetii, Francisella tularensis, Staphylococcus aureus, and Yersinia pestis. DNA amplification assays were optimized by use of Idaho Technology buffers and deoxynucleotide triphosphates supplemented with Invitrogen Platinum Taq DNA polymerase, and were subsequently tested for sensitivity and specificity on the R.A.P.I.D., the LightCycler, and the Smart Cycler. RESULTS: Limit of detection experiments indicated that assay performance was comparable among the platforms tested. Exclusivity and inclusivity testing with a general bacterial nucleic acid cross-reactivity panel containing 60 DNAs and agent-specific panels containing nearest neighbors for the organisms of interest indicated that all assays were specific for their intended targets. CONCLUSION: With minor supplementation, such as the addition of Smart Cycler Additive Reagent to the Idaho Technology buffers, assays for DNA templates from biological threat agents demonstrated similar performance, sensitivity, and specificity on all 3 platforms.

(D)-beta-Hydroxybutyrate inhibits adipocyte lipolysis via the nicotinic acid receptor PUMA-G.

As a treatment for dyslipidemia, oral doses of 1-3 grams of nicotinic acid per day lower serum triglycerides, raise high density lipoprotein cholesterol, and reduce mortality from coronary heart disease (Tavintharan, S., and Kashyap, M. L. (2001) Curr. Atheroscler. Rep. 3, 74-82). These benefits likely result from the ability of nicotinic acid to inhibit lipolysis in adipocytes and thereby reduce serum non-esterified fatty acid levels (Carlson, L. A. (1963) Acta Med. Scand. 173, 719-722). In mice, nicotinic acid inhibits lipolysis via PUMA-G, a Gi/o-coupled seven-transmembrane receptor expressed in adipocytes and activated macrophages (Tunaru, S., Kero, J., Schaub, A., Wufka, C., Blaukat, A., Pfeffer, K., and Offermanns, S. (2003) Nat. Med. 9, 352-355). The human ortholog HM74a is also a nicotinic acid receptor and likely has a similar role in anti-lipolysis. Endogenous levels of nicotinic acid are too low to significantly impact receptor activity, hence the natural ligands(s) of HM74a/PUMA-G remain to be elucidated. Here we show that the fatty acid-derived ketone body (D)-beta-hydroxybutyrate ((D)-beta-OHB) specifically activates PUMA-G/HM74a at concentrations observed in serum during fasting. Like nicotinic acid, (D)-beta-OHB inhibits mouse adipocyte lipolysis in a PUMA-G-dependent manner and is thus the first endogenous ligand described for this orphan receptor. These findings suggests a homeostatic mechanism for surviving starvation in which (D)-beta-OHB negatively regulates its own production, thereby preventing ketoacidosis and promoting efficient use of fat stores.

Developmental expression of matrix metalloproteinases 2 and 9 and their potential role in the histogenesis of the cerebellar cortex.

The development of the cerebellar cortex depends on intrinsic genetic programs and orchestrated cell-cell/cell-matrix interactions. Matrix metalloproteinases (MMPs) are proteolytic enzymes that play an important role in these interactions. MMP-2 and MMP-9 are involved in diverse neuronal functions including migration, process extension, and synaptic plasticity. We investigated the spatiotemporal pattern of expression/activity of MMP-2/MMP-9 in the developing cerebellum and their role in the histogenesis of the cerebellar cortex. The levels of transcripts of MMP-2/MMP-9 were measured with real-time quantitative polymerase chain reaction. An initial decrease in MMP-2/MMP-9 transcripts was observed between postnatal days 3 (PD3) and PD6, and the mRNA levels remained relatively constant thereafter. Zymographic analysis revealed that the expression/activity of MMP-2/MMP-9 persisted longer than their transcripts; the downregulation occurred around PD9, suggesting a mechanism of translational or post-translational regulation. The gelatinase activity was localized in the external granule layer (EGL) and the internal granule layer during PD3-PD12. The immunoreactivity of MMP-2 was mainly localized in the EGL, the Bergmann glial fibers, and the Purkinje cell layer (PCL), whereas MMP-9 immunoreactivity was detected intensively in the PCL and the extracellular space of the molecular layer. Expression of MMP-9 was relatively weak in the EGL. The immunoreactivity of MMP-2/MMP-9 became undetectable after PD21. A similar expression pattern of MMP-2/MMP-9 was observed in organotypic cerebellar slice cultures. Exposure of organotypic slices to a specific MMP-2/MMP-9 inhibitor significantly increased the thickness of the EGL and concurrently decreased the number of migrating granule neurons in the molecular layer. Thus, MMP-2 and MMP-9 play a role in the postnatal cerebellar morphogenesis.

Detection of the Bacillus anthracis gyrA gene by using a minor groove binder probe.

Identification of chromosomal markers for rapid detection of Bacillus anthracis is difficult because significant chromosomal homology exists among B. anthracis, Bacillus cereus, and Bacillus thuringiensis. We evaluated the bacterial gyrA gene as a potential chromosomal marker for B. anthracis. A real-time PCR assay was developed for the detection of B. anthracis. After analysis of the unique nucleotide sequence of the B. anthracis gyrA gene, a fluorescent 3' minor groove binding probe was tested with 171 organisms from 29 genera of bacteria, including 102 Bacillus strains. The assay was found to be specific for all 43 strains of B. anthracis tested. In addition, a test panel of 105 samples was analyzed to evaluate the potential diagnostic capability of the assay. The assay showed 100% specificity, demonstrating the usefulness of the gyrA gene as a specific chromosomal marker for B. anthracis.