Gradient Gel Research Articles

Short Communication: Polymicrobial community in teeth associated with severe early-childhood caries

This was to characterise the microbial diversity in the complex dental plaque of children with severe early-childhood caries (S-ECC), using the denaturing gel gradient electrophoresis (DGGE) technique. The DGGE technique was used as a diagnostic tool to analyse samples from the oral cavity of a patient with S-ECC. Dental plaque samples from a 3-year-old child with S-ECC were taken from the primary central maxillary incisor (biofilm on vestibular surface), primary maxillary molar (biofilm on vestibular surface), primary central maxillary incisor (dentine), primary maxillary molar (dentine) and saliva and then analysed by PCR-DGGE. Three bands occurred in all samples, moreover, 86% of similarity was observed in the pattern of bands between incisor and molar teeth biofilm samples, including four similar bands. DGGE is a valuable tool for differentiating the microbial composition of the oral plaque in S-ECC children.

Routing Misfolded Proteins through the Multivesicular Body (MVB) Pathway Protects against Proteotoxicity

The secretory pathway maintains multiple quality control checkpoints. Initially, endoplasmic reticulum-associated degradation pathways monitor protein folding to retain and eliminate aberrant products. Despite its broad client range, some molecules escape detection and traffic to Golgi membranes. There, a poorly understood mechanism termed Golgi quality control routes aberrant proteins for lysosomal/vacuolar degradation. To better understand Golgi quality control, we examined the processing of the obligate substrate Wsc1p. Misfolded Wsc1p does not use routes of typical vacuolar membrane proteins. Instead, it partitions into intralumenal vesicles of the multivesicular body (MVB) pathway, mediated by the E3 ubiquitin ligase Rsp5p. Its subsequent transport to the vacuolar lumen is essential for complete molecule breakdown. Surprisingly, the transport mode plays a second crucial function in neutralizing potential substrate toxicity. Eliminating the MVB sorting signal diverted molecules to the vacuolar limiting membrane, resulting in the generation of toxic by-products. These data demonstrate a new role of the MVB pathway in protein quality control.

Comparison of commercial DNA extraction kits for isolation and purification of bacterial and eukaryotic DNA from PAH-contaminated soils

Molecular characterization of the microbial populations of soils and sediments contaminated with polycyclic aromatic hydrocarbons (PAHs) is often a first step in assessing intrinsic biodegradation potential. However, soils are problematic for molecular analysis owing to the presence of organic matter, such as humic acids. Furthermore, the presence of contaminants, such as PAHs, can cause further challenges to DNA extraction, quantification, and amplification. The goal of our study was to compare the effectiveness of four commercial soil DNA extraction kits (UltraClean Soil DNA Isolation kit, PowerSoil DNA Isolation kit, PowerMax Soil DNA Isolation kit, and FastDNA SPIN kit) to extract pure, high-quality bacterial and eukaryotic DNA from PAH-contaminated soils. Six different contaminated soils were used to determine if there were any biases among the kits due to soil properties or level of contamination. Extracted DNA was used as a template for bacterial 16S rDNA and eukaryotic 18S rDNA amplifications, and PCR products were subsequently analyzed using denaturing gel gradient electrophoresis (DGGE). We found that the FastDNA SPIN kit provided significantly higher DNA yields for all soils; however, it also resulted in the highest levels of humic acid contamination. Soil texture and organic carbon content of the soil did not affect the DNA yield of any kit. Moreover, a liquid-liquid extraction of the DNA extracts found no residual PAHs, indicating that all kits were effective at removing contaminants in the extraction process. Although the PowerSoil DNA Isolation kit gave relatively low DNA yields, it provided the highest quality DNA based on successful amplification of both bacterial and eukaryotic DNA for all six soils. DGGE fingerprints among the kits were dramatically different for both bacterial and eukaryotic DNA. The PowerSoil DNA Isolation kit revealed multiple bands for each soil and provided the most consistent DGGE profiles among replicates for both bacterial and eukaryotic DNA.

Statins are active in acute lymphoblastic leukaemia (ALL): a therapy that may treat ALL and prevent avascular necrosis

Statins are active in acute lymphoblastic leukaemia (ALL): a therapy that may treat ALL and prevent avascular necrosis

Unforeseen decreases in dissolved oxygen levels affect tube formation kinetics in collagen gels

The availability of oxygen (O(2)) is a critical parameter affecting vascular tube formation. In this study, we hypothesize that dissolved oxygen (DO) levels in collagen gels change during the three-dimensional (3D) culture of human umbilical vein endothelial cells (HUVECs) in atmospheric conditions and that such changes affect the kinetics of tube formation through the production of reactive oxygen species (ROS). We demonstrate a decrease in O(2) tension during 3D cultures of HUVECs. Noninvasive measurements of DO levels during culture under atmospheric conditions revealed a profound decrease that reached as low as 2% O(2) at the end of 24 h. After media replacement, DO levels rose rapidly and equilibrated at ∼15% O(2), creating a reoxygenated environment. To accurately estimate DO gradients in 3D collagen gels, we developed a 3D mathematical model and determined the Michaelis-Menten parameters, V(max) and K(m), of HUVECs in collagen gels. We detected an increase in ROS levels throughout the culture period. Using diphenyliodonium to inhibit ROS production resulted in the complete inhibition of tube formation. Interference RNA studies further showed that hypoxia-inducible factors (HIFs)-1α and -2α are not involved in the formation of 3D tubes in collagen gels. We conclude that ROS affect the tubulogenesis process through HIFα-independent pathways, where the levels of ROS are influenced by the uncontrolled variations in DO levels. This study is the first demonstration of the critical and unexpected role of O(2) during 3D in vitro culture models of tubulogenesis in atmospheric conditions.

Laboratory assessment of biofilm process and its microbial characteristics for treating nonpoint source pollution

A biofilm process with the attached bacterial growth onto ceramic media was applied to remove carbonaceous and nitrogenous pollutants from nonpoint water source. The packing ratios of ceramic media were 0.05 and 0.15 (v/v). Thereafter, the reactors were operated intermittently in sequencing batch mode with different cycle periods: 0, 5, 10 and 15 d. The COD and NH4 +-N removal efficiencies were investigated under different operating conditions, such as media packing ratio, temperature and interevent period. Additionally, polymerase chain reaction (PCR)-denaturing gel gradient electrophoresis (DGGE) and INT-dehydrogenase activity (DHA) tests were conducted to observe the microbial community and activity in the biofilm. Consequently, the removal efficiency of the organic matter after 8 h remained stable, even with longer interevent periods, regardless of the packing ratio. The interevent period and packing ratio seemed to have no significant influence on the COD removal efficiency. However, stable nitrification efficiency, with longer interevent period, was only achieved with a packing ratio of 0.15. Therefore, a packing ratio above 0.15 was required to simultaneously achieve stable COD removal and nitrification efficiency. The DGGE profiles revealed that the prevalent microorganism species were changed from that of the seeded activated sludge into those detected in the sediments. Due to the prevalence of microorganisms related to the sediment, their activities did not decrease, even after a 15 d interevent period.

Functional Roles of a C-terminal Signaling Complex of CaV1 Channels and A-kinase Anchoring Protein 15 in Brain Neurons

Regulation of CaV1.2 channels in cardiac myocytes by the β-adrenergic pathway requires a signaling complex in which the proteolytically processed distal C-terminal domain acts as an autoinhibitor of channel activity and mediates up-regulation by the β-adrenergic receptor and PKA bound to A-kinase anchoring protein 15 (AKAP15). We examined the significance of this distal C-terminal signaling complex for CaV1.2 and CaV1.3 channels in neurons. AKAP15 co-immunoprecipitates with CaV1.2 and CaV1.3 channels. AKAP15 has overlapping localization with CaV1.2 and CaV1.3 channels in cell bodies and proximal dendrites and is closely co-localized with CaV1.2 channels in punctate clusters. The neuronal AKAP MAP2B, which also interacts with CaV1.2 and CaV1.3 channels, has complementary localization to AKAP15, suggesting different functional roles in calcium channel regulation. Studies with mice that lack the distal C-terminal domain of CaV1.2 channels (CaV1.2ΔDCT) reveal that AKAP15 interacts with neuronal CaV1.2 channels via their C terminus in vivo and is co-localized in punctate clusters of CaV1.2 channels via that interaction. CaV1.2ΔDCT neurons have reduced L-type calcium current, indicating that the distal C-terminal domain is required for normal functional expression in vivo. Deletion of the distal C-terminal domain impairs calcium-dependent signaling from CaV1.2 channels to the nucleus, as shown by reduction in phosphorylation of the cAMP response element-binding protein. Our results define AKAP signaling complexes of CaV1.2 and CaV1.3 channels in brain and reveal three previously unrecognized functional roles for the distal C terminus of neuronal CaV1.2 channels in vivo: increased functional expression, anchoring of AKAP15 and PKA, and initiation of excitation-transcription coupling.

Impact of chronic and acute pesticide exposures on periphyton communities

Aquatic ecosystems face variable exposure to pesticides, especially during floodings which are associated with short bursts of high contaminant concentrations that influence biological systems. A study was undertaken to highlight the impact of the herbicide diuron applied in mixture with the fungicide tebuconazole on natural periphyton during flooding events. Periphyton were grown in two series of two lotic outdoor mesocosms: one series was non-contaminated while the other was exposed to chronic contamination. After 4 weeks, one channel of each series was exposed to three successive pulses, with each pulse followed by one week of recovery. Impacts on periphyton were assessed by using Denaturing Gel Gradient Electrophoresis to characterize eukaryotic community structure. At a functional scale, photosynthetic efficiency was quantified during each pulse, and the induced tolerance to diuron was estimated by performing short-term inhibition tests based on photosynthetic efficiency. Moreover, pesticide concentrations in the water column and periphyton matrix were measured. Diuron was adsorbed in the periphyton during each pulse and desorbed 13 h after pulse end. The different pulses affected the eukaryotic community structures of the control biofilms, but not of the chronically exposed ones. During the first pulse, photosynthetic efficiency was correlated with pesticide concentration in the water phase, and there was no difference between periphyton from chronically contaminated channels and control channels. However, during the second and third pulses, the photosynthetic efficiency of periphyton chronically exposed to pesticides appeared to be less impacted by the acute pulsed exposure of pesticide. These changes were consistent with the acquisition of induced tolerance to diuron since only after the third pulse that periphyton from chronic channel became tolerant to diuron. Our experimental study indicates that the effects of pulsed acute exposures to pesticides on periphyton depended on whether the communities had previously been exposed to the same stressors or not.

Lipoprotein Lipase Is a Novel Amyloid β (Aβ)-binding Protein That Promotes Glycosaminoglycan-dependent Cellular Uptake of Aβ in Astrocytes

Lipoprotein lipase (LPL) is a member of a lipase family known to hydrolyze triglyceride molecules in plasma lipoprotein particles. LPL also plays a role in the binding of lipoprotein particles to cell-surface molecules, including sulfated glycosaminoglycans (GAGs). LPL is predominantly expressed in adipose and muscle but is also highly expressed in the brain where its specific roles are unknown. It has been shown that LPL is colocalized with senile plaques in Alzheimer disease (AD) brains, and its mutations are associated with the severity of AD pathophysiological features. In this study, we identified a novel function of LPL; that is, LPL binds to amyloid β protein (Aβ) and promotes cell-surface association and uptake of Aβ in mouse primary astrocytes. The internalized Aβ was degraded within 12 h, mainly in a lysosomal pathway. We also found that sulfated GAGs were involved in the LPL-mediated cellular uptake of Aβ. Apolipoprotein E was dispensable in the LPL-mediated uptake of Aβ. Our findings indicate that LPL is a novel Aβ-binding protein promoting cellular uptake and subsequent degradation of Aβ.

Microbial morphology and community structure in a suspended carrier biofilm reactor as a function of substrate loading rates

An aerobic suspended carrier biofilm reactor was efficient in simultaneous organic carbon and nitrogen removal, with COD removal efficiencies of 87.1–99.0% and simultaneous nitrification and denitrification (SND) efficiencies about 96.7-98.8%. The effects of substrate loading on microbial morphology and community structure were investigated by environmental scanning electron microscopy (ESEM), denaturing gel gradient electrophoresis (DGGE) and fluorescence in situ hybridization (FISH). Biofilms formed at different substrate loadings had different morphology and community structures. A higher substrate concentration resulted in denser and thinner biofilms, while a lower substrate concentration resulted in looser and thicker biofilms with significant presence of filamentous bacteria. Both sequence analysis of DGGE bands and FISH analysis indicated the dominance of β-Proteobacteria in the biofilm communities, especially Zoogloea. FISH analysis revealed that the relative abundance of β-proteobacteria ammonia oxidizing bacteria (AOB) was positively correlated with ammonium concentrations, whereas Nitrospira-like nitrite-oxidizing bacteria (NOB) were negatively affected by ammonia and nitrite concentrations. The presence of denitrifying bacteria, Hydrogenophaga spp.,Hyphomicrobium spp. and Rhizobium spp. suggested that not only the oxygen microgradients within the biofilm but also aerobic denitrifiers may be responsible for SND in the aerobic biofilm. Key words: Microbial community structure, microbial morphology, substrate loading, suspended carrier biofilm.

Poly(ADP-ribose) Polymerase-1 (PARP-1) Contributes to the Barrier Function of a Vertebrate Chromatin Insulator

The prototypic chromatin insulator cHS4 has proven effective in reducing silencing chromosomal position effects in a variety of settings. Most of this barrier insulator activity has been mapped to a 250-bp core region, as well as to several proteins that bind this region. However, recent studies from our laboratory demonstrated that an extended 400-bp core region of the cHS4 element is necessary to achieve full barrier insulator activity when used as a single copy in the context of recombinant gammaretroviral and lentiviral vectors. In this study, electrophoretic gel mobility shift assays revealed specific DNA-protein binding activities associated with the distal portion of this extended core region. Affinity purification and tandem mass spectrometry studies led to the identification of one of these proteins as poly(ADP-ribose) polymerase-1 (PARP-1). The identity of this binding activity as PARP-1 was subsequently verified by a variety of biochemical studies in vitro and by chromatin immunoprecipitation studies in vivo. Functional studies with gammaretroviral reporter vectors in cell lines and primary mouse bone marrow progenitor cultures showed that cHS4 barrier activity was abrogated upon mutation of the putative PARP-1-binding site or upon treatment with a PARP inhibitor, respectively. The barrier activity of the cHS4 element was also found to be abrogated in studies using bone marrow from Parp1-null mice. Taken together, this study demonstrates that binding of PARP-1 plays a key functional role in the barrier activity of the extended cHS4 insulator core element.

Structure of Dimeric F1F0-ATP Synthase

The structure of the dimeric ATP synthase from yeast mitochondria was analyzed by transmission electron microscopy and single particle image analysis. In addition to the previously reported side views of the dimer, top view and intermediate projections served to resolve the arrangement of the rotary c(10) ring and the other stator subunits at the F(0)-F(0) dimeric interface. A three-dimensional reconstruction of the complex was calculated from a data set of 9960 molecular images at a resolution of 27 Å. The structural model of the dimeric ATP synthase shows the two monomers arranged at an angle of ∼45°, consistent with our earlier analysis of the ATP synthase from bovine heart mitochondria (Minauro-Sanmiguel, F., Wilkens, S., and Garcia, J. J. (2005) Proc. Natl. Acad. Sci. U.S.A. 102, 12356-12358). In the ATP synthase dimer, the two peripheral stalks are located near the F(1)-F(1) interface but are turned away from each other so that they are not in contact. Based on the three-dimensional reconstruction, a model of how dimeric ATP synthase assembles to form the higher order oligomeric structures that are required for mitochondrial cristae biogenesis is discussed.

Rod/Zw10 Complex Is Required for PIASy-dependent Centromeric SUMOylation

SUMO conjugation of cellular proteins is essential for proper progression of mitosis. PIASy, a SUMO E3 ligase, is required for mitotic SUMOylation of chromosomal proteins, yet the regulatory mechanism behind the PIASy-dependent SUMOylation during mitosis has not been determined. Using a series of truncated PIASy proteins, we have found that the N terminus of PIASy is not required for SUMO modification in vitro but is essential for mitotic SUMOylation in Xenopus egg extracts. We demonstrate that swapping the N terminus of PIASy protein with the corresponding region of other PIAS family members abolishes chromosomal binding and mitotic SUMOylation. We further show that the N-terminal domain of PIASy is sufficient for centromeric localization. We identified that the N-terminal domain of PIASy interacts with the Rod/Zw10 complex, and immunofluorescence further reveals that PIASy colocalizes with Rod/Zw10 in the centromeric region. We show that the Rod/Zw10 complex interacts with the first 47 residues of PIASy which were particularly important for mitotic SUMOylation. Finally, we show that depletion of Rod compromises the centromeric localization of PIASy and SUMO2/3 in mitosis. Together, we demonstrate a fundamental mechanism of PIASy to localize in the centromeric region of chromosome to execute centromeric SUMOylation during mitosis.

Influence of Apolipoprotein (Apo) A-I Structure on Nascent High Density Lipoprotein (HDL) Particle Size Distribution

The principal protein of high density lipoprotein (HDL), apolipoprotein (apo) A-I, in the lipid-free state contains two tertiary structure domains comprising an N-terminal helix bundle and a less organized C-terminal domain. It is not known how the properties of these domains modulate the formation and size distribution of apoA-I-containing nascent HDL particles created by ATP-binding cassette transporter A1 (ABCA1)-mediated efflux of cellular phospholipid and cholesterol. To address this issue, proteins corresponding to the two domains of human apoA-I (residues 1-189 and 190-243) and mouse apoA-I (residues 1-186 and 187-240) together with some human/mouse domain hybrids were examined for their abilities to form HDL particles when incubated with either ABCA1-expressing cells or phospholipid multilamellar vesicles. Incubation of human apoA-I with cells gave rise to two sizes of HDL particles (hydrodynamic diameter, 8 and 10 nm), and removal or disruption of the C-terminal domain eliminated the formation of the smaller particle. Variations in apoA-I domain structure and physical properties exerted similar effects on the rates of formation and sizes of HDL particles created by either spontaneous solubilization of phospholipid multilamellar vesicles or the ABCA1-mediated efflux of cellular lipids. It follows that the sizes of nascent HDL particles are determined at the point at which cellular phospholipid and cholesterol are solubilized by apoA-I; apparently, this is the rate-determining step in the overall ABCA1-mediated cellular lipid efflux process. The stability of the apoA-I N-terminal helix bundle domain and the hydrophobicity of the C-terminal domain are important determinants of both nascent HDL particle size and their rate of formation.

Summer distribution and diversity of aerobic anoxygenic phototrophic bacteria in the Mediterranean Sea in relation to environmental variables

Aerobic anoxygenic phototrophic bacteria (AAP) represent an important fraction of bacterioplankton assemblages in various oceanic regimes. Although their abundance and distribution have been explored recently in diverse oceanic regions, the environmental factors controlling the population structure and diversity of these photoheterotrophic bacteria remain poorly understood. Here, we investigate the horizontal and vertical distributions and the genetic diversity of AAP populations collected in late summer throughout the Mediterranean Sea using pufM-temporal temperature gel gradient electrophoresis (TTGE) and clone library analyses. The TTGE profiles and clone libraries analyzed using multivariate statistical methods demonstrated a horizontal and vertical zonation of AAP assemblages. Physicochemical parameters such as pH, inorganic nitrogen compounds, photosynthetically active radiation, total organic carbon and to a lesser extent particulate organic nitrogen and phosphorus, and biogenic activities (e.g. bacterial production, cell densities), acted in synergy to explain the population changes with depth. About half of the pufM sequences were <94% identical to known sequences. The AAP populations were predominantly (~80%) composed of Gammaproteobacteria, unlike most previously explored marine systems. Our results suggest that genetically distinct ecotypes inhabiting different niches may exist in natural AAP populations of the Mediterranean Sea whose genetic diversity is typical of oligotrophic environments.

Biodegradation of methyl tert-butyl ether by newly identified soil microorganisms in a simple mineral solution

Methyl tert-butyl ether (MTBE) is a widely used fuel ether, which has become a soil and water contaminant. In this study, 12 microbial strains were isolated from gasoline-contaminated soils and selected because of their capacity to grow in MTBE. The strains were identified by 16S/ITS rDNA gene sequencing and screened for their ability to consume MTBE aerobically in a simple mineral solution. Solid phase microoextraction and gas chromatography were used to detect MTBE degradation. High levels of MTBE biodegradation were obtained using resting cells of the bacteria Achromobacter xylosoxidans MCM1/1 (78%), Enterobacter cloacae MCM2/1 (50%), and Ochrobactrum anthropi MCM5/1 (52%) and the fungus Exophiala dermatitidis MCM3/4 (14%). Our phylogenetic analysis clearly shows that bacterial MTBE biodegraders belong to the clade of Proteobacteria. For further insight, MTBE-degrader strains were profiled by denaturing gel gradient electrophoresis (DGGE) of PCR-amplified 16S rRNA gene sequences. This approach could be used to analyse microbial community dynamics in bioremediation processes.

The impact of zero-valent iron nanoparticles on a river water bacterial community

Zero-valent iron (ZVI) nanoparticles are of interest because of their many potential biomedical and environmental applications. However, these particles have recently been reported to be cytotoxic to bacterial cells. The overall objective of this study was to determine the impact of 100mg/L ZVI nanoparticles on the diversity and structure of an indigenous river water bacterial community. Response during exposure for 36 days was determined by denaturing gel gradient electrophoresis (DGGE) analysis of bacterial 16S rRNA genes, amplified from extracted DNA, and viable and total cell abundances were determined by plate counting and fluorescent microscopy of DAPI-stained cells. Changes in river water chemistry were also monitored. Addition of ZVI nanoparticles led to a rapid decrease in oxidation–reduction potential (ORP) (+196 to −281mV) and dissolved oxygen (DO) concentration (8.2–0.6mg/L), both of which stabilized during the experiment. Interestingly, both viable and total bacterial cell abundances increased and pH decreased, characteristic of an active microbial community. Total community structure was visualized using rank-abundance plots fitted with linear regression models. The slopes of the regression models were used as a descriptive statistic of changes in evenness over time. Importantly, despite bacterial growth, addition of ZVI nanoparticles did not influence bacterial community structure.

Proof of oligomeric state of frog rhodopsin: Visualization of dimer and oligomers on gels after BN- and HRCN-PAGE using antibodies to rhodopsin and by retinylopsin fluorescence

Staining by antibodies to rhodopsin (Rh) and fluorescence of N-retinylopsin (RO) have shown that digitonin (DIG)- , dodecyl-β-D-maltoside (DM)- , and sodium dodecyl sulfate (SDS)-solubilized frog Rh after BN- and HRCN-PAGE is situated in the gradient gel in the state of dimer with a slight content of higher oligomers (trimer, tetramer, etc.). With increasing detergent harshness (DIG < DM < SDS), the proportion of higher oligomers in extracts becomes more prominent. Formation of RO in rod outer segments (ROS) in the presence of 0.7 M NaBH(3)CN at pH5.0 occurs only when Rh is simultaneously photolyzed during reduction. Dithiothreitol at the concentration of 0.005M failed to induce RO production. Formation of a stable C-N bond between all-trans-retinal and opsin in RO is accompanied by decrease in the dimer share and increase in the share of the higher oligomers due to secondary dissociation-aggregation of solubilized opsin. The position of the Rh dimer in relation to the anode during both native electrophoreses is determined not only by its molecular mass, but probably also depends on unfolding degree (or form): the harsher the detergent, the closer to the anode the dimer is located. Treatment of ROS by agents modifying the cholesterol component of lipid membrane (MβCD, filipin III, nystatin, saponin) did not change the character of Rh oligomerization, thus showing that integrity of the cholesterol component of photoreceptor membrane is not a crucial factor for oligomerization of opsin. It is supposed that the dimer-oligomer "portrait" of frog Rh, which has been found by two methods of native electrophoresis in three detergents with different degree of harshness, corresponds to a physiological state of this protein in native photoreceptor membrane.

An intrinsic gut leptin-melanocortin pathway modulates intestinal microsomal triglyceride transfer protein and lipid absorption

Fat is delivered to tissues by apoB-containing lipoproteins synthesized in the liver and intestine with the help of an intracellular chaperone, microsomal triglyceride transfer protein (MTP). Leptin, a hormone secreted by adipose tissue, acts in the brain and on peripheral tissues to regulate fat storage and metabolism. Our aim was to identify the role of leptin signaling in MTP regulation and lipid absorption using several mouse models deficient in leptin receptor (LEPR) signaling and downstream effectors. Mice with spontaneous LEPR B mutations or targeted ablation of LEPR B in proopiomelanocortin (POMC) or agouti gene related peptide (AGRP) expressing cells had increased triglyceride in plasma, liver, and intestine. Furthermore, melanocortin 4 receptor (MC4R) knockout mice expressed a similar triglyceride phenotype, suggesting that leptin might regulate intestinal MTP expression through the melanocortin pathway. Mechanistic studies revealed that the accumulation of triglyceride in the intestine might be secondary to decreased expression of MTP and lipid absorption in these mice. Surgical and chemical blockade of vagal efferent outflow to the intestine in wild-type mice failed to alter the triglyceride phenotype, demonstrating that central neural control mechanisms were likely not involved in the observed regulation of intestinal MTP. Instead, we found that enterocytes express LEPR, POMC, AGRP, and MC4R. We propose that a peripheral, local gut signaling mechanism involving LEPR B and MC4R regulates intestinal MTP and controls intestinal lipid absorption.

Ribosomal 18 S RNA Processing by the IGF-I-responsive WDR3 Protein Is Integrated with p53 Function in Cancer Cell Proliferation

Insulin-like growth factor-I (IGF-I) signaling is strongly associated with cell growth and regulates the rate of synthesis of the rRNA precursor, the first and the key stage of ribosome biogenesis. In a screen for mediators of IGF-I signaling in cancer, we recently identified several ribosome-related proteins, including NEP1 (nucleolar essential protein 1) and WDR3 (WD repeat 3), whose homologues in yeast function in ribosome processing. The WDR3 gene and its locus on chromosome 1p12-13 have previously been linked with malignancy. Here we show that IGF-I induces expression of WDR3 in transformed cells. WDR3 depletion causes defects in ribosome biogenesis by affecting 18 S rRNA processing and also causes a transient down-regulation of precursor rRNA levels with moderate repression of RNA polymerase I activity. Suppression of WDR3 in cells expressing functional p53 reduced proliferation and arrested cells in the G(1) phase of the cell cycle. This was associated with activation of p53 and sequestration of MDM2 by ribosomal protein L11. Cells lacking functional p53 did not undergo cell cycle arrest upon suppression of WDR3. Overall, the data indicate that WDR3 has an essential function in 40 S ribosomal subunit synthesis and in ribosomal stress signaling to p53-mediated regulation of cell cycle progression in cancer cells.