text: Hunting Stories Story : Posted 12-18-2014 Kansas Accidental Opportunity John Butler was sitting in the "Hangover Stand,'' a name given to the stand just 50 yards from the clubhouse. For obvious reasons, it would be the first choice of anyone unable to get out of bed on time, but up in time to actually hunt. Read More Story : Posted 12-18-2014 What Is the Second Rut? Whether you hunt in the Northeast or Midwest, around this time of the year you will often hear hunters begin to talk about seeing the "second rut." Scrapes open up, and like in early November, bucks can be seen dogging does. Read More Story : Posted 12-17-2014 Nothing Like Deer Camp Deer camp is one of the most cherished traditions in the hunting community, and for good reason. It's a gathering place for multiple generations of family and friends, many of whom see each other only during that special time. Read More Story : Posted 12-16-2014 Sooner Quail Hunters Reporting Better Results Several hunters have reported that quail populations seem to be improved, and they are having success finding and harvesting bobwhites and scaled quail in some sections of Oklahoma. Read More Story : Posted 12-12-2014 Compassionate Convictions I recently caught a spotlighter who had hidden his rifle and light off in the weeds just before I encountered him. He was a young man about to get married and with his first child on the way. Read More Story : Posted 12-12-2014 Pleased to Meat You Although he doesn't hunt anymore, Andy Shaw saw more deer this fall than most. Of course, none were alive. Read More Story : Posted 12-10-2014 Targeting Rib-Eyes from the December Sky They are called "rib-eyes from the sky," the flocks of sandhill cranes that annually wing their way down the Central Flyway into Oklahoma, calling rhythmically with their eerie guttural sounds emitting from high above. Read More Story : Posted 12-05-2014 Indiana Rut: Hoosier Daddy? After spending three days at a deer camp for the opening of the Indiana rifle season, I am baffled that the Hoosier state is lost in this conversation. Read More Story : Posted 12-04-2014 'Weather' or Not You'll Bag a Buck There might not be any worse weather element I hate hunting in than wind. Whether it's because I can't hear anything else other than the wind, or because I've never had success when it's windy, the bottom line is when wind is in the forecast, I cringe. I almost feel defeated before the hunt even begins. Read More Story : Posted 12-02-2014 Give the Gift of the Outdoors Catalogs, emails and commercials touting must-have gifts this holiday season fill our mailboxes, flood our inboxes and splash across our television screens as the biggest shopping weekend of the year approaches. Read More Page 10 of 98 Prev ...78910111213... Next Categories News < Stories Recipes Tips Gear Videos Photo Galleries Viewer Photo Galleries Features Deer Season Outdoors 365 with Lynn Burkhead Everything Deer with Jeremy Flinn Short Stories by Larry Potterfield Opening Days Live Hunts Explore the United States. Find information about and activities within your state. Get Started


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OCEAN CITY — Concerns over the political involvement of committee members in today's debate for candidates in the city's upcoming municipal election have forced a change in the event's format.
The event, organized by the Ocean City Community Association, was originally designed to devote one hour to the five men vying for three open at-large council seats and an hour to the two candidates running for mayor. Following a specially convened OCCA board meeting late Monday, the organization informed the candidates that the format had been changed from question-and-answer to a meet-and-greet that would feature short speeches by the candidates.
The event will start at 6 p.m. with each of the five council candidates giving a five-minute speech. The remainder of the hour will be open to the audience approaching the candidates for one-on-one discussions. The format will be repeated at 7 p.m. with the two mayoral candidates.
The change in format was forced by the associations held by three of the four debate committee members. OCCA President Bob Barr is serving as incumbent council candidate Keith Hartzell's campaign manager, board member Drew Fasy is serving as incumbent Mayor Jay Gillian's campaign manager, and board member Jim Tweed is hosting a meet-and-greet for mayoral challenger Ed Price and council challenger Eric Sauder at his home one day after the debate.
Fasy resigned a while ago from his position on the debate committee, although the exact time he did so is unclear.
Barr, who originally solicited questions for the candidates to his personal email address, said he had sufficiently separated himself from the debate Q&A by forwarding any input he received to Tweed. Hartzell said last week that Barr was not taking an active part in the debate process and that Barr's involvement was primarily ceremonial. Also, the men said there would be an announcement prior to the start of the debate as to their affiliations with the candidates.
Tweed said last week that he had erected what he called a "firewall" between questions forwarded by Barr and Barr's candidate by putting those questions in the mayoral debate pool. The point became moot when Tweed's computer crashed over the weekend and he lost all his data for the debate. Additionally, the board felt that Tweed, by scheduling a coffee klatch for two candidates and allowing two yard signs on his property, presented a concern considering his role as the sole person charged with formulating and asking questions of the candidates.
The event has been beleaguered from the start, with an initial announcement that $10, representing the cost of OCCA membership, would be charged for attendance. That was later amended so that no requirement to join the association would be imposed but that priority seating at the event would be reserved for members.
Contact Cindy Nevitt:

subset: commoncrawl


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Travel Deals
Coming to campus? Be sure to take advantage of some of the great travel deals available.
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These offers are limited, though, so if you want to experience all Western has to offer, plus the best inbounds extreme terrain in the lower 48 this winter, now is the time to book your trip.
Call Gunnison-Crested Butte Reservations at 855.256.0771.


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Mice double deficient in LAMP-1 and -2 were generated. The embryos died between embryonic days 14.5 and 16.5. An accumulation of autophagic vacuoles was detected in many tissues including endothelial cells and Schwann cells. Fibroblast cell lines derived from the double-deficient embryos accumulated autophagic vacuoles and the autophagy protein LC3II after amino acid starvation. Lysosomal vesicles were larger and more peripherally distributed and showed a lower specific density in Percoll gradients in double deficient when compared with control cells. Lysosomal enzyme activities, cathepsin D processing and mannose-6-phosphate receptor expression levels were not affected by the deficiency of both LAMPs. Surprisingly, LAMP-1 and -2 deficiencies did not affect long-lived protein degradation rates, including proteolysis due to chaperone-mediated autophagy. The LAMP-1/2 double-deficient cells and, to a lesser extent, LAMP-2 single-deficient cells showed an accumulation of unesterified cholesterol in endo/lysosomal, rab7, and NPC1 positive compartments as well as reduced amounts of lipid droplets. The cholesterol accumulation in LAMP-1/2 double-deficient cells could be rescued by overexpression of murine LAMP-2a, but not by LAMP-1, highlighting the more prominent role of LAMP-2. Taken together these findings indicate partially overlapping functions for LAMP-1 and -2 in lysosome biogenesis, autophagy, and cholesterol homeostasis.
One crucial role of the membrane limiting late endosomes and lysosomes is to separate the potent activities of lysosomal acid hydrolases from other cellular constituents. Protein components of the lysosomal membrane mediate a number of essential functions of this compartment, including the acidification of the lysosomal lumen, transport of amino acids, fatty acids, and carbohydrates resulting from the hydrolytic degradation as well as other nutrients generated by lysosomal hydrolases. In addition, lysosomal membrane proteins may be involved in the interaction and fusion of the lysosomes with themselves as well as with other cell components, including endosomes, phagosomes, and the plasma membrane (Fukuda, 1991 ). Several highly glycosylated proteins of the lysosomal membrane have been identified (Peters and von Figura, 1994 ; Hunziker et al., 1996 ) but their specific functions are largely unknown.
Lysosome associated membrane protein-1 (LAMP-1) and LAMP-2 are major protein components of the lysosomal membrane. They are type I transmembrane proteins with a large luminal domain, one transmembrane domain, and a C-terminal cytoplasmic tail. The conserved cytosolic tails of LAMP-1 and -2 are 11 residues long and contain necessary information for their intracellular targeting after biosynthesis (Hunziker et al., 1996 ). Despite their 37% amino acid sequence homology, LAMP-1 and -2 are distinct proteins which most likely diverged relatively early in evolution as evidenced by their localization on different chromosomes (Fukuda, 1991 ). To address the specific functions of LAMP-1 and -2, we previously generated mice deficient in each of these proteins. LAMP-1-deficient mice were viable and fertile. Apart from a mild regional astrogliosis and altered immunoreactivity against cathepsin D in the brain, all tissues of these mice were normal (Andrejewski et al., 1999 ). Although LAMP-1 is an abundant protein component of the lysosomal membrane, lysosomal properties including enzyme activities, pH, osmotic stability, density, morphology, subcellular distribution, and lysosomal enzyme processing were similar to controls in LAMP-1-deficient cells. An upregulation of LAMP-2 protein was observed in kidney, spleen, and heart of LAMP-1-deficient mice, whereas the levels of another lysosomal membrane protein, LIMP-2, were unaffected. This upregulation was also evident in tissues lacking only one LAMP-1 allele, suggesting compensation of LAMP-1 by LAMP-2.
In contrast to the relatively mild phenotype in LAMP-1 knockout mice, deficiency of LAMP-2 caused a more severe phenotype. Fifty percent of the mice died at the age of 20-40 days (Tanaka et al., 2000 ). Electron microscopy revealed a massive accumulation of autophagic vacuoles in several tissues including liver, pancreas, spleen, kidney, skeletal muscle, heart, capillary endothelium, intestinal wall, lymph nodes, and neutrophilic leukocytes. Both skeletal and cardiac muscle cells showed an accumulation of large autophagic vacuoles. Autophagy is an intracellular bulk degradation pathway that plays an important role in the cellular protein economy (Mizushima et al., 2002 ). Autophagic vacuoles originate from isolation membranes that finally close to form sealed autophagosomes, which then fuse with late endosomes/lysosomes (Mizushima et al., 2003 ). The physiological importance of LAMP-2 is supported by the finding that LAMP-2 deficiency is the primary defect in Danon disease (Nishino et al., 2000 ), a lysosomal glycogen storage disease with normal acid maltase activity (Danon et al., 1981 ).
Reduced degradation of long-lived proteins, which mainly occurs via autophagy, and quantitative electron microscopy indicated that retarded consumption rather than increased formation of autophagic vacuoles was the cause for the accumulation of autophagic vacuoles in LAMP-2-deficient hepatocytes (Tanaka et al., 2000 ; Eskelinen et al., 2002a ). We further detected an elevated secretion of a subset of lysosomal enzymes and impaired processing of cathepsin D in LAMP-2-deficient hepatocytes. We proposed these defects to be due to a reduced half-life of the 46 kDa mannose-6-phosphate receptor (MPR). In addition, we found that the localization of this receptor was shifted from the trans-Golgi network (TGN) toward autophagic vacuoles.
One of the three isoforms of LAMP-2, LAMP-2a, was proposed to be the receptor for a lysosomal pathway of selective uptake and degradation of cytosolic proteins (Cuervo and Dice, 1996 ). This pathway is known as chaperone-mediated autophagy because it requires various chaperones for the transport through the lysosomal membrane (Agarraberes and Dice, 2001 ). Chaperone-mediated autophagy was shown to be activated in confluent cells including human lung fibroblasts by serum withdrawal and in rat tissues including liver by prolonged starvation (reviewed in Dice, 2000 ).
We now describe the generation of mice, double deficient in both LAMP-1 and LAMP-2. The double-deficient mice died between embryonic days 14.5 and 16.5, with a variable overall pathomorphology mainly featuring facial dysgenesis. Electron microscopy revealed that most tissues including Schwann cells and endothelium accumulated autophagic vacuoles. In this article we address the specific functions of LAMP-1 and -2 in lysosomal biogenesis, autophagy, chaperone-mediated autophagy, and cholesterol homeostasis using single- and double-deficient fibroblast cell lines isolated from the knockout mouse embryos.
LAMP-1-deficient mice (Andrejewski et al., 1999 ) and LAMP-2-deficient mice (Tanaka et al., 2000 ) were mated. Doubly heterozygote (LAMP-1+/-; LAMP-2+/-) females of the following generation were used for further breeding with LAMP-1-deficient males. Neonatal and postnatal offspring of this generation were genotyped using established PCR protocols for the targeted LAMP-1 and -2 gene loci (Andrejewski et al., 1999 ; Tanaka et al., 2000 ). In addition immunoblot analysis was performed to confirm the complete absence of LAMP-1 and -2 protein.
Mouse embryonic fibroblasts (MEF) were isolated from 12.5-13.5-day-old embryos by trypsin digestion. Independent cell lines derived from individual embryos were used for all experiments. Primary cells, at passage below 10, were used in most of the experiments. Some of the cell lines underwent a spontaneous immortalization around passage 10-20. Some cell lines were immortalized by transfection with a plasmid containing the Simian Virus 40 large T antigen. At least two independent cell lines with identical genotype were used for most experiments. Cells were grown in Dulbecco's MEM (DMEM) containing 10 or 20% fetal calf serum and antibiotics. Amino acid starvation of cells was performed using serum-free EBSS medium (Invitrogen, Karlsruhe, Germany) or Krebs-Henseleit buffer.
Antibodies used in this article have been described earlier (Eskelinen et al., 2002a ). In addition, we used rabbit anti-LC3 (Isei Tanida and Takashi Ueno, Juntendo University, Tokyo, Japan), rat anti-mouse LAMP-1 and rat anti-mouse LAMP-2 (Developmental Studies Hybridoma Bank, Iowa), rabbit anti-rat LIMP-2/LGP-85 (Kuronita et al., 2002 ), rabbit anticathepsin L (Yoshitaka Tanaka), mouse antilysobisphosphatidic acid (Jean Gruenberg, University of Geneva, Switzerland), rabbit anti-mouse NPC1 (William Garver, University of Arizona), rabbit anti-human NPC2 (Shutish Patel, New England Biomedical Research Center, Newington), rabbit anti-rab7 (Suzanne Pfeffer, Stanford University), and rabbit antidinitrophenol (ICN ImmunoBiologicals, Lisle, IL).
Cells were grown on coverslips and fixed in cold methanol or 4% paraformaldehyde in phosphate-buffered saline (PBS). After aldehyde fixation, cells were permeabilized in 0.1% Triton X-100 or 0.2% saponin in PBS. Primary and secondary antibodies were diluted in 3% bovine serum albumin (BSA) in PBS and incubated on the cells for 1 h. Goat anti-rabbit, -rat, or -mouse conjugated to Alexa Fluor 488 or 594 (Molecular Probes, Eugene, OR) were used as secondary antibodies. For filipin or Nile Red staining, paraformaldehyde-fixed cells were incubated with 0.5 mg/ml filipin (Sigma, Munich, Germany) or 5 μg/ml Nile Red (Molecular Probes) in PBS for 60 min and washed with PBS. After filipin staining, immunofluorescence labeling was performed without a separate permeabilization step. Acidic compartments were labeled by incubating cells in Lysotracker Red or DAMP (N-(3-((2,4-dinitrophenyl)amino) propyl)-N-(3-aminopropyl) methylamine) (Molecular Probes). The coverslips were mounted with Mowiol containing the antifading reagent DABCO (1,4 diazobicyclo-(2.2.2) octane), and viewed with a Zeiss Axiovert 200M fluorescence microscope (Göttingen, Germany) with or without an Apotome device for optical sectioning.
For electron microscopic investigation three controls (LAMP-1+/-, LAMP-2+/-) and three double-knockout embryos at embryonic day 12.5 were used. They were fixed by immersion in 6% glutaraldehyde (in 0.1 M phosphate buffer, pH 7.4), dissected in transversal or sagittal plain, postfixed with 2% osmium tetroxide, and embedded in Araldite according to standard methods. Semithin sections were stained with toluidine blue. For ultrathin sections the tissue blocks were trimmed to obtain sections from the following organs: neural tube, dorsal root ganglia, and roots of spinal nerves, heart, and liver. Ultrathin sections were stained with uranyl acetate and lead citrate and viewed with a Zeiss EM 900.
Cells were grown on 6-cm cell culture dishes to subconfluency. Fixation was performed with 2.5% glutaraldehyde in 0.2 M HEPES, pH 7.4, for 2 h. The cells were scraped off the dish after 30-min fixation and pelleted. Postfixation was done using 1% osmium tetroxide in water for 1 h. The pellets were then dehydrated and embedded in Epon. The number of autophagic vacuole profiles per cell area was counted under the microscope using 12,000 × g magnification. Cell area was estimated by point counting from negatives taken at 400 × g magnification, covering the area of one grid square. Two to five grid squares were counted for each sample.
Tissue homogenates of fibroblasts were prepared in 0.25 M sucrose, 3 mM imidazole/HCl, pH 7.4. Postnuclear supernatant (0.8 ml) was applied onto 11.2 ml of Percoll solution to obtain a final Percoll concentration of 27%, and the gradient was centrifuged for 30 min at 20,000 rpm in the vertical rotor VTi 65.1 (Beckmann Instruments, Berkeley, CA). Density as well as horseradish peroxidase (HRP) and β-hexosaminidase activities were determined in the collected fractions. LIMP-2/LGP-85 content was estimated by Western blotting by combining two adjacent fractions into one sample.
Cells were extracted using Tris-buffered saline (TBS), proteinase inhibitor cocktail (Roche, Mannheim, Germany), and 1 or 0.5% Triton X-100. For LC3 Western blotting, cells were extracted with PBS containing 2% NP-40, 0.2% SDS, and proteinase inhibitor cocktail. After SDS-PAGE, the proteins were transferred to PVDF membranes using a semidry blotting system. Transfer efficiency was checked with Ponceau staining. The membranes were subsequently blocked with 5% skimmed milk powder and incubated with the primary antibody. After washing in PBS, 0.1% Tween 20, the blots were incubated with HRP-coupled secondary antibodies. Signals were visualized using the ECL-Detection System (Amersham, Freiburg, Germany). LC3 Western blotting was done using 15% SDS-PAGE according to (Kabeya et al., 2000 ). Quantitation of the bands was performed by densitometry using AIDA 3.2.1 and Gel-ProAnalyser 3.1 Software.
The amount of cholesterol was measured in cell extracts prepared using 50 mM Tris, pH 8.0, 2 mM CaCl2, 80 mM NaCl, 1% Triton X-100, with the Amplex Red Cholesterol Assay kit (Molecular Probes). For the fluorometric quantification, 25 μl of cell extracts and cholesterol standards were incubated in 96-well plates according to the instructions of the manufacturer, and fluorescence was detected using Fluorolite1000 (Dynatech Laboratories, Alexandria, VA).
Growth curves were determined for each cell culture before and parallel to the protein degradation experiments. To label proteins, MEFs were incubated at 37°C for 48 h in complete fresh DMEM (Sigma) containing 20% (vol/vol) FCS (fetal calf serum, Invitrogen) and with 5 μCi/ml l-[3H]leucine or 5 μCi/ml l-[3H]valine (Amersham Pharmacia Biotech Europe, Freiburg, Germany). Before the proteolysis experiments were sarted, the cells were washed once with PBS containing 2 mM l-leucine or 10 mM l-valine and chased at 37°C for 24 h in complete DMEM containing 20% (vol/vol) FCS and 2 mM l-leucine or 10 mM l-valine to eliminate short-lived proteins. For prolonged starvation, the cells were washed and chased as above but in DMEM without FCS.
Proteolysis experiments and measurements of intracellular protein degradation were carried out as described previously (Fuertes et al., 2003a , 2003b ). According to previous results with human fibroblasts (Fuertes et al., 2003a , 2003b ) and with MEFs (our unpublished results), NH4Cl (20 mM) + leupeptin (0.1 mM) were used to inhibit all lysosomal proteolytic pathways and 3-methyladenine (3MA; 10 mM) to specifically inhibit macroautophagic degradation. The contribution of the nonmacroautophagic lysosomal pathways (i.e., microautophagy, chaperone-mediated autophagy, etc.) was calculated by subtracting the inhibition obtained with 3MA from the inhibition obtained with NH4Cl + leupeptin. The contribution of the nonlysosomal pathways to intracellular protein degradation was calculated from the degradation remaining after inhibition with NH4Cl + leupeptin. Protein degradation was analyzed 1 h after the addition of the different inhibitors and for a period of only 3 additional hours to ensure optimal inhibition and to avoid possible secondary effects of the inhibitors. All experiments were performed at least six times with duplicate or triplicate samples and using three independent cell lines (for control and single-deficient cells) or one cell line (for double-deficient cells). Student's t test analyses were performed using GraphPad Prism version 4.0 (GraphPad Software, San Diego, CA).
Mouse NPC1 in pEGFP-N3 (Clontech, Palo Alto, CA) and mouse NPC2 in pcDNA3.1 (Invitrogen, Carlsbad, CA) were from Matthew Scott, Stanford University. Dog rab7 in pEGFP-C1 (Clontech) was from Cecilia Bucci, University of Lecce, Italy. Rat-LAMP1 cDNA in pcDNA 3.1 was from Stefan Höning, University Göttingen, Germany. Mouse LAMP-2a cDNA was prepared by RT-PCR from mouse liver and subcloned into pcDNA 3.1. Transfections were performed with Fugene 6 (Roche, Mannheim, Germany) according to the manufacturer's instructions.
We used LAMP-1- and LAMP-2-deficient mice for the generation of mice lacking both LAMP proteins. LAMP-1-/- females were mated with LAMP-2y/- males. In the offspring of these matings LAMP-1+/-, LAMP-2+/- mice were obtained and females were further used to breed with LAMP-1-/- males. In the next generation eight different combinations of LAMP genotypes were observed (Supplement 1A). In one of eight offspring (12.5%) LAMP double-deficient (LAMP-1-/-, LAMP-2y/-) mice were expected. However, after monitoring and genotyping 90 mice at ~2-3 weeks of age, no viable LAMP-1-/-, LAMP-2y/- offspring were observed (Supplement 1B), indicating that the lack of both LAMP proteins resulted in embryonic lethality. We also observed that the number of male mice with only one intact LAMP-1 allele (LAMP-1+/-, LAMP-2y/-) was much lower than expected. All three mice obtained with this genotype died within 10 days of age suggesting a dose-dependent effect of LAMP expression on the viability (Supplement 1B). To determine the stage where embryonic development ceased, pregnancies were terminated starting at day 13.5 pc, the last day of organogenesis in mouse development, to 16.5 pc. At day 13.5 pc, 5 (11.3%) of 44 embryos were double deficient. Also at day 14.5 pc, 11 (12.5%) of 87 embryos were LAMP-1-/-, LAMP-2y/-. At this age the majority of these embryos (7 of 11) looked less developed compared with control or single-deficient littermates. At day 16.5 pc, no viable LAMP-1-/-, LAMP-2y/- embryos were identified. Taken together these data suggested that LAMP double deficiency lead to prenatal death between embryonic days 14.5 and 16.5. To verify the absence of LAMP proteins, we derived embryonic fibroblast cell lines from day 12.5 and 13.5 pc embryos. The absence of both LAMP proteins was confirmed by immunoblot experiments (Figure 1A) and immunofluorescence labeling (our unpublished results).
LAMP-1/LAMP-2 double-deficient mouse embryos exhibited a variable macroscopic phenotype, ranging from virtually normal (compare wild type in Figure 1, B and F, with LAMP-1/LAMP-2 double deficient in Figure 1, C and D) to a complex pattern of predominantly craniofacial (Figure 1, E and G) abnormalities. In the latter case, the main features consisted of a foreshortened forebrain and head and a severely reduced length of both the maxillary and mandibular portions of the jaws combined with the absence of detectable vibrissal anlagen. In several cases, the closure of the ocular choroid fissure appeared incomplete (Figure 1, E and G), and in these "severe" cases the developing external ear was likewise missing.
The general microscopic appearance of the organs was essentially similar in controls and double knockouts (Figure 1H). However, on the cellular level, double-knockouts showed a much higher frequency of cytoplasmic vacuoles, which could often be identified as autophagic vacuoles. These vacuoles were most regularly seen in vascular endothelium (Figure 2B compared with a control embryo in Figure 2A) and Schwann cells (Supplement 2A), furthermore they occurred in the neuroepithelium (Supplement 2, B and C) and occasionally in hepatocytes (our unpublished results), whereas they were not detected in the myocardium.
To get a deeper insight into the cellular functions in fibroblasts lacking both LAMP-1 and -2, we analyzed these cells morphologically and biochemically. First we investigated whether the autophagic accumulation observed in situ was also preserved in fibroblast cells derived from LAMP-double-deficient embryos. Quantitative electron microscopy revealed that after 2 h of starvation in serum- and amino acid-free medium, ~2.5 times more autophagic vacuoles accumulated in the double-deficient cell lines than in control cell lines or single-deficient cell lines (Figure 3A). Also without starvation the number of autophagic vacuoles was higher in the double-deficient cells as compared with single knockout and control cells (Figure 3A). Interestingly, in the double-deficient cells the amount of early autophagic vacuoles was comparable to control cells, but the amount of late vacuoles, containing partially degraded cytoplasmic material and also a fluid-phase endocytic marker (Figure 3B), was increased. This suggested that although initial maturation of autophagosomes including fusion with endosomes was functional, the final maturation step of the late autophagic vacuoles was retarded. The autophagic vacuole accumulation was confirmed by analyzing the levels of the autophagosome marker protein LC3 by Western blotting (Kabeya et al., 2000 ). The ratio of the cytoplasmic form (LC3I) to the membrane-bound form (LC3II) was comparable in all genotypes with and without starvation (Figure 3C). However, in both starved and nonstarved LAMP-1/LAMP-2 double-deficient cells (Figure 3D), and also in LAMP-2 single-deficient cells (our unpublished results), the absolute amounts of LC3I and LC3II were increased as compared with controls. We showed earlier LAMP-2 single-deficient liver and heart muscle tissues to have an excessive autophagic vacuole accumulation (Tanaka et al., 2000 ). In liver tissues LC3II level was clearly increased compared with controls, whereas in heart muscle, where a less severe accumulation of vacuoles was observed by electron microscopy, LC3I and LC3II levels were not increased (Figure 3E). This suggested that electron microscopy was the most sensitive way to detect autophagic vacuole accumulation, particularly when mainly late autophagic vacuoles were accumulating.
To clarify whether a decreased proteolysis caused the accumulation of autophagic vacuoles in double-deficient MEFs, we analyzed the degradation of long-lived proteins in starved and nonstarved MEFs in the presence or absence of 3MA, an autophagy inhibitor (Figure 3F). Surprisingly, LAMP- deficiencies did not affect the overall rates of protein degradation. The rate of 3MA-sensitive protein degradation in amino acid-starved cells (which represented autophagic degradation) was also unaltered. Thus, it is unlikely that decreased proteolysis was the cause for accumulation of late autophagic vacuoles.
In agreement with the normal proteolytic rates in the double-deficient MEFs, we observed no differences in the steady state protein levels of two major lysosomal proteases, cathepsin L and cathepsin D (our unpublished results). Lysosomal enzyme activities were also unaltered (our unpublished results) in LAMP-single and double-deficient MEFs. Further, the processing of newly synthesized cathepsin-D appeared to be normal as evaluated by pulse-chase labeling followed by immunoprecipitation from cell extracts and medium (our unpublished results). Acidification of the lysosomal compartments also appeared normal as judged by vital staining with the acidotropic drug Lysotracker Red, or by DAMP (N-(3-((2,4-dinitrophenyl)amino) propyl)-N-(3-aminopropyl) methylamine) followed by immunofluorescence staining with antidinitrophenol antibodies.
Lysosomal hydrolases are targeted from the TGN to endosomes by mannose-6 phosphate receptors (MPRs). We therefore studied the steady state levels of the 46 and 300 kDa MPRs (MPR46 and MPR300, respectively) by Western blotting. Both receptors mediate Golgi-to-endosome transport, whereas MPR300 only is able to mediate transport from the cell surface to endosomes. The levels of MPR300 in the double knockout MEFs were similar to those in control and single-deficient cell lines (our unpublished results). MPR46 levels tended to be lower in primary MEF lines deficient in LAMP-1/LAMP-2, but this difference was lost in double-deficient lines immortalized with the SV large T antigen (our unpublished results). However, MPR300 subcellular localization was altered in LAMP-double-deficient MEFs. In control and single-deficient cells MPR300 localized to a vesicular-reticular network around the nucleus (Supplement 3, A-C). In the double-deficient cells MPR300 tended to localize to more vesicular structures in the perinuclear area (Supplement 3D), although control cell-like staining pattern was also observed in some of the cells. The immunofluorescence localization of MPR46 was not affected in any of the knockout MEF lines (Supplement 4).
We investigated lysosome distribution and morphology in LAMP-deficient cells using immunofluorescence staining. Labeling of LIMP-2/LGP-85 (Figure 4, A-D), cathepsin-D (our unpublished results), or lysobisphosphatidic acid (LBPA), a late endosomal/lysosomal marker (see Figure 7, E and F, red), revealed an altered size and distribution of late endosomes/lysosomes in LAMP-1/LAMP-2-deficient fibroblasts. In these cells (Figure 4D) lysosomes were slightly larger and more peripherally localized than the endo/lysosomes in the other genotypes. However, electron microscopy revealed that typical dense lysosomes, containing electron-dense grainy material and some internal lamellar membranes, still existed in the LAMP-1/LAMP-2 double-deficient cells. These structures were additionally identified as lysosomes by accumulation of BSA-gold, a fluid-phase endocytic marker fed to the cells for 2 h (Figure 4E). The only obvious abnormality of the LAMP-deficient endo/lysosomes was a slightly increased content of internal lamellar membranes (Figure 4E).
We used Percoll gradient centrifugation to study the lysosomal density of LAMP-1/LAMP-2 double-deficient cells. Control and LAMP double-deficient cells were fed with HRP for 5 min and then chased for 3 h to allow transport of HRP from early endosomes to lysosomes. The cells were then homogenized and postnuclear supernatants were centrifuged in 27% Percoll gradients. Activities of HRP and the lysosomal marker enzyme β-hexosaminidase were determined from the collected fractions. Western blotting was used to estimate the amount of the lysosomal membrane protein LIMP-2/LGP-85. In control cells HRP and β-hexosaminidase activities, as well as LGP-85, were located at the bottom fractions (Figure 5, A and B), indicating transport of HRP to dense lysosomes. LAMP-1 and -2 single-deficient cell lines gave results similar to the control cells (our unpublished results). However, in LAMP-1/LAMP-2-/- cells both the lysosomal markers and HRP were located in the light fractions of the gradient (Figure 5, A and B). Only a small fraction of LIMP-2/LGP-85 was found in the dense fractions from the double knockout MEFs (Figure 5B). These results suggested that the lysosomal density was decreased in LAMP-deficient cells compared with the control and single-deficient cells.
LAMP-2a has been suggested to act as a receptor for chaperone-mediated autophagy (Cuervo and Dice, 1996 , 2000 ). We therefore wanted to check whether the activity of this pathway was decreased in the single- and double-deficient cell lines. There are no known specific inhibitors of this pathway. However, all lysosomal proteolysis pathways can be blocked using a combination of a weak base such as NH4Cl and an inhibitor of lysosomal cathepsins such as leupeptin (Fuertes et al., 2003a ). Therefore, these inhibitors, together with the macroautophagy inhibitor 3MA (Seglen and Gordon, 1982 ), allow a separate analysis of the contribution of macroautophagy, other lysosomal pathways (which should include chaperone-mediated autophagy), and the nonlysosomal pathways, as we have shown with human fibroblasts (Fuertes et al., 2003a ). No significant differences in the degradation of long-lived proteins (Figure 6A), nor in the protein degradation by nonlysosomal (Figure 6D), macroautophagic (Figure 6B), or other lysosomal pathways (Figure 6C) could be found under any conditions and for any cell line tested, except for a moderate increase in total, lysosomal, and nonlysosomal degradation in LAMP-1-deficient cell lines. Even under conditions where chaperone-mediated autophagy was supposed to be more active (prolonged starvation in confluent cells, the last group of columns in Figure 6C), we were unable to find any significant difference in the protein degradation by lysosomes in LAMP-2-deficient cell lines or in a LAMP-1/LAMP-2 double-deficient cell line, when compared with the control cell lines. The difference found in exponentially growing LAMP-1-deficient cells subjected to serum withdrawal (Figure 6A) appeared to be due to a general increase in the protein degradation because it affected all pathways of protein degradation (Figure 6). Taken together, these data with LAMP-2-deficient and LAMP1/LAMP2 double-deficient cell lines did not support a role for LAMP-2 as a receptor for chaperone-mediated autophagy in MEFs.
Because we observed decreased lysosomal density but no significant swelling of lysosomes in the double-deficient cells, we hypothesized that accumulation of lipids may have influenced the lysosome density. Therefore we analyzed the lipid distribution in more detail. Filipin staining was used to detect unesterified cholesterol. The staining pattern of LAMP-1 single-deficient cells was comparable to the control cells (Figure 7, A and B), whereas LAMP-2 single-deficient cells (Figure 7C) showed increased staining in small vesicles scattered around the cytoplasm. Interestingly, LAMP-1/LAMP-2 double-deficient cells (Figure 7D) showed a massive accumulation of brightly stained large vesicles around the cytoplasm. This vesicular filipin staining pattern was observed in all LAMP double-deficient cell lines tested including primary, spontaneously immortalized, and SV40 large T antigen immortalized MEF lines. The filipin-stained vesicles in double-deficient cells showed a partial colocalization with the late endosomal marker lysobisphosphatidic acid (LBPA; Figure 7F, yellow) as well as with cathepsin D or LIMP-2/LGP-85 (our unpublished results). In addition, the filipin-positive vesicles showed colocalization with transfected GFP-rab7 (Figure 8, G and H, yellow), as well as with endogenous rab7 (our unpublished results). In many vesicles rab7 signals were surrounding the filipin-positive contents (Figure 7, G and H, insets). This indicated that in LAMP double-deficient cells cholesterol accumulated in rab7 containing late endosomal/lysosomal compartments. Interestingly, filipin-positive structures almost completely colocalized with MPR46 (Supplement 4A) in control fibroblasts, whereas much less colocalization with MPR46 was found in LAMP-1/LAMP-2 double-deficient cells (Supplement 4B). Similar results were obtained in double labeling of MPR300 with filipin. Although the distribution of MPR300 tended to be more vesicular in the double-deficient MEFs (Supplement 3D), there was no colocalization of MPR300 with filipin in these vesicular structures (our unpublished results). These findings suggested that although in control cells highest concentrations of cholesterol were located to the MPR46/MPR300-positive organelles including TGN and late endosomes, in LAMP double-deficient cells cholesterol accumulated in endo/lysosomal compartments, which were negative for MPRs and positive for rab7.
Filipin-labeled membrane cholesterol is also visible by electron microscopy as typical membrane deformations (Punnonen et al., 1989 ). In agreement with the light microscopical analysis, in wild-type fibroblasts filipin-induced membrane formations were detected in small vesicles close to the Golgi apparatus (Figure 8A). In double knockout cells labeling was observed in both the limiting and internal, typically concentric, membranes of endo/lysosomal vesicles, indicating that unesterified cholesterol accumulated in both the limiting and internal membranes (Figure 8B). This labeling pattern was reminiscent of the cholesterol distribution in endo/lysosomes of NPC1 patient fibroblasts (Garver and Heidenreich, 2002 ).
To confirm that the cholesterol accumulation was due to the deficiency of LAMPs, we transiently transfected LAMP double-deficient cells with a mouse cDNA for LAMP2a. The proportion of cells containing filipin-positive endo/lysosomal vesicles was quantitated. Vesicular filipin staining was observed in 7% (37 of 533) of control wild-type cells and in 80% (1051 of 1314) of nontransfected LAMP double-deficient cells (Figure 9A). However, only 18% (69 of 381) of the double-deficient cells reexpressing LAMP-2a had vesicular filipin staining; instead, filipin staining was similar to control cells (Figures (Figures7A7A and 9, A and B). A significantly smaller rescue effect was observed when a similar experiment was performed using LAMP-1 cDNA, 69% (34 of 49) of double-deficient cells reexpressing LAMP-1 still showed vesicular filipin staining. This suggested that LAMP-2 deficiency was the major cause for the endo/lysosomal cholesterol storage observed in LAMP-1/LAMP-2 double-deficient MEFs.
We next analyzed the cholesterol levels per milligram of cell protein in whole cell extracts. In LAMP-1/LAMP-2-deficient primary MEFs the cholesterol concentration was about twofold higher than in control cells (Figure 9C). In LAMP-2 single-deficient cells an intermediate level of cholesterol was observed (our unpublished results). The difference in total cholesterol content was lost in many of the spontaneously immortalized or SV40-transformed double-deficient cell lines (our unpublished results); however, these cell lines still showed endo/lysosomal cholesterol accumulation in filipin staining. Thus the cholesterol localization was altered in all double-deficient cell lines, although the primary cell lines only showed an additional increase in the total cholesterol levels.
We also tested whether the double-deficient cells could be cleared of accumulated cholesterol by culturing them in the absence of exogenous cholesterol. Culturing the cells for 2 days in a medium containing lipoprotein and cholesterol-free serum (LPDS) did not change the filipin staining pattern or the overall cholesterol content of the double-deficient cells (our unpublished results). This indicated that the stored endo/lysosomal cholesterol could not be cleared in the absence of exogenous low-density lipoprotein (LDL) cholesterol. During culture in LPDS medium cells upregulate the LDL receptor levels to enhance uptake of extracellular cholesterol. This regulation is mediated by cholesterol levels in the endoplasmic reticulum (ER; Brown and Goldstein, 1986 ). Upregulation of LDL receptor in LPDS medium was detected in both control and LAMP double-deficient cells (Figure 9D), which suggested that intracellular LDL receptor regulation via ER was functional in the LAMP double-deficient cells. However, although some variation was observed between the individual deficient lines, the unstimulated LDL receptor levels tended to be higher in the double-deficient cell lines than in the control cell lines (Figure 9D). This might have been due to impaired transport of cholesterol from the lysosomal compartment to the ER, where the cholesterol sensors are located. Decreased cholesterol in the ER in turn is known to lead to upregulation of LDL receptor levels.
Fibroblasts derived from Niemann Pick-Type C patients show endo/lysosomal cholesterol storage (Garver and Heidenreich, 2002 ) similar to what we observed in LAMP double-deficient cells (Figures (Figures77 and and8).8). Mutations in the transmembrane protein NPC1 (Loftus et al., 1997 ) or in the soluble lysosomal protein NPC2 (Naureckiene et al., 2000 ) cause the cholesterol storage of this disease. We therefore investigated the expression of these proteins in the double-deficient cells. Using Western blotting we found that the steady state levels of NPC1 and NPC2 proteins were not affected in LAMP-deficient cells (our unpublished results). Further, transient overexpression of GFP-tagged NPC1 or myc-tagged NPC2 did not rescue the cholesterol storage phenotype of LAMP-deficient cells (our unpublished results). However, the localization of endogenous NPC1 as well as transfected NPC1-GFP in LAMP-1/LAMP-2 double-deficient cells was different from that of control cells. In control cells NPC1-GFP localized to fine punctate, filipin-negative structures (Figure 10A), whereas in many of the LAMP double-deficient cells, enlarged, often ring-like structures were observed. In these structures NPC1-GFP colocalized with filipin (Figure 10B, arrowheads and inset). The altered distribution of NPC1 in double-deficient fibroblasts could be rescued by reexpression of LAMP2a (Figure 10, C-E).
Rab7 overexpression has been reported to abolish the endo/lysosomal cholesterol accumulation in NPC1 patient cells (Choudhury et al., 2002 ). In LAMP double-deficient cells, however, transient expression of GFP-rab7 did not alter the filipin staining pattern (Figure 7, G and H). Quantitation showed that 77% of cells expressing GFP-rab7 still had vesicular filipin staining. Instead the localization of both endogenous (our unpublished results) and transfected rab7 was altered. In control cells rab7 was found in the perinuclear region where it showed some colocalization with filipin (Figure 7G), whereas in LAMP double-deficient cells rab7 was almost exclusively localized in filipin-positive vesicles, which were often found in the peripheral cytoplasm (Figure 7H). We also compared the rab7 expression levels in control and LAMP double-deficient cells by Western blotting and found them to be identical. These results indicated that rab7 localization was severely altered in the LAMP double-deficient cells.
We next wanted to investigate whether localization of neutral lipids and cholesterol esters was affected. These lipids are components of lipid droplets, and Nile Red has been used to stain these structures (Greenspan et al., 1985 ). In almost all control cells we observed large spherical structures around the nucleus (Figure 10F). In contrast, no spherical structures were seen in many of the LAMP-1/LAMP-2 double knockout cells, and instead a diffuse plasma membrane staining was observed (Figure 10G). This suggested that synthesis of lipid droplets was impaired in cells deficient in both LAMPs. This may be connected to the accumulation of unesterified cholesterol in lysosomes, which likely leads to decreased cholesterol esterification. Esterified cholesterol is one of the lipids stored in lipid droplets (Aoki and Massa, 1975 ).
Several functions have been suggested for lysosomal membrane proteins, such as sequestration of numerous acid hydrolases, maintenance of an acidic intralysosomal environment, transport of degradation products out of lysosomes, and specific interaction and fusion events between lysosomes and other organelles (Fukuda, 1991 ; Peters and von Figura, 1994 ; Eskelinen et al., 2003 ). LAMP-1 and -2, the two most abundant lysosomal membrane proteins, have been estimated to contribute to ~50% of all proteins of this membrane (Hunziker et al., 1996 ). The presence of LAMP molecules is one of the major definitions of the lysosomal compartment (Kornfeld and Mellman, 1989 ). What then was the consequence of total absence of both major lysosomal membrane proteins on lysosomal structure and function? Surprisingly, we observed that typical lysosomes, as defined by ultrastructural appearance, and capacity to take up fluid phase markers and degrade proteins, still existed in LAMP-1/LAMP-2 double-deficient MEFs. It is likely that other lysosomal membrane proteins including LIMP 1 and LIMP-2/LGP-85 were able to take over the protective function against lysosomal hydrolases.
However, the generation of LAMP-1/LAMP-2 double-deficient embryos described in this study demonstrated that these proteins fulfill at least partially overlapping functions in vivo. The tight functional association of the proteins is also highlighted by the observation that mice with only one functional LAMP allele were more susceptible to early postnatal mortality. Although no gross impairment of organ development was encountered, LAMP-1/LAMP-2 double-deficient embryos were characterized by a massive accumulation of autophagic vacuoles in many tissues, especially endothelial cells. It is possible that further embryonic development beyond embryonic day E15 was prevented due to these cellular alterations and impaired delivery of nutrients.
We showed earlier that autophagic vacuoles accumulate in LAMP-2 single-deficient liver tissue and isolated hepatocytes (Tanaka et al., 2000 ; Eskelinen et al., 2002a ). This accumulation could be attributed to a decreased lysosomal degradation of long-lived proteins. In this study we observed an accumulation of late autophagic vacuoles in the LAMP double-deficient cells. Surprisingly, in LAMP-1/LAMP-2 double-deficient MEFs the protein degradation rates under all conditions tested were similar to control cells, suggesting that the accumulation of autophagic vacuoles was not due to retarded protein degradation. Thus, the increased accumulation could be due to impaired degradation of other macromolecules including lipids or possibly to defective export of degradation products from autophagic vacuoles to the cytoplasm. It is also possible that in the double-deficient cells, retarded protein degradation per autophagic vacuole was compensated by the increased amount of vacuoles, and therefore we did not see a difference in protein degradation rates between control cells and the double-deficient cells.
One of the three LAMP-2 isoforms, LAMP-2a, has been suggested to be the receptor for the chaperone-mediated autophagy, a selective uptake and degradation of cytosolic proteins by lysosomes (Cuervo and Dice, 1996 ). This pathway was shown to be active in confluent lung fibroblasts and Chinese hamster ovary cells cultured in serum-free medium (Cuervo and Dice, 1998 ). In this study we analyzed MEFs deficient in LAMP-2, LAMP-1, or both. In LAMP-2-deficient cells all LAMP-2 isoforms including LAMP-2a were disrupted. MEF cells deficient in LAMP-2 or both LAMP-1 and -2 did not show changes in the lysosomal protein degradation rates under six different conditions. These results suggested that deficiency of LAMP-2 or both LAMP-2 and LAMP-1 did not alter lysosomal protein degradation rates even in confluent cells under prolonged (28 h) serum starvation, a condition where chaperone-mediated autophagy is supposed to be more active. Thus, it is possible that LAMP-2 is not the only receptor in chaperone-mediated autophagy or that the pathway is not active in mouse embryonic fibroblasts, like in other cells. However, the latter alternative appears unlikely because the proteolytic rates measured in our MEFs resembled those of human fibroblasts, where the pathway is known to be active. Another possibility is that other proteolytic pathways compensate for the defective activity of chaperone-mediated autophagy in LAMP-2-deficient mouse fibroblasts. Although our experiments with various inhibitors excluded this for macroautophagy, proteasomal degradation, and nonlysosomal proteolytic pathways different from proteasomes, the possibility still exists that a lysosomal pathway that is different from macroautophagy (such as microautophagy) is activated to compensate for the deficiency in chaperone-mediated autophagy.
We observed an altered subcellular distribution of MPR300 in LAMP double-deficient MEFs. Despite this, cathepsin D delivery to the lysosomal compartment and uptake of arylsulfatase A, a mannose-6-phosphate-containing ligand, from the culture medium were not affected (unpublished data). This was in contrast to LAMP-2 single-deficient hepatocytes where we observed that lysosomal biogenesis, MPR46 expression levels (Eskelinen et al., 2002b ) and MPR300 function in endocytosis (unpublished data) were affected. This may be explained by different expression levels of LAMP proteins and different requirements for lysosomal functions in embryonic fibroblasts and adult hepatocytes.
Intriguingly, we found altered lipid localization in LAMP-1/LAMP-2 double-deficient and LAMP-2 single-deficient MEFs. Prominent storage of unesterified cholesterol in endo/lysosomal compartments of LAMP-1/LAMP-2 double-deficient cells was observed. Transfection with wild-type LAMP-2a constructs reversed the cholesterol storage phenotype, whereas LAMP-1 transfection had a much smaller effect, which demonstrated the major importance of LAMP-2 for the cholesterol metabolism. The decreased amount of Nile Red-stained lipid droplets in the double-deficient MEFs also indicated that lipid metabolism was altered. Recent studies have demonstrated that lipid droplets are not passive storage structures but have an active role in lipid metabolism (Liu et al., 2004 ).
The pattern of cholesterol storage in LAMP-1/LAMP-2-deficient cells resembled that in fibroblasts from Niemann-Pick type C patients (Garver and Heidenreich, 2002 ). Similar to LAMP double-deficient cells, these cells accumulated unesterified cholesterol in late endosomal/lysosomal vesicles. Mutations in NPC1 or in NPC2 have been described to be responsible for the cholesterol storage phenotype (Garver and Heidenreich, 2002 ). It has been reported that after transfection of NPC1 to cells deficient in this protein, the protein first localized to the limiting membranes of cholesterol-loaded endo/lysosomes. After cholesterol had been cleared from endo/lysosomes, the localization of the reexpressed NPC1 changed to small late endosomal filipin-negative vesicles, which was identical to the localization of NPC1 in control cells (Zhang et al., 2001 ). We observed that in LAMP double-deficient cells NPC1 localized in the cholesterol-loaded endo/lysosomes, whereas in control cells NPC1 localized in small filipin-negative vesicles. Thus, it is likely that in the double-deficient cells NPC1 was recruited to the correct localization for cholesterol clearance, but it was not able to fulfill this function. The altered distribution of NPC1-GFP could be reversed by parallel expression of NPC1-GFP and LAMP2, indicating that LAMP-2 directly or indirectly influenced the trafficking or function of NPC1. Similar to NPC1, rab7 also localized in the cholesterol-loaded endosomes in the LAMP double-deficient cells. It has been reported that cholesterol accumulation in the lysosomal compartment increases the amount of membrane-associated rab7 (Lebrand et al., 2002 ) and this was suggested to interfere with the rab7 function. Thus, it is possible that at least some of the alterations observed in the LAMP double-deficient cells, such as altered localization of lysosomal vesicles in the peripheral cytoplasm, were due to impaired function of rab7. Further studies are however necessary to clarify the mechanistic reasons for the accumulation or cholesterol and decreased amount of lipid droplets in MEFs deficient of LAMP-1 and -2.
In conclusion, the viability of mice deficient in either LAMP-1 or LAMP-2, as well as the embryonic lethal phenotype of LAMP-1/LAMP-2 double-deficient mice indicated that these two major lysosomal membrane proteins share common functions in vivo. However, LAMP-2 seemed to have more specific functions because LAMP-2 single deficiency had more severe consequences than LAMP-1 single deficiency. In embryonic fibroblasts, mutual disruption of both LAMPs was associated with an increased accumulation of autophagic vacuoles, altered lysosomal distribution and appearance, and disturbed cholesterol metabolism, whereas protein degradation rates were not affected. These results clearly show that the LAMP proteins fulfill functions far beyond the initially suggested roles in maintaining the structural integrity of the lysosomal compartment.
We are grateful to Ellen Eckerman, Anegret Schneeman, Jenny Schröder, Stefanie Jäger, Marlies Rusch, and Katharina Stiebeling for technical assistance and to Mathew Scott, Stefan Honing, Cecilia Bucci, Isei Tanida, Takashi Ueno, Jean Gruenberg, William Garver, and Shutish Patel for plasmids or antibodies. This study was funded by a grant from the Deutsche Forschungsgemeinschaft (SA683/1-3) to P.S., the Ministry of Labor, Health, and Welfare of Japan and the Ministry of Education, Science, Sports, and Culture of Japan to Y.T., the Ministerio deCiencia y Tecnología of Spain (BMC2001-0816 and SAF2002-00206) to E.K., the German Niemann-Pick Parents Foundation, the Fonds der Chemischen Industrie and the Hensel-Stiftung, Kiel, Germany.
Article published online ahead of print. Mol. Biol. Cell 10.1091/mbc.E04-02-0103. Article and publication date are available at www.molbiolcell.org/cgi/doi/10.1091/mbc.E04-02-0103.
Abbreviations used: Avi, early autophagic vacuole; Avd, late autophagic vacuole; BSA, bovine serum albumin; ER, endoplasmic reticulum; FCS, fetal calf serum; GFP, green fluorescent protein; HRP, horseradish peroxidase; LAMP, lysosomal associated membrane protein; LIMP, lysosomal integral membrane protein; LBPA, lysobisphosphatidic acid; LDL, low-density lipoprotein; LPDS, lipoprotein-deficient serum; 3MA, 3-methyladenine; MEF, mouse embryonic fibroblast; MPR, mannose-6-phosphate receptor; PBS, phosphate-buffered saline; TGN, trans-Golgi network.
Online version of this article contains supporting material. Online version is available at www.molbiolcell.org.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


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text: Just wondering , does anyone pull a #14 +/- along with their pipe on Zone line's. There is always a tracer line along the mainline out to the valves. Obviously because of the valve wiring. But were wondering if anyone continues with a tracer wire along all the zone lines as well. The benefit I see is if a customer may want to add landscape later on down the road. There would be no concern of hitting a sprinkler line. Of course if he did he ht a zone the damage wouldn't be horrible, However I hate having to repair ML breaks as all the valves get contaminated Ken


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Balls 6 and new tail flash
Images of military aircraft and military life from around the world.
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Balls 6 and new tail flash
I saw Balls-6 at the Boeing hangers today and snapped these pictures. Note the red and white tail marking. I wonder why red and white?
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Finished in dark walnut, this classic bedroom set has a graceful sleigh bed. The matching night stand, dresser and chest are accented with antique brass hardware.Includes
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You know what they say, "Once a cheater, always a cheater." But while this statement may prove true, that doesn't mean all people choose to acknowledge it. After we found out that Shay Carl was caught cheating on his wife of 14 years with an adult webcam performer, we couldn't help but think of all the YouTube-star cheating scandals of the past. While most happened between those who were simply dating, a handful occurred in an actual marriage.
We're not saying cheating is ever okay, but breaking the supposed lifelong vow made before god, family, friends, etc., takes the disgust to a whole new level. And you know what's probably even more disturbing? Most of the YouTube couples on the below list actually stayed together despite the fact that the husband admitted to cheating. To each their own, we guess, but it's important to remember that respect is a precious thing.
Shay Carl
Shay's infidelity is the most recent of the YouTube world, and perhaps the most shocking, too. Adult video performer Aria Nina exposed the 37-year-old father of five last month when she posted explicit private messages he sent her over the course of several months.
While the YouTuber never actually admitted that he was was a cheater, he wrote a lengthy Twitter note in which he explained that his alcoholism had returned, causing him to "hurt the ones [he] loves most."
We haven't heard from Shay, or his wife, Colette, since the drama went down, but reports say the founder of The Shaytards is currently in rehab getting help for his addiction.
Photo: Instagram (@shaycarl)
Sam Rader
While Sam didn't physically cheat on his wife, Nia, he came pretty darn close. Weeks after the couple suffered a miscarriage (which, BTW, some people say was fake), the 31-year-old was one of the celebs who was exposed in the AshleyMadison.com hack.
The leak of info revealed that the YouTuber made two $189 payments to the infidelity site, which he confirmed in a video called "FORGIVEN." Basically, Sam said he wanted to cheat on his wife and made the account to do so, but never actually went through with the plan.
"[Nia] has forgiven me for this mistake that I made in opening the account," he said. "I've sought forgiveness from god and he's forgiven me."
Photo: Instagram (@sampaulphoto)
Austin Null
Like many YouTube stars on this list, Austin's affair was exposed thanks to a social media leak. In early January 2016, videos of Austin Null, a married family vlogger, masturbating hit the Internet. While the YouTuber could've kept quiet, he made a video coming clean.
Basically, a girl reached out to the 27-year-old online and sent him inappropriate pictures the year prior. "Instead of blocking her and moving on, [I] decided to flirt with the idea of responding, and basically had an online affair for like 5 months…" he admitted.
Austin eventually told his wife, Brittany Null, everything, the couple went to counseling and they're currently raising their three young children together.
Photo: Instagram (@thenivenulls)
This YouTuber's situation is pretty confusing, but it's an example of cheating nonetheless. Onision married fellow YouTuber Laineybot in 2012 and shortly after the girl told him she was bisexual and wanted to explore her sexuality. The 31-year-old encouraged her to find a girlfriend and soon after Billie Dawn Ingle entered the picture.
Things were going fine, until Onision gave the 19-year-old a sensual massage and cuddled her, which the 22-year-old considered to be cheating. This event caused the married couple to separate and seek therapy. Eventually, they got back together.
Photo: Instagram (@onision)
Charles Trippy
While some people say Charles' actions were an act of infidelity, the YouTuber claims he was totally innocent. Here's the scoop — In April 2014, CT announced that he and his wife, Alli Speed, were going their separate ways after three years of marriage. The YT guy immediately got hit with rumors that he was unfaithful, which he quickly refuted on Twitter.
"When we were together I was 100% faithful. This is sad. Point blank. I did NOT cheat while we were together, being separated doesn't mean what u do next is cheating. If u think that then that's dumb."
What the 32-year-old was referring to in the latter part of his statement was his new relationship with Allie Wesenberg, who he started dating in May 2014. Technically speaking, we guess critics had a point — Charles was still LEGALLY married to Alli when he got a new GF. However, the former husband and wife were planning to divorce.
CT and AW got engaged on March 6, 2016, and are planning to get married soon.
Photo: Instagram (@charlestrippy)
Bryan Lanning
They say the proof is in the pudding, which is why we're not convinced that the Daily Bumps guy is actually a cheater. Multiple male Craigslist users claim that they responded to the YouTuber's ~personal~ ads on the site, meeting up with his for sex while he was married to the mother of his two boys, Missy Lanning.
Bryan has never talked about the allegations and we must remind you how easy it is for people to make up stories about other people.
Photo: Instagram (@bryanlanning)


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Because you are.
Most Helpful Girl
Because you are.
Have an opinion?
Did you know you are a liar?
Because you are.
How is he lying? I think you're super pretty. I'm sure you're a good person too
@lynneishappi eh eh eh, thank u
Awww, and you're handsome. Am I helping? XD
I'm mediocre at best
You are beautiful. Take his words
thank you :)
I am beautiful not just because people tell me.. I feel beautiful some days LOL :D
I strongly disagree..
You are too. In my opinion everyone has at least one beautiful aspect. Physical or mental.
Aww. Sometimes I feel like I do, but mostly I don't. You are too. 😊
I did but it's always nice to hear :P
@Not-helping... U r not helping.. -_-
oh wait! you weren't talking to me.
Damn it
Well I was. Hai beautiful ^.^
As beautiful as a forest on fire!
Yes, they are. :)
I'm well aware of that but thank you.
Now I do!!!
Thanks bro. I'm tearing up
Awwh, thank you! <3
You're beautiful too dude. c:
I would say about average
thank you senpai for noticing me
I think most women are far naturally sexier than they give themselves credit for-and i don't mean just looks
thanks mate i know you are too :)
Why you are not helping?
You cannot undo this action. The opinion owner is going to be notified and earn 7 XPER points.


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|Full name
|Galleria Buz Pisti
|Ataköy, Bakırköy, Istanbul, Turkey
|Galleria Buz Pateni Pisti
Galleria Ice Rink (Turkish: Galleria Buz Pisti) is an indoor ice skating and ice hockey rink located within the Galleria Ataköy shopping center located in Bakırköy district of Istanbul, Turkey. It was opened in 1989. The rink is run by a the company "Galleria Buz Pateni Pisti", a subsidiary of the shopping mall.[1]
The venue hosts all kinds of ice sports events in Istanbul including Turkish ice hockey leagues for men's, women's and junior's. It is home to the ice hockey club Istanbul Paten Kulübü.
By April 2010, the director general of the Galleria shopping mall announced that the ice rink will be closed to make space for additinal recreational places and new stores within the renovation project of the shopping mall.[2]
See alsoEdit
- ↑ Galleria Buz Pateni Pisti (Turkish). Türk Ticaret Net. Retrieved on 2011-03-20.
- ↑ "Galleria'nın 22 yıllık buz pisti yeşile dönüyor", 29191-04-12. Retrieved on 2011-03-20. (Turkish)
|This page uses content from Wikipedia. The original article was at Galleria Ice Rink. The list of authors can be seen in the page history. As with Ice Hockey Wiki, the text of Wikipedia is available under the Creative Commons Attribution-Share Alike License 3.0 (Unported) (CC-BY-SA).


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Dominik is co-founder of the Bitcoin Frankfurt Community as well as a co-founder of Bitcoin Vietnam Co. Ltd., the company that launched the first Bitcoin exchange in Vietnam back in March 2014. The team behind Bitcoin Vietnam recently also launched Vietnam's first live trading platform, "VBTC."
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Author | exchange4media Mumbai Bureau | Monday, Dec 06,2004 8:04 AM
Gujarat Tea Processors and Packers Ltd. (GTPPL) along with Triton Communications have signed on Rajkumar Hirani to direct their latest TV commercial for their brand Wagh Bakri. This would be the first TVC for Wagh Bakri after Triton was signed up as the creative agency.
Said Ali Merchant, Director, Triton Communications, "It was with a great deal of faith and expectation that we took the script to Rajkumar Hirani and after seeing the final product, I am confident that this commercial will go a long way in strengthening the essential equity of the Wagh Bakri brand, which is centered around relationships. I see this commercial making an impact in the market from day one."
Renton D' Sousa, National Creative Director, Triton Communications, explaining why Hirani was chosen, said, "Once we had the script ready we were looking for a director who understood the delicate nuances of portraying relationships. I had seen 'Munnabhai MBBS' and once the strategy and the script were in place, I instinctively felt that Rajkumar Hirani was the person best suited to do the commercial and add value to it. The best part is that the client shared the same belief."
Hirani has the rare distinction of being among the few Bollywood directors to have an adaptation of his film in Hollywood. Known for his focused control over a story-line and the ability to extract remarkable performances from his cast, he brings a fresh approach to directing TV commercials with an emphasis on small moments and subtle nuances which tend to lift a script to greater heights.
Emphasizing the need for a grand scale TVC to re-energize the brand, Parag Desai, Director, GTPPL, observed, "Wagh Bakri is a leader in its category and has an excellent brand loyalty among tea drinkers who value relationships. The TVC, woven around a simple idea of relationship building, aims to drive home the association with an emotional twist."
Desai said the company was excited about working with Hirani. "We are sure the Wagh Bakri magic will spread through India just like the magic of Munnabhai MBBS. We had a warm, charming and a lighthearted script and translating this to film and adding value is Hirani's forte. We saw a synergy between Triton's Hirani's thought process and were convinced that this synergy is what would create magic for Wagh Bakri."
The TVC for Wagh Bakri is now in the final stages of post-production and would be ready for broadcast in the next few weeks.
Programming HeadRadio Mango


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Love and monogamy doesn't have to be synonymous. It's just that most prefer it that way.
I don't quite understand how you can be in love and not commit :/ Seems like those are just supposed to be together. If one is missing, it all falls apart
Yes it's possible, one of my friend it like that, he can't commit to person he loves (he is afraid of getting hurt) so he just just never dates them, he loves them from afar while dating other girls that he doesn't really like.
Not possible if it's real love. He can only be infatuated and filled with lust to not commit.
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Functions & Events at the Red Lion, Cromer
When planning an functions or events there can be a plethora of variables to consider; how many guests, who are the guests and most importantly when and where?
Situated on the beautiful North Norfolk coast and boasting a function room which can hold up to 80 guests, the Red Lion is the perfect location for your event. With the capacity to tailor the room and menu for your requirements whether it be a family celebration, a corporate meeting or a funeral gathering, we can offer a personalised experience for your function.
If you would like any further information or to discuss the options for your own function please do not hesitate to contact us either by filling out the form below or calling us directly on 01263 514 964.


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Hazel Reeves
Hazel Reeves is a specialist portrait sculptor working to commission. Obsessed by faces, portraits are at the core of her artistic practice. She is fascinated by how emotions and expressions flicker across the face, yet a person's character resides more permanently, captured in the way features of the face form and age. How to capture this is what obsesses her. Capturing a likeness, seen by the sitter and family or peers, is the wonderful challenge of portrait commissions. But her interests are moving beyond this, to seek to understand how society defines beauty or decides whose face is worthy of capturing in bronze. And to seek to challenge this by sculpting those who would normally never be sculpted let alone cast in bronze and placed in public – those who are excluded because of their race, their gender, age, class or dis/ability.
Hazel has received two prestigious awards: the Society of Portrait Sculptors' Master Class Prize in 2010 and 2012 and the Falle Fine Art 'Bronze' Award. She is on the Council of the Society of Women Artists and a Fellow of the Royal Society of Arts, exhibiting her work at London galleries and across the South of England and Wales. Her commissioned bronze sculpture of Sadako Sasaki was blessed during a ceremony on the World Day of Peace, 21st September 2012, in Wales. Hazel is an experienced tutor and loves teaching portrait sculpture.
Courses Taught
Hazel teaches on Short Courses


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By Dominic Toner
Erik Lamela was named in the team of the round for the EFL Cup after his exceptional performance against Gillingham.
Tottenham completed a comfortable 5-0 victory in the third-round and set up a huge cup match against Liverpool in the last 16.
Lamela, 24, scored one goal and grabbed three assists against the League One side as Mauricio Pochettino opted to rotate his squad.
The Argentinian midfielder will be hopeful of playing against Middlesbrough on Saturday, after being used as a substitute in the previous two league matches.
Lamela was the only Spurs player named in the starting XI, which comprised of six Premier League and five Championship representatives.
The official EFL Cup twitter account released their official team of the round.
TEAM OF THE ROUND: Our selection of stand-out stars from #EFLCup Round Three >> https://t.co/xdv41rHpmm. pic.twitter.com/nMYbYxuhIn
— EFL Cup (@EFLCup) September 23, 2016
Spurs remain unbeaten domestically this season, with their only defeat coming to Monaco in the Champions League, and currently sit third in the league table.
Mauricio Pochettino faces a selection headache for his next game about who will fill the three attacking positions.
Lamela will be competing with Dele Alli, Christian Eriksen, Moussa Sissoko, Heung-Min Son and Georges-Kevin N'Koudou for a starting position.
He has made 114 appearances for Spurs since his arrival in 2013 and scored 29 goals in that time.
In other Tottenham news, Mark Lawrenson has predicted the outcome against Middlesbrough.


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Time might have robbed David Cone of his best fastball, but time has also granted him a sense of perspective, which he badly needed after the Anaheim Angels beat him thoroughly Wednesday night in his first start of the year.
He faced 18 batters and 10 reached base before Cone was relieved in the third inning. And when his pitching line was complete, he had been charged with eight runs in two and two-thirds innings in the Yankees' 12-6 loss to the Angels.
The Yankees were to take a day off today before beginning a three-game series in Seattle on Friday.
The Yankees took two of three games from the Angels in the series, and some of the young players who might derive confidence from a good start performed well.
Jorge Posada, beginning his first season as a full-time, front-line catcher, went 4 for 4 Wednesday to increase his three-game batting average to .500, and Manager Joe Torre has been pleased with how Posada is handling the pitchers. Shane Spencer has two home runs, and after going hitless in the first two games, Ricky Ledee contributed a pinch-hit single.
Cone glumly reviewed his performance afterward, pausing between sentences as if he were chewing on thoughts before presenting them. But he recalled that two years ago he had pitched poorly in the season's first game at Yankee Stadium, allowing nine runs and 11 hits in four and one-third innings. And in his first two starts combined, Cone gave up six walks in nine and two-thirds innings. ''Ugly,'' was his succinct appraisal of his work then.Continue reading the main story
Cone pitched so poorly in April 1998, in fact, that he had very brief thoughts of retiring, but they were quickly dismissed.
What Cone will undoubtedly try to remind himself before he starts the Yankees' home opener next Tuesday, is that the raw quality of his stuff Wednesday night was pretty good. A radar gun posted behind home plate repeatedly flashed 88 to 91 miles an hour when he threw his fastball; it was as good as any reading Cone had last year.
His slider and split-fingered fastball, his two most reliable pitches in spring training, moved well when he warmed up in the bullpen before the game.
''He had good stuff,'' Posada said.
Cone might as well try to forget the other details of his outing, because he was terrible, his control failing him time and again. He fell behind the two balls and no strikes to the very first hitter, Darin Erstad, before misfiring on a slider. Cone meant to throw it over the outside corner, but it drifted over the plate and Erstad launched a home run.
''They probably sensed early that I wasn't getting my stuff over,'' Cone said, ''and they were going to force me to throw it for strikes.''
The pattern was established: Cone fell behind in the count, could not effectively pinpoint his slider or splitter and when he wasn't issuing walks -- he had five in his brief outing -- the Angels' hitters successfully seized on his lack of command, driving the ball.
There were several moments when a called strike on a close pitch might have helped Cone minimize the damage, but it just so happened that Paul Schrieber, the home plate umpire, was an unfamiliar presence to Cone, and there were no fringe benefits. Schrieber, wearing the new umpires' hat with the Major League Baseball logo above the bill, called a textbook strike zone.
Erstad, who accumulated nine hits in the three-game series, hit a two-run single in the second inning before Anaheim added five runs in the third. Mo Vaughn singled, Tim Salmon walked, Garret Anderson singled, Troy Glaus walked, and with two outs, Gary DiSarcina singled and Torre relieved Cone.
When Cone returned to the dugout, Don Zimmer, the bench coach who is bothered by bad knees, made a point of getting off the bench and greeting Cone with pat on the back.
''It was a combination of them having a really good approach, falling into a lot of hitters' counts and me missing a lot,'' Cone said. ''My inability to get my breaking stuff over for strikes really cost me. The trick is to make a quick adjustment, which is usually my strength, and I wasn't able to do that tonight. It seemed like I was just missing or was up too much. It's a helpless feeling when you feel good and your stuff is moving all over the place, and you can't quite find the zone.''
Cone's e.r.a. after his first start is 27.00.
''It's tough,'' he said. ''I've been in this position before, having a couple of bad starts. I'm encouraged with the way that I felt, I'm just disappointed with the results.''
With left-hander ANDY PETTITTE pitching Friday and probably generating a lot of ground balls, Manager JOE TORRE said that CLAY BELLINGER will play third base. He has more experience at the position than ALFONSO SORIANO, who was called up Wednesday to replace the injured SCOTT BROSIUS (rib cage strain) on the roster. Soriano will likely play Saturday and Sunday.Continue reading the main story


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New Delhi:Ahead of the November 21 by-election to Ratlam-Jhabua Lok Sabha constituency in Madhya Pradesh, Chief Minister Shivraj Singh Chouhan on Wednesday asserted that there would be no rethink on the issue of reservations.
"As long as Narendra Modi is the Prime Minister and I am the CM, nobody can end reservations," Chouhan said at campaign meetings at Bori and Baledi villages in the district. Ratlam-Jhabua constituency, where the by-election was necessitated by the death of BJP's Dilip Singh Bhuria, is reserved for the scheduled tribes.
The row over the issue of reservations had erupted during the Bihar Assembly elections after RSS supremo Mohan Bhagwat's statement calling for a review of the policy. Chouhan on Wednesday said that Congress distorted Bhagwat's statement and was "playing politics" over it.
Congress and other opposition parties have made the issue a major poll plank during the campaign here. Chouhan sought votes for BJP for a rapid and co-ordinated progress of the area, reminding the audience that BJP was in power both at the Centre and the state. He pointed out the welfare measures of his government such as one kg of rice at Re one for the poor and agricultural loans at zero percent interest.


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Diana has appeared in over 60 films/television shows since she started her acting career in the mid seventies.
She graduated from the American Academy of Dramatic Arts.
In mini-movies, she won an Emmy nomination playing 'First Lady' Bess Truman in "Truman" and later played Rose Kennedy in "JFK: Reckless Youth."
For her roles in "Inside Moves" and "Mommie Dearest," she became the first actress ever to go from being an Oscar nominee to a Razzie "winner" in just one year.


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After completing a two-year application process, NBAA's Certified Aviation Manager (CAM) program received accreditation last week from the National Commission for Certifying Agencies (NCCA), an independent body that developed the standards for professional credentialing programs. NCCA has accredited some 300 programs, including the Certified Financial Planner (CFP) and Certified Human Resource Professional (CHRP) designations.
According to the association, the NCCA accreditation demonstrates that the CAM credential, which was launched in 2001, provides a "valid measure of readiness" for a person to be an aviation manager. "Through certification, these individuals demonstrate their level of expertise and commitment to the aviation industry, and are tested in five subject areas: leadership, human resources, operations, technical and facilities services, and business management," NBAA said.
The CAM governing board had to create more than 700 pages of documentation on meeting 21 NCCA standards to complete the accrediting application process. NCCA accredited the CAM program for a five-year period through Oct. 31, 2018.
"We commend the work of the CAM governing board in reaching this major milestone for the program," said NBAA president and CEO Ed Bolen. "NBAA and the business aviation community acknowledge CAMs as professionals who have demonstrated an exemplary level of industry knowledge and management skills, and this accreditation serves to underscore their outstanding achievements and leadership potential."


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A CHARITY night organised by a mum with ovarian cancer and based on one of her favourite TV shows 'Peaky Blinders' has raised more than £1,000.
Edna Pomeroy, of Llandudno Junction, hosted the 1920's Gangster and Molls charity night last month for Cancer Research UK.
The 53-year-old was first diagnosed with perineum cancer - a form of ovarian cancer in December 2015 – and recently discovered her cancer had returned, making her even more determined to reach keep fundraising.
The event took place at Maenan Abbey in Llanrwst on Friday, March 24 and has currently raised more than £1,000 for the cancer charity – with more still being counted.
Mrs Pomeroy said: "I had just come of out hospital with a diagnoses of the return of my cancer which has been a bit of a shock, but it was not going to stop me attending the event. David at the Maenan Abbey and his team went out of their way to help us."
Edna in the centre with her husband Stephen on her right and the five Peaky Blinders re-enactors
The evening included an auction of promises with prizes donated from the likes of Zip World; Surf Snowdonia, Clogau Gold, and Tanners merchants.
There was also a raffle and five Peaky Blinders re-enactors who joined the party to bring the era to life.
Mrs Pomeroy - who is a re-enactor of the Napoleonic Era alongside her husband Stephen, added: "What a fantastic evening we had.
"I thank everyone who came and made the evening such a success, Maenan Abbey Hotel, our wonderful Peaky Blinder boys and organist, Kieran Eastwood- Mulvaney who was an absolute star."


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* We do not pass your details to third parties
Rix Petroleum is one of the largest independent family owned fuel distributors in the UK. Our competitively priced haulage fuel delivery service has been providing hauliers across the industry with high performance, efficient fuel for decades.
With huge demands on the haulage sector to deliver within tight deadlines, we know you need a fast and flexible supplier you can rely on with readily available fuels. We hold ISO 90001:2008 Quality Management and ISO 50001:2011 Energy Management accreditations.
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We supply everything from diesel to Q8 lubricants and AdBlue, so whether you operate a small fleet of haulage vehicles or have a national operation, you can rely on Rix to provide you with a complete end to end fuel solution.
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If you would like Rix Petroleum to take care of your haulage fleets fuel then please contact one of our dedicated haulage customer advisors at your nearest depot for more information.
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- Anónimo
- Inglaterra, Homem, 25-30
- Nómada principiante
- 1 comentário
PRO'S: Great location, friendly staff, good wifi. CON'S: Mould around taps, door to bathroom comes completely off hinges, poor aircon that does not cool the room, got bitten to shreds as mozzies coming through in numerous locations. Overall ok place but definitely would pay a few pounds more for a much better place.
- Anónimo
- Inglaterra, Mulher, 41+
- Nómada principiante
- 2 Comentários
The hotel is somewhat tired looking and in need of renovation. Had to ask to pay no staff around. Clean linen. Within walking distance to city 15 mins
- joanne27
- Canadá, Casal, 25-30
- Nómada principiante
- 3 Comentários
The room has a distinct old smell from the carpet, the shower area has molds on the tiles and glass door. The bed sheet on one of the beds in the room has a small menstrual blood stain upon our arrival to the room. The fan needs to be fixed, a missing screw needs to be replaced. The place is quiet throughout our stay, wifi and cable connection is excellent. It is a short walk to Cairns train station and marina and all other restaurants.
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text: Home inspiration for a long weekend By Carly Jacobs January 08, 2016 This content is only available in Chinese. Tags: family living backyards and gardens Carly Jacobs Carly is the editor of Smaggle and a freelance writer and presenter. Her writing has appeared in Cleo, Cosmoplitan, The Age and The Sydney Morning Herald. She almost got run over by Myf Warhurst whilst out jogging once and natural yogurt is her favourite food.


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Cook Insurance Agency, Inc. is an independently owned and locally operated insurance agency that has been serving Bates and the surrounding counties for over 50 years. We believe that local insurance agencies can best serve your insurance needs. We will assist you in finding just the right combination of tailored, personalized insurance coverage at just the right price. We offer low down payments and monthly payment options to simplify your insurance purchase. We invite you to complete an online quote or to call us to review coverage options for auto, home, life, health, business, commercial, renters, boat and more. We provide free, no hassle and no obligation quotes to let you see what we have to offer.
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Be the first NJ.com user to add photos or videos of Freehold Public Library
Cute little library in downtown Freehold. Would have been a great place to get some work done except for the staff's constant conversation with a patron. I learned all about the fatal accident a...
The Freehold Public Library was the second Carnegie library built in New Jersey. The library is independent of the Monmouth County Library system and is free for Freehold Borough residents. Citizens of other Monmouth County municipalities, including Freehold Township, can become a member for a $10 annual fee.
The Freehold Public Library has a garden area in the back.