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In the liver, sterol 27-hydroxylase (CYP27) participates in the classic and alternative pathways of bile acid biosynthesis from cholesterol (Chol). In extrahepatic tissues, CYP27 converts intracellular Chol to 27-hydroxycholesterol (27OH-Chol), which may regulate the activity of 3-hydroxy-3-methylglutaryl CoA reductase (HMG-CoA-R). This study attempts to better define the role of CYP27 in the maintenance of Chol homeostasis in hepatic and extrahepatic cells by overexpressing CYP27 in Hep G2 cells and Chinese hamster ovary (CHO) cells through infection with a replication-defective recombinant adenovirus encoding for CMV-CYP27. After infection, CYP27 mRNA and protein levels increased dramatically. CYP27 specific activity also increased two- to fourfold in infected cells (P ≤ 0.02), with a marked increase in conversion of [14C]Chol to [14C]27OH-Chol (∼150%; P ≤ 0.01). Accumulation of 27OH-Chol in CHO cells was associated with a 50% decrease in HMG-CoA-R specific activity (P ≤ 0.02). In infected Hep G2 cells, the significant increase in bile acid synthesis (46%; P ≤ 0.006), which prevented the accumulation of intracellular 27OH-Chol, resulted in increased HMG-CoA-R activity (183%; P ≤ 0.02). Overexpression of CYP27 in Hep G2 cells also increased acyl CoA-cholesterol acyltransferase (71%,P ≤ 0.02) and decreased cholesteryl ester hydrolase (55%, P ≤ 0.02). In conclusion, CYP27 generates different physiological responses depending on cell type and presence or absence of bile acid biosynthetic pathways.
- liver
- bile acid
- 3-hydroxy-3-methylglutaryl CoA reductase
- Hep G2 cells
- acyl CoA-cholesterol acyltransferase
- cholesteryl ester hydrolase
- adenovirus
- gene
hepatic cholesterol homeostasis is maintained largely through the coordinate regulation of cholesterol uptake, esterification, biosynthesis, secretion, and degradation to bile acids. In humans, conversion of cholesterol to bile acids accounts for approximately half of daily net cholesterol elimination from the body, and most of the remaining 50% is eliminated in the form of biliary cholesterol secretion (36).
In the liver, the 27-hydroxylation of cholesterol by mitochondrial sterol 27-hydroxylase (CYP27) is the initial step in an alternative or acidic pathway of bile acid biosynthesis (Fig.1). Once formed, 27-hydroxycholesterol (27OH-Chol) can compete with cholesterol for binding and further oxidation. Under nonsaturating substrate concentrations, 27OH-Chol can be further oxidized by mitochondrial CYP27 to form 3β-hydroxy-5-cholestenol and finally 3β-hydroxy-5-cholestenoic acid (22). Both 27OH-Chol and 3β-hydroxy-5-cholestenoic acid are substrates for the newly discovered microsomal oxysterol 7α-hydroxylase (CYP7B1). Recent in vivo studies in the rat (37) and in primary rat hepatocytes (23, 28) have suggested that this pathway may be responsible for generating up to 50% of bile acid biosynthesis (Fig. 1).
CYP27 is also responsible for the 27-hydroxylation of bile acid intermediates (5β-cholestane-3α, 7α-diol, and 5β-cholestane-3α, 7α, 12α-triol) in the neutral pathway of bile acid biosynthesis, a pathway initiated by cholesterol 7α-hydroxylase (CYP7A1) (Fig. 1) (7). CYP27 has been found in peripheral tissues (i.e., extrahepatic tissues), where it has been shown to convert cholesterol to 27OH-Chol and 3β-hydroxy-5-cholestenoic acid (3, 4, 5, 17). These metabolites efflux from the peripheral cells and are incorporated into the lipoprotein fractions, which are then transported to the liver where they are presumably metabolized via the alternative pathway into bile acids. This mechanism of cholesterol elimination from peripheral cells may represent a second reverse cholesterol transport pathway to the liver (3, 4, 5, 17).
The addition of 27OH-Chol, the product of this reaction, to various cell lines has resulted in repression of 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA-R), the rate-determining enzyme in cholesterol biosynthesis (2, 12). On the basis of these observations, it has been hypothesized that circulating 27OH-Chol may be a potent negative feedback regulator of HMG-CoA-R in peripheral and possibly liver cells (2, 5): 27OH-Chol has also been shown to stimulate acyl-CoA-cholesterol acyltransferase (ACAT) activity, thereby increasing the esterification of itself, cholesterol, and other potential regulatory oxysterols.
The mechanism of regulation of CYP27 is just beginning to be elucidated. Studies in the rat (both in vitro and in vivo) have shown that CYP27 is downregulated at the transcriptional level by hydrophobic bile acids (28, 34, 35). In contrast, in the rabbit, CYP27 mRNA is neither repressed by bile acids nor stimulated by cholestyramine. Cholesterol stimulates rabbit CYP27, whereas bile drainage has no effect on mRNA levels (1). Furthermore, CYP27 in the rat has been shown to undergo diurnal variation, is upregulated by glucocorticoids, and is downregulated by insulin (28, 33, 35).
A genetic defect in CYP27 in humans leads to the development of cerebrotendinous xanthomatosis (CTX), a condition characterized by progressive neurological dysfunction, xanthomatosis, and accelerated atherosclerosis (25). As a result of point mutations in the CYP27 gene, patients with CTX exhibit a marked decrease in bile acid synthesis and reduced bile acid pool size (in particular chenodeoxycholic acid) (16). In mice with targeted disruption of CYP27 (CYP27−/−), there is reduced metabolism of cholesterol to bile acids, with increased cholesterol synthesis (24). However, CYP27−/− mice demonstrated no accumulation of cholestanol or bile alcohols. CYP27−/− mice showed no CTX-related pathological abnormalities, suggesting that some of the consequences of CYP27 deficiency are specific for the species being studied. The presence of a 25-hydroxylase pathway in the mouse, as shown by Honda et al. (13), offers an explanation for the lack of a specific mouse phenotype. However, an increased death rate was observed when CYP27−/− mice were fed an atherogenic diet, suggesting that CYP27 may be an antiatherogenic agent (24).
In the current study, we used a replication-defective recombinant adenovirus encoding cytomegalovirus (CMV)-CYP27 to overexpress CYP27 in hepatic and Chinese hamster ovary (CHO) cells and attempted to define the role of this enzyme in the maintenance of cellular cholesterol homeostasis in CHO cells (representative of extrahepatic cells), Hep G2 cells, and primary human hepatocytes.
Hep G2 and CHO cells were obtained from the American Type Culture Collection (Rockville, MD). All cell culture materials were obtained from GIBCO BRL (Grand Island, NY) unless otherwise specified. The CsCl, agarose, and RNA ladder used to size CYP27 mRNA were also purchased from GIBCO BRL. FBS was obtained from Bio Whittaker (Walkersville, MD). Tissue culture flasks were purchased from Costar (Cambridge, MA). The chemicals used in this research were obtained from Sigma Chemical (St. Louis, MO) or Bio-Rad (Hercules, CA) unless otherwise specified. The secondary antibody (horseradish peroxidase-conjugated goat anti-mouse IgG) was also purchased from Bio-Rad. All solvents were obtained from Fisher (Fair Lawn, NJ) unless otherwise indicated. All radionucleotides, Aquasol solution, and enhanced chemiluminescence reagents were purchased from Dupont NEN (Boston, MA). Waters Silica Sep-Paks were obtained from Waters (Milford, MA). The β-cyclodextrin was purchased from Cyclodextrin Technologies Development (Gainesville, FL). The testosterone and 27OH-Chol were obtained from Research Plus (Bayonne, NJ). LK6 20 × 20-cm thin layer chromatography (TLC) plates were purchased from Whatman (Clifton, NJ). Mevalonate was obtained from Aldrich Chemical (Milwaukee, WI). Nylon membranes were purchased from Micron Separation (Westborough, MA). Ammonium persulfate was obtained from Amresco (Solon, OH). Finally, nonfat dry milk was purchased from Carnation (Glendale, CA).
Generation of recombinant adenovirus encoding rat CYP27.
The recombinant adenovirus AdCMV-CYP27 was generated by homologous recombination in HEK-293 cells. A rat cDNA encoding CYP27 (1.9 kb) was obtained from N. G. Avadhani (31) and cloned into plasmid (AdCMV, 7.7 kb) constructed from pGEM 4z (Promega) and pcDNA3 (Invitrogen). The CYP27 gene was cloned into the AdCMV vector at the EcoR I restriction site. Transfection into HEK-293 cells was used to confirm expression by the recombinant adenovirus.
Propagation and purification of AdCMV-CYP27.
Large-scale production of recombinant virus was performed by infecting confluent monolayers of HEK-293 cells grown in 15-cm tissue culture dishes with stock adenovirus at a multiplicity of 1 plaque-forming unit (pfu)/cell. After 2 h of infection, the virus was removed and replaced with DMEM with 2% FBS. The infected monolayers were harvested by scraping when >90% of the cells showed cytopathic changes and were centrifuged at 2,700 g and 4°C for 10 min. The infected cellular pellet was suspended in DMEM-2% FBS and subjected to 5 cycles of freeze/thaw lysis to release the virus. Cell debris was removed by centrifugation at 7,700 g and 4°C for 5 min. To purify, the crude viral supernatant was carefully layered over a two-step gradient containing 3 ml of CsCl [d (density) = 1.4 g/ml] in TD buffer (0.14 M NaCl, 5 mM KCl, 19 mM Tris, pH 7.4, and 0.7 mM Na2HPO4) layered over 3 ml of CsCl (d = 1.25 g/ml) in Tris buffer, and centrifuged at 155,000 g and 20°C for 1 h. The viral band was removed, layered over 8 ml of CsCl (d = 1.33 g/ml) in TD buffer, and centrifuged at 155,000 g and 20°C for 18 h. The pure viral opalescent band was removed and dialyzed against 10 mM Tris, pH 7.4, 1 mM MgCl2, and 10% glycerol overnight at 4°C. The virus was aliquoted and stored at −70°C. The virus titer (pfu) was determined by plaque assay, and viral particles were determined by optical density using spectrophotometry (λ = 260 nm).
Cell medium.
Hep G2 cells were grown in MEM containing nonessential amino acids, 0.03 M NaHCO3, 10% FBS, 1 mM l-glutamine, 1 mM sodium pyruvate, and 1% Pen/Strep and incubated at 37°C in 5% CO2. CHO cells were grown in DMEM containing 0.04 M NaHCO3, 10% FBS, and 1% Pen/Strep and incubated at 37°C in 5% CO2.
Isolation of primary human hepatocytes.
Primary human hepatocytes were isolated according to the method of Strom et al. (29, 30). Before plating, cells were judged to be >90% viable by using trypan blue exclusion. Cells were then plated onto 150-mm plastic Petri dishes previously coated with rat tail collagen and were incubated in 20 ml of William's E Medium supplemented with insulin (0.25 U/ml) and penicillin (100 U/ml) in a 5% CO2 atmosphere at 37°C. Unless otherwise indicated, culture medium contained 0.1 μM dexamethasone and 1.0 μMl-thyroxine.
Infection of cells with AdCMV-CYP27.
Hep G2 cells and CHO cells were grown in 162 cm2 (25 ml) tissue culture flasks until they were 80–90% confluent. In control flasks (i.e., no adenovirus), the old medium was removed and replaced with 25 ml of fresh medium. In flasks to be infected, the old medium was removed and replaced with 12 ml of fresh medium. Hep G2 and CHO cells were infected with AdCMV-CYP27 or the control adenovirus without the CYP27 gene (AdCMV+1) at a multiplicity of infection of 1 to 10. After 3 h of infection, an additional 13 ml of fresh medium was added to the flasks and cells were allowed to incubate at 37°C in 5% CO2 for 48 h. Cells (both control and infected) were harvested for the isolation of mitochondria, microsomes, cytosol, and RNA.
Primary human hepatocytes were plated in 152 cm2plates until they were 80–90% confluent. Twenty-four hours after plating, culture medium was removed and 2.5 ml of fresh medium was added. Cells were then infected with AdCMV-CYP27 at a multiplicity of infection of 10. After 2 h of infection (with gentle shaking every 15 min), the media were discarded and 2 0 ml of fresh medium was added. The cells were then allowed to incubate at 37°C in 5% CO2 for 48 h before harvesting.
Mitochondrial and microsomal preparation.
Mitochondria and microsomes were isolated from cell culture as described previously (28, 35).
Purification of cholesterol.
Cholesterol was purified by the method of Winegar et al. (39). Cholesterol, dissolved in hexane (0.6 mg/ml), was loaded dropwise onto a dry Waters Silica Sep-Pak. Cholesterol was eluted with 8 ml 2% isopropanol in hexane, and the eluant was dried under a nitrogen gas atmosphere. For the CYP27 specific activity assay, 375 μl of β-cyclodextrin were added to the dry extract and vortexed vigorously until the cholesterol was completely dissolved.
For the determination of bile acid and 27OH-Chol synthesis, 10 μCi of [14C]cholesterol (250 μl) was dried down under nitrogen gas and dissolved in 2 ml hexane. The radiolabeled cholesterol was purified by use of a Waters Silica Sep-Pak as described above. The dry extract was taken up in 250 μl ethanol and added to 50 ml of complete Hep G2 or CHO culture media. Purity was determined by running a small aliquot of the purified [14C]cholesterol (0.05 μCi) on TLC using the buffer system mentioned below for determination of 27OH-Chol synthesis.
CYP27 activity assay.
CYP27 specific activity was assayed by the method of Petrak et al. (21), with a few modifications. The assay was performed on 300 μg of freshly prepared mitochondrial protein, to prevent loss of activity due to freeze/thaw. Purified exogenous cholesterol (∼80 nmol) in β-cyclodextrin was added to each reaction tube to increase the concentration of substrate available to the enzyme. Testosterone, at a concentration of 0.003 μg/μl, was added to each reaction tube as an internal standard. Control reactions were produced in which 0.003 μg/μl 27OH-Chol was substituted for mitochondrial protein. Samples were suspended in 100 μl of 5% isopropanol in hexane before HPLC analysis. An aliquot of 50 μl was analyzed via normal-phase HPLC on a Beckman ultrasphere silica column (4.6 × 250 mm) by using a mobile phase of hexane-isopropanol-glacial acetic acid (96.5:2.5:1 vol/vol/vol). All assays were carried out in duplicate including the appropriate controls. All 27OH-Chol concentrations were corrected for the amount of testosterone recovered, and CYP27 activity was calculated as nanomoles of product produced per milligram of sample per minute. Finally, CYP27 activities were expressed as percentage of control values.
ACAT specific activity assay.
ACAT activity was determined by the methods of Burrier et al. (6) with the following modifications. ACAT reactions were carried out using 40 μg microsomal protein in a buffer containing 0.1 M potassium phosphate, 9.8 mM β-mercaptoethanol, and 0.01 μM BSA in a total volume of 50 μl. Purified exogenous cholesterol was added to incubations from cyclodextrin stock solutions in 5-μl aliquots (1.2 mg purified cholesterol/0.375 ml β-cyclodextrin). After a 15-min preincubation, [1-14C]oleoyl-CoA (NEN; 55 μCi/μmol) was added to a final concentration of 7.2 pM. Thirty minutes after addition of [1-14C]oleoyl-CoA, reactions were terminated by direct application of a 20-μl aliquot of each sample to silica gel TLC plates (20 × 20 cm; Whatman LK6). Oleoyl-CoA and cholesterol oleate were plated as standards. The chromatograms were developed in hexane-diethylether-glacial acetic acid (85:15:1 vol/vol/vol). [14C]Cholesterol oleate formation was detected by using a PhosphorImager (Molecular Dynamics), and the substrate and cholesterol oleate bands were scraped into scintillation vials containing biodegradable counting scintillant (Amersham, Arlington Heights, IL) and counted in a Beckman liquid scintillation counter. Cholesterol oleate concentration was calculated as a percentage of oleoyl-CoA substrate to correct for possible loading errors, and values for samples were expressed as a percentage of control values.
Cholesterol ester hydrolase activity assay.
Four hundred micrograms of cytosolic protein, obtained when isolating microsomes for the ACAT assay, were used in the cholesterol ester hydrolase (CEH) assay. CEH activity was measured by the methods of Ghosh and Grogan (10).
HMG-CoA-R activity assay.
Microsomal HMG-CoA-R specific activity was assayed essentially by the method of Shefer et al. (26).
Quantitation of CYP27 mRNA levels.
Methods for the isolation of RNA and the determination of mRNA levels by Northern blot have been previously described by Pandak et al. (20). The cDNA probe used in these experiments has also been described previously (31).
Bile acid and 27OH-Chol synthesis.
Labeled bile acids and 27OH-Chol were extracted from both media and cells by using the method of Folch et al. (9). Bile acid biosynthesis was measured as conversion of [14C]cholesterol to 14C-labeled bile acids (14). [14C]27OH-Chol was isolated from the cell extract or media CHCl3 phase on TLC (hexane-isopropanol-glacial acetic acid; 95:3:2 vol/vol/vol). [14C]27OH-Chol (Rf = 0.19) and [14C]cholesterol (Rf = 0.36) bands were scrapped from the TLC plates, and radioactive counts were obtained using a Beckman liquid scintillation counter. 27OH-Chol concentration was calculated as a percentage of the cholesterol substrate to correct for possible loading errors. Both bile acid biosynthesis rates and 27OH-Chol concentrations for infected samples were expressed as a percentage of control values.
Immunoblot analysis of CYP27 protein levels.
Mitochondrial and cytosolic proteins (30 μg) were separated on a 10% SDS-polyacrylamide denaturing gel according to the method of Laemmli (15). Following electrophoresis, proteins were electrophoretically transferred to Immobilon-P membranes by using a Millipore rapid transfer system. The membranes were blocked overnight (4°C) in blocking buffer (PBS, pH 7.4, 0.1% Tween, and 5% nonfat dry milk). They were then incubated with for 2 h (25°C) with a mouse CYP27 monoclonal IgG (1:1,000) as previously described (31). After membrane washing, a secondary antibody (horseradish peroxidase-conjugated goat anti-mouse IgG; 1:3,000) was added to the blocking solution (25°C, 2 h). Protein bands were detected using the Dupont Renaissance enhanced chemilluminescence kit.
Results are reported as means ± SE where possible. Statistical significance was determined where appropriate by Student'st-test.
Figure 2 is a representative Northern blot showing CYP27 mRNA levels in Hep G2 and CHO cells before and after infection with AdCMV-CYP27. As shown, messenger RNA was isolated from Hep G2 and CHO cells harvested at several time points (24, 48, and 72 h) after infection. CYP27 mRNA levels increased with time in both cell lines, from undetectable levels in control cells to easily detectable levels in infected cells. To standardize RNA loading as well as quantitate relative CYP27 mRNA levels, cyclophilin levels were determined as an internal loading standard. The cyclophilin levels shown in this figure were similar in each lane, showing that similar amounts of RNA were loaded into each well.
As a second set of controls, AdCMV+1 was added to cell cultures. No visible changes in CYP27 mRNA levels were observed compared with no-addition controls (Fig.3). Blots in which control and infected samples were separated by several blank lanes required additional exposure to detect CYP27 mRNA levels in control Hep G2 cells. By using densitometry, it was then estimated that CYP27 mRNA levels increased at least 1,300-fold (P ≤ 0.002; n = 6) following AdCMV-CYP27 infection (data not shown).
CYP27 protein levels in mitochondria were determined by immunoblot analysis by using a mouse CYP27 monoclonal antibody. As seen with CYP27 mRNA levels, mitochondrial CYP27 protein levels increased from undetectable levels in control cells under the conditions employed to easily detectable levels in infected Hep G2 and CHO cells (Fig.4). Using Hep G2 cells as an example, we have shown an increase in CYP27 protein levels with time following AdCMV-CYP27 infection (Fig. 4). No CYP27 protein was found in the cytosol, indicating that the protein was localized to the mitochondria, where it is known to be active (data not shown).
The increases in CYP27 mRNA and protein levels observed in cells infected with AdCMV-CYP27 were associated with a significant increase in mitochondrial CYP27 specific activity. Illustrated in Fig.5 are the effects of adenoviral infection on CYP27 specific activity. Forty-eight hours after infection with AdCMV-CYP27, CYP27 specific activity increased significantly (P < 0.02) in both Hep G2 and CHO cells (Hep G2, from 0.018 to 0.050 nmol · mg−1 · min−1; CHO, undetectable to 0.033 nmol · mg−1 · min−1). No increase in CYP27 specific activity was observed in cells infected for 48 h with a control virus (Fig. 5).
Infected CHO cells showed a marked increase in conversion of [14C]cholesterol to 14C-labeled 27OH-Chol, as evidenced by an increase in intracellular and extracellular 27OH-Chol levels (∼150%; P ≤ 0.01; Fig.6 and Table1). In contrast, accumulation of 27OH-Chol in Hep G2 cells was only 16% greater after infection with the CYP27 adenovirus (Fig. 6).
The effect of CYP27 overexpression on the rates of bile acid synthesis in both Hep G2 and primary human hepatocytes is shown in Fig.7. CYP27 overexpression in Hep G2 cells led to a significant increase in bile acid biosynthesis (46%;P ≤ 0.006; n = 4) (Fig. 7). In one experiment in primary human hepatocytes, the increase in bile acid synthesis was 61% following CYP27 overexpression.
In CHO cells, endogenous accumulation of 27OH-Chol (0.23 ng/1 × 106 cells), due to infection with AdCMV-CYP27, was coupled with a 50% decrease (P ≤ 0.02) in HMG-CoA-R activity (Fig.8). HMG-CoA-R specific activity in control CHO cells was 5.2 ± 0.47 nmol · mg−1 · h−1(n = 3). The exogenous addition of 27OH-Chol to CHO cells in concentrations up to 100 times the cellular levels found following infection with the AdCMV-CYP27 had no effect on HMG-CoA-R specific activity. In Hep G2 cells, HMG-CoA-R specific activity increased dramatically (183%; P ≤ 0.02) following infection with AdCMV-CYP27 (Fig. 8). HMG-CoA-R specific activity in control Hep G2 cells was 2.13 ± 0.96 nmol · mg−1 · h−1(n = 3). No change in HMG-CoA-R specific activity was observed when Hep G2 and CHO cells were infected with control virus (data not shown).
The effects of CYP27 overexpression on other enzymes involved in the regulation of cellular cholesterol homeostasis are shown in Fig. 8. ACAT specific activity increased (71%; P ≤ 0.02), whereas CEH specific activity decreased (55%; P ≤ 0.02) when Hep G2 cells were infected with the AdCMV-CYP27. Overexpression of CYP27 in CHO cells had no significant effect on ACAT or CEH specific activity. Cells infected with a control virus showed no change in either ACAT or CEH specific activity (data not shown). Paired control specific activities in Hep G2 cells for ACAT and CEH were 7.8 ± 0.85 and 0.38 ± 0.03 nmol · mg−1 · h−1, respectively (n = 3). Paired control specific activities in CHO cells for ACAT and CEH were 9.4 ± 1.4 and 1.33 ± 0.15 nmol · mg−1 · h−1, respectively (n = 3).
The overexpression of CYP27 in both CHO and Hep G2 cells led to different alterations in cellular cholesterol metabolism in the two types of cells studied. The observed changes appeared to be a result of increased conversion of cholesterol to 27OH-Chol, which was coupled with an increase in CYP27 specific activities. The cell-specific responses to increased CYP27 specific activities seemed to depend on whether or not the cell in question had a bile acid biosynthesis pathway capable of metabolizing 27OH-Chol. The most important finding of the present study was the observation that overexpression of CYP27 in Hep G2 cells resulted in a significant increase in CYP27 steady-state mRNA levels, protein specific activity, and bile acid synthesis. These results demonstrate that the alternative pathway is fully capable of increasing the rates of bile acid synthesis and hence increasing the rates of cholesterol elimination.
A large discrepancy in the magnitude of increase in CYP27 mRNA and protein levels versus that in CYP27 specific activities was observed in cells infected with AdCMV-CYP27. Even though the increases in CYP27 specific activities were not large, they were consistent following infection and coupled with significant increases in bile acid synthesis. The reason for the disparity in specific activity levels and in protein and mRNA levels is being explored. One possible explanation is the insufficient delivery of the substrate, cholesterol, to the enzyme. Another is lack of sufficient ferridoxin/ferridoxin reductase availability in the mitochondria.
CYP7B1 catalyzes the second step in the alternative pathway by adding a hydroxyl group to the C-7 position. It has been suggested that, as in the neutral pathway of bile acid synthesis, this 7α-hydroxylation step could represent the rate-determining step in this pathway. In preliminary experiments, overexpression of CYP7B1 increased CYP7B1 mRNA and specific activities but failed to increase bile acid synthesis. Furthermore, coinfection of Hep G2 cells with AdCMV-CYP27 and AdCMV-CYP7B1 did not increase bile acid synthesis over CYP27 overexpression alone. These findings suggest that CYP27, not CYP7B1, is more likely to be the rate-determining enzyme in the alternative pathway of bile acid synthesis.
Reasons for the diverse tissue localization of CYP27 have been previously discussed (5, 8). It has been suggested that CYP27 is important in the elimination of cholesterol from extrahepatic (i.e., peripheral cell) tissues through its conversion to the more polar 27OH-Chol and 3β-hydroxy-5-cholestenoic acid (5, 8,38). Effluxed 27OH-Chol and 3β-hydroxy-5-cholestenoic acid are then incorporated into lipoproteins and translocated to the liver, where they are metabolized to bile acids presumably via the alternative bile acid biosynthetic pathway. Assuming that all the 27-oxygenated products taken in by the human liver are converted to bile acids, it has been suggested that ∼4–10% of total daily bile acid biosynthesis could stem from metabolism of circulating 27-oxygenated metabolites (17). Recently, studies have shown that 27OH-Chol competes with cholesterol for high density lipoprotein-mediated efflux from peripheral cells, whereas albumin facilitates the efflux of 3β-hydroxy-5-cholestenoic acid (4). Therefore, CYP27 metabolism of cholesterol to 27OH-Chol and 3β-hydroxy-5-cholestenoic acid may represent a second pathway of reverse cholesterol transport.
In the present study, CHO cells (i.e., no bile acid biosynthesis pathway) in which CYP27 had been overexpressed for 48 h were characterized by increased CYP27 activity, as manifested by increased conversion of [14C]cholesterol to 14C-labeled 27OH-Chol and increased efflux of labeled 27OH-Chol into the culture medium (Figs. 5, 6, and 8 and Table 1). Despite the increased formation of 27OH-Chol, the ratio of intracellular 27OH-Chol to medium 27OH-Chol remained the same as in control cells. Therefore, efflux of 27OH-Chol from the cell is not arbitrary but appears to be under tight regulatory control. Westman et al. (38) also found that the cellular efflux of 27OH-Chol was proportional to total sterol cellular content in human monocyte-derived macrophages.
Numerous oxysterols, most notably 27OH-Chol, have been shown in in vitro experiments to be potent repressors of HMG-CoA-R and cholesterol biosynthesis (2). On the basis of these findings, it has been hypothesized that circulating 27OH-Chol may be a potent negative feedback regulator of HMG-CoA-R activity both in liver and in the peripheral cells (19). Incubation of cells with control virus did not alter any of the cholesterol homeostatic parameters measured. We believe that the adenovirally mediated increase in CYP27 expression represents a more physiological method of studying the role of 27OH-Chol as a regulator of HMG-CoA-R activity than the addition of 27OH-Chol to the culture medium. It is feasible that intracellularly generated 27OH-Chol may exert different regulatory effects from exogenous addition of 27OH-Chol.
Although in our experiments efflux of 27OH-Chol into the medium increased with adenoviral infection, intracellular 27OH-Chol accumulated in CHO cells (0.23 ng/1 × 106 cells). The intracellular accumulation of 27OH-Chol following overexpression of CYP27 was coupled with decreased cholesterol biosynthesis (∼50% HMG-CoA-R activity). Our results suggest a mechanism by which CYP27 may act as an antiatherogenic enzyme by simultaneously mediating the elimination of exogenous cholesterol from peripheral tissues and by inhibition of peripheral cholesterol synthesis.
Recently, it has been shown that 7α-hydroxylation of 27OH-Chol by CYP7B1 eliminates the ability of 27OH-Chol to downregulate cholesterol biosynthesis (19). This finding suggests that 27OH-Chol would have little or no effect on cholesterol biosynthesis in liver cells, where it is rapidly converted to the cholest-5-ene, 3β, 7α, 27-triol (by microsomal CYP7B1) and subsequently to bile acids. This proved to be the case in Hep G2 cells. Microsomal 7α-hydroxylase activity specific for 27OH-Chol has been found in Hep G2 cells by our lab (unpublished data) and others (18). Even though CYP27 specific activity increased almost twofold, 27OH-Chol levels increased only ∼16% (P < 0.01) over control levels in Hep G2 cells following infection with AdCMV-CYP27(Fig. 6). The small accumulation of 27OH-Chol in infected Hep G2 cells had no downregulatory effect on HMG-CoA-R specific activity. In fact, HMG-CoA-R specific activity in Hep G2 cells increased almost twofold following infection with AdCMV-CYP27. The increase in HMG-CoA-R specific activity occurs most likely because of a compensatory mechanism of depletion of cholesterol from the intracellular pools resulting from an increased CYP27 specific activity/bile acid biosynthesis. On the basis of these data, it would seem highly unlikely that under physiological conditions 27OH-Chol levels in hepatocytes could accumulate to the point at which they would downregulate hepatic cholesterol biosynthesis.
Increased 27OH-Chol levels in CHO cells had no effect on ACAT activity, implying that 27OH-Chol is not a potent regulator of ACAT activity. In Hep G2 cells, ACAT activity increased with the increase in HMG-CoA-R activity. This upregulation in ACAT specific activity is consistent with the observed increase in newly synthesized cholesterol (183% increase in HMG-CoA-R specific activities), which supports previous observations suggesting that microsomal cholesterol availability is a potent regulator of ACAT activity (32).
CEH specific activities have been closely tied to cholesterol availability. Cholesterol feeding and mevalonate infusion both lead to decreased CEH specific activity, whereas inhibition of cholesterol synthesis with lovastatin stimulated CEH activity. An increased cholesterol flux observed with cholestyramine or chronic biliary diversion has also been associated with increased CEH specific activity (11). The increase in bile acid synthesis coupled with higher HMG-CoA-R specific activity following CYP27 overexpression in Hep G2 cells seems to most closely follow these last two experimental models.
In summary, we have successfully overexpressed CYP27 in both hepatic and nonhepatic cells in culture. This type of endogenous overexpression should represent a more physiological method of studying the effects of increased CYP27 specific activity and the role of 27OH-Chol in cholesterol and bile acid metabolism. In Hep G2 cells, overexpression of CYP27 resulted in an increase in bile acid biosynthesis with a compensatory stimulation of cholesterol biosynthesis. In CHO cells, where there is no bile acid biosynthesis pathway, CYP27 overexpression resulted in an increase in the cellular levels of 27OH-Chol with subsequent downregulation of HMG-CoA-R specific activity and cholesterol biosynthesis, as well as increased efflux of 27OH-Chol into the culture medium (Fig. 9).
These results suggest that CYP27 could act as an antiatherogenic enzyme in a number of tissue-specific ways. More specifically, CYP27 mediates the removal of excess cholesterol from peripheral tissues through its conversion to 27OH-Chol and 3β-hydroxy-5-cholestenoic acid. The ability of peripheral cells to increase CYP27 specific activity and thus the intracellular concentrations of 27OH-Chol may also allow for downregulation of cholesterol synthesis. Within hepatocytes, overexpression of CYP27 facilitates the removal of cholesterol from the body by acting as an upregulator of bile acid biosynthesis. These in vitro data suggest that CYP27 generates different physiological responses in the body depending on the cell type and the presence or absence of bile acid biosynthetic pathways.
The primary human hepatocytes were a gracious gift from Dr. Robert Fisher, Department of Transplant Surgery at the Medical College of Virginia, Virginia Commonwealth University. We also thank the following people for their technical assistance: P. Bohdan, E. Gurley, and K. Redford.
This work was supported by a National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) Program Project Grant (P01-DK-38030) and Merit Review Grants from the McGuire Department of Veterans Affairs Medical Center. Dr. Hall is the recipient of a National Research Service Award (5-F-32-DK-09821) from the NIDDK and a Liver Foundation grant.
Address for reprint requests and other correspondence: W. M. Pandak, McGuire DVAMC, GI Section (111 N), 1201 Broad Rock Blvd., Richmond, VA 23249.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
- Copyright © 2001 the American Physiological Society


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Mobile Learning Symposium
June 1, 2011
IM to co-sponsor "Mobile Learning" symposium June 10
Bradley University's Department of Interactive Media will co-sponsor a symposium on "mobile learning" Friday, June 10, at the Caterpillar Global Communications Center.
The department is teaming up with Float Mobile Learning of Morton, which strives to help companies and individuals understand how effective mobile learning can be. Float believes needed information should be accessible anywhere and anytime. The company's definition of mobile learning is simple: "mLearning is the use of mobile technology to aid in the learning of, reference of or exploration of information useful to the user at that moment or in a specific use context."
"This event marks the first time that a mobile-focused event has happened on Bradley's campus," said Chad Udell, managing director of Float Mobile Learning.
Anyone interested in mobile learning technology is encouraged to attend the event. It will be geared toward marketing, learning, and information technology professionals and focus on mobile design, development and strategy. The symposium will host a variety of speakers including Jim Ferolo, chair of the Department of Interactive Media at Bradley, and professionals representing TMobile, O'Reilly Press, Magic+Might, and i5 Research. Udell will also speak at the event.
"The caliber of speakers and expertise we have assembled is more akin to a conference line-up you would see on the coasts, not in Central Illinois. We're bringing great talent to the department with this day and giving faculty and students an opportunity to see industry leaders and respected authors who will provide valuable thought leadership right here on campus for a meager price," Udell said.
Jim Ferolo sees opportunity in the rapidly advancing field of mobile technology. IM students and faculty at Bradley have been working on a variety of mobile games and applications.
"We recognize that this is the fastest growing platform out there right now. We believe that our students can become thought leaders by following the mobile trend," Ferolo said.
"This symposium allows for students and business people to come together. We are hoping that this can be the first in a series of symposiums," Ferolo said.
Two different sessions have been planned for the symposium. Those who attend will have the option of choosing between "Business and Strategy" and "Design and Development." A continental breakfast and lunch will be provided. Students who attend can earn course credit. The registration fee is $179.

subset: commoncrawl


text: In an attempt to help your FM transmitter "blend into your car's dashboard," Green House has produced and released a pretty simple FM transmitter that looks like a shift-knob. It plugs into the standard 12V DC power outlet and includes an audio jack for plugging in an MP3 player and a USB port for charging supported devices. The real hilarious hijinks come when you accidentally try to aggressively shift with this device and tear the hell out of your dash. That, or you put it in a vehicle with a "three on the tree" and confuse the hell out of the driver. We can already imagine the LOLs. [CG]


text:
Exclusive: 'Morning Glory' Movie Posters Debut!
Paramount Pictures based production outfit Bad Robot isn't known for romance. The company, headed by J.J. Abrams and Bryan Burk, has carved a comfortable niche in the entertainment industry for itself as a prolific purveyor of action and science fiction, with genre hits in film and television like Lost, Cloverfield, Alias, Fringe and Star Trek under its belt. Of course, you can't have all your eggs in one basket, so the company has begun to branch out with Morning Glory, a romantic comedy about a hotshot television producer tasked with the challenge of reviving a struggling morning show program despite the constant feuding of its high-profile anchors. Needless to say, the film, directed by Notting Hill's Roger Michell, is a bit of a departure for Abrams and co.
It's also a bit of a departure for star Harrison Ford, who plays egocentric curmudgeon Mike Pomeroy in the November 12th release. The last comedy that Ford worked on was 2003's Hollywood Homicide, and that was funny for all the wrong reasons. On the flipside, the last time he took on a "romantic" role was in 1999's Random Hearts, so longtime fans like myself are anxious to see his suave side and sly smile on the big screen once again. And is there any better way for Ford to get back in touch with his inner heartthrob than doing it alongside Diane Keaton and Rachel McAdams? I think not.
So to get you feeling all warm and fuzzy inside, we're exclusively debuting the character posters for Paramount Pictures Morning Glory! Check out Ford, Keaton and McAdams in character as Pomeroy, Colleen Peck and Becky Fuller, respectively, and be sure to see Morning Glory when it hits theaters on November 12th!
Check out the posters below or click for high resolution versions.


text:
HAWAIIAN GAZETTE, FRIDAY, AUGUST 2X -SEMi-WEEKLY.
NAMFh FflD QPHfini Q,F,0W Through
llfilllLU I Ull UUNUULU
Board of Education Accomplishes
Immense Amount of Detail
Work At Regular Meeting Held
No More Buildings Tq Be. Erected
Except On Land Owned Or
Leased By Department, Super
intendent Kinney Says
An immense amount of detail work I
mi eoinuleted at tbp reviiler nicotine 1
of the board ui education yesterday
including the official appointment or
transfer of about one thousand teach
pr, the complete elimination of (ler
niHii fioin the S'Lu'j of the Territory,
tlie din ipline of Home teachers and tin
pii.iiM.liuii of others and, especially, the
amendment of the rules of the depart
n t by whi. h the marking by teachers
rt ri,l the board of examiners of the pH
pers of pupils becomes more standard
ieil; ill-... a committee of review ii
pinvide.l for when the markings of
teachers and the examiners (lifter.
"I am very much pleased with the
work accomplished today,' remarked
Supei iiitemleiit Henry W. Kinney, of
the .(i pin I oi. 'lit of public i nsl ruct ion,
who pienided at the meeting. " Kvery
thing went smoothly and much import
ant work was done, especially
r), !
gaid to the new rules for marking of
credits. ' '
All of the commissioner were pre
ent and uere greatly interested in an
announcement made by Super intcinlent
Kiiincv when the meeting was tirst rail
ed to order. (
War Changes Situation
"Considerable-'changes have taken
place iu the situation as to teacher,
since on r last meeting, due to war con
ditions," said Mr. Kianey. "This is
due to so many man being called in the
draft, or who buve gone to the main
land, and the families left behind have
changed their plans. There is no trou
ble in getting teachers from the main
lnnd; the real trohble is in the trsu
portal ion problem. However, I have
Ixen notified thut the Matsi.n Naviga
tion Company may put ou a special
steamer to accommodate the three hull
tired or Hi. ire teachers now trying to
get here from the ('oust.
"A great many of these teacher
have been nimble fo get transportation
to the islands. . I faaae aotitie.l thaiu
that their cs.itracts would be extrmt
ed, but thut they must notify thU
dVpoPtme-nt'Df itifcTl'dr ther delay an-,
make every effort to get here as soon
as possible."
Mr. Kiu.iey announced that at the
begiiiuiiig of tin1 heason I.e had provid
ed for a margin of thirty five teachers,
but now this luis been reduced to four
This year there hae been employed
about I'MI luiite teachers fruii. t lit:
mainland than in I. inner years. It wa
announced tlmi it is now a I must in.
possible to c.et uien tpuchers.
The snperiiitcniient stated Hint son.e
tiling; new had been sprung mi th.
boai I by I he .-."-i" ti ii t ton nf one of the
Kauai liih school teachers by cable
jind the hcciiiiiii; nf a new one imu.e
tiiately by a cable older.
('o.u.u.sMoper W. li. r-inith, of Fast
Hawaii, uid that Miss Virginia Hursl
had been sec:iie.l tin I lie lillo lli'
SehiMil st iff I. nt that Kara, ha I rubbed
Hilo, It win It 1 i.ii:. ii Issii iici Knudsel.
..f Kjiiii.i, ;i in .-d, I. -i l.. i. ..t .iefeli..
himself or Ins island.
It as staled bv the . nt cli. lent
illllinji the j.:i-T,illl as to win. should
lie l. nine. I f.ii tie I i ,li n ;, s.hu.,1, tlia'
nt one liiri1 it i-a s I . r I in .-1 tt :i'-her
fur this si hoiil. but now the s- lmol sn
a most popular one. lie -aid Hint Mrs
.1. Knox Hod.-I. wife of the chaplain ot
the Second Hawaiian InianiM, ha'
been nfTeied a place there so she could
be near her husband, Put that she
now on the i'i.-ist and if was uncertain
if she won'-1 return it. time to fill 1 1.
posit inn.
New School Buildings
At this stae.e of the proceedings the
Oiestioll of new hi-IidoI In. il. lines cum.
np and the super i -it endent nin.le a d
laration whi.h will iiii-an a clini.ee
attitude by the depa i ! nient He stated
that a g eat .leal of lau.l occiit.ied In
territorial s, hmils does not belong to
the school depa-tmcnt , while tnuch was
not even leased and the department
had no claim of auv I-not to land njion
wliteh expensive schoolhouses lia.1 been
"Hereafter no more schools will be
l.nitt," declared the superintendent, j
"except upon land owned or leased l .
j ...i
the department.
The question of a petition from Ka
liului. Maul, for a nc school was re i
f 'rred to bv Mr. Kinney, iu this con . i xam.ii.i." board differed they should
liection, and he said that he had writ i." rew,..l b a committee consisting of
tun to the petitioners that there wiir''lie n-miiii :ut fioin i- eh island, the
f'HHIt) available for the building, but it 1 supervising pnm ipal and the princi
V ould not be erected until the laud ! pal of the high s. hool or normal s lnnd,
n alter bad bii straighteiied out.
f omaiisstoner Kiiudsei. brought up
tlie qteation of supplying a teacher fo'
a school of tubercular children at Ka
J sS, Kauai, but it was stated that til.
Territory wus so short of teacher
that M was hanl t fill such places
if it was shnwn there would be
fifteen children to attend such a school
an extra effort Mould be made to se
nire teacher.
This discussion led to more tnlV aboo'
children nnd Mrs. Theodore Kicliard
said that over on Niihau the scl.oio
children semnied able to talk only in
Hawaiian, and she thounht this ens
wrong. Mr. Kuudsen remarked thuh
this was only because the children were
bashful, and that they could spe.il.
Knglish. It was determined that a
more careful supervision uf these Ha
walian schools should be had
Principal Deserts Post
Knne'ieleli.le.-t K ' ., '.' , t, '
tf, -enh C;l ' Illllii. Ii'li ...il .,f siiui'l
School ou Maui, had .lc-eile.1 lu-i post
of Beer Will '
Sewers of Honolulu
Brewing Company Has Thou
sands of Bottles On Hand
Which Prohibition Forbids It To
Dispose of By Sale Or Gift
Tlir contents of thousands of battles
nf iocnlly made beer will be turned int.)
the sewer or otherwise dumped, offici
als of tli Honolulu Hrewing & Wait
ing Company, believe, fur with the
clamping down of thp prohibition lul
lr.st night, the brewery cnTin.it sell, shin
or give, serve or barter nnv of its
piesent supply.
Having but sixty days in which to
dispose of its product, thp brewery
as unable to unload its putirp stock
ml the storeroom nf thp hrcwerv still
hoi. I n limit ten thousand dollars ivoita
I he officials see no way nut except
to uncork the bottles and allow the
rontents to trickle Into the rit." an I
storm basins and permit the fluid to
tlow out into the broad I'acitic Ocean.
St. C. Sayres. manager of the com
innv, Is now in Hilo am) in not expect
ed brick n,il Hiiturday. .Inst whit dis
position the official have in mind for
their plant, the buildings and beer
making equipment, has not been mad '
public. The company bns manufactured
no beer for two months. It has been
manufacturing ice and part nf ikj
"1dtnrng equipment is used by local
concerns for the storage of meats and
fher perishable foodstuffs
It was suggested that the company
turn over its plant Into a soda water
manufactory, but it was pointed nut
the building and equipment are alto
'tether too large nnl expensive to ns
f.ir this purpose nnd thp operation
would nndouh'edlv cause a loss.
w. a. a.
HonoluJii is one of four c.i.ies outsi.lp
of Japan selected by Yasudn, president
of the Yastida and Third Banks of
Tokio, for the (establishment of a
branch bank. Y a sod a is regarded as
one of the most prominent bankers and
money mngnares or .mpan. ine oiuer i follown: Ferreira vs. Ferre.ra, or
cities selected by Ynsuda for branches !,(,.,. to Bnow rase in divorce case, and
of his bank are London, New York and r.ipm.jrrer in equity suit of J. F. Col-
Iudley l'ratt, son of Dr. J. S. B.
I'ratt, will enter Yale this Fall. He
has received notice of this admission
without conriUiona. He was prominent
ut Fuuahou in athletics and dramatics
and was a feuder'n. student activities,
lie wan captain of the rhnmpionshi)
football team of last" Fall.
w ithout p rmissnin w hen lie heard i.e
'"" i
, , , . -.1- l-.
a.. ueeu iiia.reu, , vm, ...s re,aiies
an. I bill then, ta.ewell. Hut he was re I
jccieu ,.y me oraii ..oaro anu " '
returned to his school. The board en
loisc.l the action of the superintendent
hi 1 1 ansf crri ng 1. 1.11 to another school
and niukmo a simple teacher of hliu.
but nt the same salary; a mild disci
pline, rein.'u lie I one commissioner.
It was announced that it was against
the rules to appoint a principal for a
ao loom school, unless the teacher held
a certificate. It was filially decide'!
that an "acting" principal might be
appointed, and a resolution to this
nect m.lioiied the superintendent to
lame them.
Of especial interest to Hilo was the
.ii.tei.ie.it of the superintendent that
he .i..ii - of Hawaii were ap
parently a leep. tor although he had
ippeuied I.. Uien several times they
ia.1 not provided the land for the new
il,ools needed t In-1 f. lie stated that
'l.e.e was a fund of 'l."i.uull for an nihil-
ion to the I 'mon School at Hilo, also a
no for the laud
but the
land had not been secured.
"It is up to the supervisors of tin-
vnii," icn arkel Mr. Kinney, and turn
in to Coinniissioni t'. H. Smith add
.it: ''Vour supcrv.JoVs seem to nave
.one to -leep altoeet liVr. ' '
When the commissioners n'ft iu the
ifternoon session .csoltit ions were pass
el pt ohi tut i ui; the teaching of (iermtin
.. the pulilii s.'hools. as told elsewhere.
ii.lloAiiie this imp., i taut ai'tiou the
I board we.i int.. executive se-tHiou lis II
! committee of the whole to consider the
atinys of tea hers and to take uct.on
tMpon the marking of tiapers.
Praise a id Censure
The lepoit of the committee censur
ing some teachers an.) piaisiug oth.s
wus endorsed bv the commissioners and
Mie superintendent wits instructed to
M.m. i it, contents of admonition, and
o foith, and the report to each indi
. i . 1 1 i;i I teacher and piincipal involved
. u the censure or prn ise.
It was also leiolved that when the
marks imii bv a teacher and b the
' whichever the pilptl desires to enter,
in the island where the pupil is g:adu
j a ted.
I II was also de.-iiled thut In the fll
, tare the daily no.k of a pupil will
.oi, ni foi fifty percent, with the ex
i in i ii a : i n ma U to count fifty pe'eeut
to be , -.muted separately; with the ex
' e ! on. lo.we. that tlie passing mark
-.! 7 . pe; i ent must be reached in the
I '-iiul'sh '.-i a eiiage, cotiipi. sit i"ii, cram
ur" an I spelling.
Tins minor! ant rule i-aiise.l cons'der
I able discuss,,,!., but Wis felt to be a
icce-sity under the i 1 1 vi insl ;i in es dn.'
to tin osuiopidit a n 'haiacier of the
t err 1 1 oi in I schools.
Other inlcs passed ale to the etTe-
that liTeafiei bool. agents or canvas
' sars will not be allowed to enter school
premises dm mo school hours.
1 More ,i si i etii.n was also allowed the
superintendent to grant haves of :ib
in., to iia)ich..is with t'-iv. ai d tie wa
ji.lso given etia authority to in:. he ap
p,,l n t men I s. '
HRI ISH ARMY MAN 'American
w aamai was
Major Henderson of Royal Engi
neers Owned Hawaiian Se
curities Worth $210,000
A petition was filed in the office of
Henry Hm.ith, clerk of the flrst circuit
court, yesterday by A. fAcwis, Jr., nom
inee of the domiciliary administrator,
for probate of the local aetata of tbe
Inte Major Henry May Henderson, of
Red House, County of Pembroke, Fng
land, who died oil Cnre.h 10, 1917. At
the time of hi death tajor Henderson
was In command of th British Royal
, ,. .... i
hngmeers in t ranee. The petition for
adininistratirm will be, heard on Sep
fembcr 27.
Major Henderson's estate, consisting
of bonds, stocks and other collateral in
Hawaiian corporations. is valued at
''09,12K.fl. the heirs being his father
and mother, John Ilanhnm Henderson
and Mrs. H. A. Henderson.
The Hawaiian estate of the deceased
is as follows:
Held In name of B. A. Henderson
and Oe.pt. John H. Henderson, trustees
for Henry Nf. Henderson: forty-seven
shares C. Brewer k Co. and twenty-five
"hares Onhil Railway 1-and Comjiany.
Bit AJriomiit In Bank
Held in the name of Mr. B. A. Hen
derson, trustee for Henry May Hender
son: One California (las Klectric
Company bond, valued at H17.50, and
savings bank account, L'tl,1M." 51.
In name of Henry May Henderson:
!'H) shares C- Brewer Co., 0 shares
Hawaiian Agricultural Company, seven
shnrcs Onhu Railway & Land Company,
nnd thrpe bonds, Inst mentioned vulued
at .1itfi5.
Titilge Kdings yesterday grnnted n
divorce to Avelinn Nakai from lilwrt
Nukai on thp ground of nonsupport, the
mother being given the cirstody of the
three minor children of the couple. The
decree will take effect on August 31.
The 8nnl aerounts of Oeorge Robin
son, administrator of the estate of
it. vv. .Ionian, oeceasea, were approveu
by Judge Kdinga yesterday. .
I'nrler 200 bond, Judge Kdings yes
terday appointed Benjamin N. Kahale
puna as guardian of James Maulinla,
Aftvr hearing ,tho order to show
cause in the divorce suit of Victoria
I.e Roy vs. Christeen I.e Roy, which
cume to nothing, Judge Kdings yester
day set the case for trial at two o'clock
tomorrow afternoon.
Tnd re Kdintrs' calendar for today is
bun vs. Knpiolani Kstate.
Suit for damuges in the sum of 400,
an action arising over a wrecked auto
mobile which was insured, was insti
vi.t,l.v bv Ihp l'hoenii As- I
s.irance Company of I.ondan and Frank
Santos vs. John Ferry.
tin motion lil.'d by the plaintiff. Judge
F.dii.irs yesterday ordered discontinued
the suit nnd discharged the attachment i
in the cuse of the California Feed Com-
. 1V- ('..-.I.. ,1.,;,,,. l.oulnos. I
..Ull V Vs. . i,, uiixuim. uui.it "un.,i.
- p j- rom,iny. This
. . ,i.i ,1.1,0 !
M,rrl.ee . paUure
Filed on August 11, the papers in
the divorce suit of Mileka Puni Sui
HooM vs. St.i Hoon were returned from
service yesterday, the libellee being
served at Kaalaea, this island. The
couple were married in Honolulu on
February 24, l!Ho, by Rev. Henry K.
I'oi'i Mrs. Sui charges nonsupport.
Four suits for divorce were filed yes
ter.lav, making twentv six instituted
in Honolulu this month: Rachel Don
nelly s. William II. Donnelly, cruelty;
Roseliua A. l.uke vs. Charles A. I. like.
nonsiippoi t : t.lina Pod res vs. perg i
Pmlrc", cruelty and nonsupport and I
n ,.'., Caraballo vs. Klisa Caraballo,
desiri o'i '
Street Improvement Suit j
Claiming that it will be damage 1 to I
the extent of l.ri71.75 should its prop-;
' . . , , . i
ertv be condemned for street widening I
purposes, ttie ( lty jiiii s ompany, onv
.of tl
uiany defendants in the suit ol .
the 'itv and County of Honolulu vs.
... ,.-i-.ffi..irv ..... .1.
the iiai.u uai.way ol i.ann onipuiiy mm
otl""" hied it i answ er yesterday.
Manv slioiilations were also tiled yes
ter.lav in Ihe same suit, granting addi
tioi'-l '.me in which the defendants
tnent.oiie.l are given to plead, as follow-:
Inter Island Steam Navigation
Company, August .11-1 Oahu Railway A
Laud I' .uipnuy, S. (I. Kibler Estate,
'l a w n i in ii Tiust Company and Rev.
Father II Valentin, trustees under the
will of John Una, deceased, Hawaii
an I. and Improvement Company, Ha
waiian Pineapple Company and Amer
ican Can Company, Sentember 15;
I. ewers & Cooke and V. Aliin, Septem
ber L'lt.
- , . I. a.
l s1rrJ;ftKM ftmKD
P.y In. er I si In li rl Mt"no.r M n
'i Hawaii slid Mailt ports. Aniens! '!"-
I'roin Ihiwutl Ur. Mil rut n. S. Shlmails.
' '.e rii. oil-. 1 II (iljnoli.v. Mrs. W. T.
linrdeti Mi: null Mrs. I.. Newton. I. Kil
t.".ilet v.iss V. .Inrili.n. Owirire i Sell
Mr ami Mrs. V W. T. Hnltolllh-y. Mr Slid
Mrs I II VVInslnnl Mr. it". I Mrs. leo
Mown.' i. N K Wlllliiins. I-' l ' Ath-r
ton. Ho I: Montis nt. Mrs V . I! Mc
Neil I'. .' 1 .11 Mill' h X I! Itose. Mrs. .1
Ii. Wlillieuiore I-: Casleiir I'. A ulearv.
lico II ii v Ml s T vu.iil Ml s Kekl.
I'. S Mien Mrs S It Allen Mr. Slid
Ms V On. .' s: via... s- V li, H,
"' Ino I' s,,. is I' TsuJI. Mrs. A.
Kiiiinii Miss l: Mala' ana. Mis II. S
Ihi.IIii. . I M .Ionian M k v..(-...
i -,. l ' .se v' ii MUs ft
Johnson .s A liiirtlev. uit. A ('.
Cot, In ll MaJ II .1 Mielilotl. .link lleshn.
,, I VI lit:'" 1 ' ' I 'i ill ' "I
ills l,,,,.L'.. Mol'i'I Miss Klllltel. Miss
hiiin.'il Mis i; i, iih nnd ihlld. Miss 'I'
Ashl.i'ook .VI i ami Mrs :- I -'-til Mrs
" ' I It" v ,1 i ll" W. It MllleV .
I-' il .oi. It W I or It. II II It I'orler
i I,,.. :, I' It VI i to... Miss II III. I
en.,"' 1... n It VV V. . VI t II I l',,ll j
vers. I Ihi'ld V. . II S I til t I I'll . :
I o : ,. VI' s I' .l, VM . I
I ki.'d... I. lllllll'.litl'-l .1 S It Itoh I
el! VI, -- I I'.Mtlol'. Miss A llolllelo i
Mi-- V I . i in .1 Vim -in ii I,.... Man I
I ' 1 1 1 H . i II VI V Ill W I III I
1-:. I. mn
rl Ka:
Mis KeknhlM nnd
Mr iiinl Mis K
i .o v. T
li'ol M'-s I I'. .si I
h.iliiliill. Mrs l-'ul-
i -I'vrei v T
mi s v Ho. tie a.
s , i i n est ru in Minn
,;...l.... III- a
II M.i 1. r
Mrs VI., iv
,,i Mr
.-la. M
Mi .s
'I c:
M-s. ,
l lil..C
i I II
nl. Ik. -'I.
K i , i 1,1
Ii l.nf
Factors Is
Six Hundred and Sixty-one appli
cations For Shares Received;
Afl Classes of Sacieiv R 2Dre "
SCnte((--Litti3 felloWS TO GS.
FirSt CfiafiCt
MoVe tl liri a m.ili.n nnd half do!
wb'i oversnlis. i il.ed for the shares
of the new A1..1
sureessots to tl
n i'.n lors, Limited,
I 11. Ilackteld ti
the t u on i sting ma
1 ,, . -,,, ,,
i0., 1. mined, nl.i
chines of Trent T i.si (dinpany finish
ed the work last mIi! ot listing the
naiues .i .he .uli -. mem, tlie xhaie up
plied for, and t.ie amounts johere.l ...
rasa, or in l.ibeity Bonds.
ti. hundred ami sixty one subscribers!
i t..r .in .kfl..,. r
market value ol l.-.o each, represent
ing a total of H,0!i.-..l00, although only I
.W.lKM) shares are otiered for sa.e oi a I
total value of a;,.-,ini,ii00. The over
scrlptlon of sha.es totaled lO.fKU.
Doctors; law vers, s, hool . teachers,
cIptks, ts.tehsHii.V csipitali ts and lHt,or
ers, elemosyiia.y institutions, a, id largo
vim siiinii runiiii i ninp .iri .uuii); ,ni.i
subwribera to the sto. k, as llsteM per-
ally last ni-dit by R. H. Trent, re
itative.in the Hawaiian IslanOs
of V""iis! odiaa of Knemv I'roiierty I'alm
er, of Washington, a;id ('. U. Heiser,
who Is aseisitiiig Mr. Trent in American
Factors, affairs.
Fev From Mainland
The Subscribers ::re from "II pa-ta
of the Islands; less than fifty are
t rn.d the ii.aiula ml.
The clerical l..-ce of Trent T:nst
Company worked until nejily midnight
last night on the tahiilati.u of the
silb-.rMif ins. p ."isifltur' to ge'ii'ir
Out allo'tment notices without delay,
and it was announced last night thnt
the name will lie in the mails in Ihm
hours from the clos
. . At . ' , . .. ,
As previously announced by
announced by Mr
Trent, small subscribers will lie given
the first consideration in the allott.ne.it
of shares, and they will be allotted the
fnll number subscribed for unlesa in
particular cases there be reasons why , lan,,p' rorim telling of action taken by
this should not be done. Some of the I fhe Pxutive committee in opposition
Inrger subscriptions will have to be 1 to t,e teaching of German ii the
sealed down ns much as twentv percent ! whools of the Territory by the pas
or more, f6. permif this policv being ""P' of the following resolution, which
carried out
l.lMirtV KfllUlB IllTArJIl
Subscribers whose too liberal offering
of I.ihertv Ronds mv. .eem n inrlicst..
an nnrea sonable dpsiVe to unload gov '
ernment Hecurlties will be given an op
t.nrtrini- H ..ibiititi.ta larirae nm. 1
portionate chsh pavmeht in demonstra .
tinn of their natriotie alnceritv. nr else
reduce or wVl.draw their siibscriutiens.
The I.ihertv Hnnd offered in "nnv.
ment of shares" applied for amounted ""' H"' resolution requests " assert
to 1.100,0fib, or one-third of the total ?. Knperuitendent hiiiney, who is bim
of subscriptions.
One hnlf "of the subscriptions,
l.l.l.l..l ..... .!.! ... c.
on' III, in linin I . , I 'DU, BIT I II I
shares or less, and of the total of
, . ,, ... , .
There are onlv aixtv three snbscriii
Hons for mo-e than 100 slmres, and
these varyfrom 12S shares to i
shares, ther'e beinir seven subscribers
for the last named number.
w. a. a.
BOOZE Ta SAILORS
R-ar-Admiral R. IL Doyle, I,. S. N.,
coniiimndant of Peurl Harbor Naval
station, yesterday made public new
,, ,jrastiC(, regulations just received
. '
fro mthe nnvy department, proliibiting
' 1
1, uP.,;n ool of nl,'..l..,lii. Honors
to any oflicer or memher of tlie nnval
tr(.,. in r 0tsiue the nuvy depart-
ment area
No leeway is given under the new
instructions which withhold the use
of liquors at any place within the
Cited ISutes by officers and enlisted
men of the navy. No officer or member
of the forces under Admiral Doyle's
command may be furnished any liquor
by any person. Inasmuch as liquor can
not be serv"d to an officer or enlisted
nil. n, the regulations strictly prohibit
one officer ur navy man giving or serv
ini' honor to another member of the
The publicity which Admiral Doyle
gives to the order, coordinates with the
general prohibition proclamation of the
President, effective (rt every part of the
Territory at midnight lust night. Ad-
miral Doyle, in a letter dated August
2(1 and addressed to the editor of The
Advertiser says on the subject:
"The commandant is in receipt of
n formation from tne secretary or the
navy to the effect thut the (leneral
Onler legirding the giving or selling
of intoxicating liquors to members of
Naval Forces within certain zones has
beon modified by inserting the follow
ing provision:
" 'Outside of said zones, alco
holic liquors, including beer, ale
and wine, either alone or with any
other article, ahull not, directly or
indirectly, be sold, bartered, given,
served, or knowingly delivered to
any oflicer or member of the naval
forces, within the I'nlted Htates,
their territories pr possessions, or
any place under their Control, ex
cept when administered for mCdicar
purposes by or under the direction
of a regularly licensed pliysiciuu
or met Heal officer of the Cnited
Stutes. '
"The Pensr'ment desires that this
be given the widest publicity."
VK7.0 OINTMENT ia guaranteed to
cure bli ui, bleeding, ItrhL.g or pro
truling PII.KS in - iu l- lay o
nisinev rrlutii'id Al mot, lun bv
tbt ' AH I.i M I llK'n-'H Cvi ,St k.oi.is
1 a. a.
TURN THUuiCSDQWfl
I Oil IIUIILME
j ,i- t 1 ' ,v Kl'l(AVI I I
With action itarteA bf the Hawaiian
Vigilance Corps the cause of "kulfcor"
received a serious solar plexnt blow
veste.dav. when the hoard of suDer-
visors, as aovernora of th Island of
u, prohibited thtuse of the German1 1
language iii the im.idings under control i
of the county; also when the school
...niissioner. of the Territory resolve
lo ..ui t.lc leachn.K i.i Lfiumn in alt
the );u!diir schools of th Territory. Ha
V , , . ' t-ape' ftflrhukal 0 the I'ujia Coast,
. Zt'A th; "nnT' " ,,p'rV,"nlime 'rytrrif6tv.'' With the cftft?
was sta-ied when a letter was read , , , TriWr1on
from Mayor Joe whleh he , f J .
i !
" mBV'' ' '" rounly
a ... i I i . a
" " i
"'",r" m " "00 ""
iimter the care ami
dihI control or tne rxiaro
h"M be used for any purpose it.
. or 10 "". tw!-
" Wman. .
aub-."""" , , .
"While the course of atiidv is a mat-
,pr tor, territorial department of
";l'-ition, through its commUsioneri..
V'"'f Vty "" T' "h.ottM,"b' .af'
'ord 0;pir -rd fnc I'tles for the con-
. , . , i
."''hmg of the lonie of the
'V " ov e ""'" "BI- !
Z t i
enemies frtm the curriculum of . meri '
crn edo "tional cer'era. j
"I edvise the serious consideration ;
of Viis enemv langitflfe nurstlon and j
iian.su inr iroTT'irai laniriicire or wuriu n i
ask that the action Of .this board he
taken, in as far as its power goes, so
as n bar German being spoken in ,
ithe Tuib'ic buildings under the control j
'of ti r c'.tv nod courty. "
Hey .t-.;rTn rp-"-'-." out the si";ges
of Mavor r.'-n " e-e pushed en'd (
";l vnvp tbe effect ef n-p'-er' fi.
' ,. r..r- ' r e hc01-
' ' p : :iv of ;!.c co-ati- choo!
....i.. .urd Rm rurman
The first thing done when the board
of education for the Territory met at
its aftprnoon session yesterday after
noon was when Superintendent Kinney
read a letter from the Hawaiian .VIgl-
j Colleges ami the College of Hdwnil'f
n ..a nu,i win ti, -i. Lfiuiif aim vauu
"That it is the sense nf this eom-
m.ttee that those pupils who have parti
nll.v prepared thpmsplves for entrance
I college exni'iinations in Oerroen should
He given turtner instructions in tnat
lHn".n"' f,,r ,nt pnrp"". but that
orner insi' iciions in Herman snouiu
i'jciinrnged. "
1 ne.ieva m point? even Tnrtpe'
eir a Herman scnniar, ana eurring
it out altogether. '
No Compromise!
Commissioner I.. 0. Blsckman, prin
cipal of the Honolulu Military Acade
me, immediately took up the matter in
' ni" 'n,,Mt emphatic stv.e and mane tae
official motion that the teaching of
German in the public schools be done
n" ay w ith entirely ami w ithout any
I compromise, stating that to continue
t its study gives German altogether ah
I ntipearnnce of too much importance in
the eves of the pupils.
"i o epee- it" of beinjt familiar
with flermnn " added Commissioner
PVickmati. "There are few occasions
when ore would have to (to to the.
original Herman scientific text bookS
to secure ideas for study, as good trans'
' la'.i .in be In, I ef nil worth whi'e
In the future there will be onlv two
lant'uages for thp commercial and liter
arc world, Knglish and French."
Commissioner Blackman then submit
f. '. his re-oliiion and included Spanish
as the lanouage to be taught in the
p'ace of I'.pi.imn. There was some op
position to making Spanish eomnulsory
such as (icrmnu was in some cases, and
, it was made an elective study, ,with
which amendment the resolution niseed
unanimously, eliminating the study of
Herman in the schools of the Territory'.
w. i. a. -1 ' . .
11. llachfeld & Compnnv, Limited,
w ho yesterday concluded ststy nine
years as a business firm in Honolulu,
sent out circular letters to its' patrons
informing them of the officinl change
from Haihfeld t Company, to Amir
, i.-an Knctuis, Limited.
A second circular letter wn-i also
I m.dosc I from the American Factors.
Limited, signed by Allen W. T. Hot
ti mley. pn s.dent and manager, tfiv
tug notice of the new name snq an
nonncing the names of the dinvti rs,
i fticers and eieiutive ftaft.
I he HaiUt'eld farewell announcement
coin hides ns follows:
"The transfer is made as of August
'Jti, I'HK, ami the new corporation hai)
acquired all or tne assets ami asaumeii
all of th
i liabilities of this company;
.tiling from the business in
"On ictiiiiig
which we have been engaged for the
pa-t sivty nine years, we wish to con
vey to you an expression of our ap
preciation of the support accorded to
us and to bespeak for our successors1
the American Factors, Limited a eon.
1 1 ii 1 1 it t i ii of your esteemed good will."
The American Factors Bnno'utice their
status as follows
In in a k i l. o thin nnnouiicement. w
wish to state that it will 'lib the aiul
of this company and its management
to successfully continue the business
whiih we have taken over, in mch a
wav that the wishes of the federal
government for the amerlcanizatiou of
j the company may be truly realised, and
! In that end we respectfully solicit your
kind cooperation. We believe that our
1'ir.irls to i'"v out n policy along
these lines will prove of mutual ad
vantage, ' '
mil IN 7MRIT0M
rP quarters
r 1-rob Iblt ion workers todny v. ill fcnve
fw s . ,
tit the Haa1fpil Btyd IsHnrl -reaVrVa-
tion to tli farthest Kdnthesst point on
..?., ai.ge la map shadings is,
YlAaWsaM-sVaS ' a41sV M Ww.Msfr.A k Mria. i r. i
" v-v si a . vi l J H 1 UIUUI VI HITt HI'IIUV
ra'the UbiU ihA WhtaNla which' mst
rome o th,. alrlagliff 0.rrjn of the
S.17,00() populavt1oa of Hawaii, whether
, l w .. n.,i v-i. i
thf jugnfl, Ulr 'Wa BJauilh or
(johu. ).
tesferdev jroif ennld jrive vour
rsierrtev von eniiin jtve vour
frieriil k Wi'tllr hot" If vou felt 'hha-
ritnbly Inclined. Mi It tiiitlay ah'd von
will violate the Kuhio Hhepptrd Ha
Prohfliitioa Art which has brousrht
the rlftrtige in the -fiKihrtiil ion ina
r.ii,,,n bcii pur'enos ine enange in
th, hatita and diatoms of two rliirds
fiaiiinps aert por'ends the change
of the Island population,
n .il "
. , ..
rn Blbifioq rahie to Chn witt out
"a Ja-r.' 8 thii Island has Ireen in
"Hry territory since the presidential
"r,"4nn.aion went into effect on April
1 But the nesting of John Hfirtcvervm
from this Territory, nt leai imtll the
Tar is ended,' must have left dim
memories tlllfi tnorniog of a cV!li-ioa
on Vn HmTHftWnrt where the hnhtt
f . al''inem-e "hi stifl to' tie aerrrfi8d,
lieeirllv SI mnrlt n..lnl mm 1, ITn.
I li-Sai rti. .1 lit Vr.t . i.ir.i l
- .i tit 111- , u.l DIIU WHI'-I! nilVv
-.:ise.1-fl ti'ntfti raSea fOi' tLe- thiiity
traveler pn he $ifc Inland. '
Theoretically al Hawaii today is
''bcrne dty,' and not even a doctor's
preactlpHoh legally "ea get a feeble
patient a "shot of "straight stuff,"
even nnder the (tiod provision of the
prohlblllbh art' whleh rftill permits al
eohol' to b nser"r6r Wdfelrial prtr
. Tbf aon thereof la that the liquor .
boards which nave federal nower dele-
. .. - - - - "J f
gated to tbeni thront'Ii the Governor
for the' KeeoHn'r ot-the" iihporttlryn
and. distribution - ot liquor for rhedl-
cium, aacrameniai, meenanirai anu sc.-
entitle 'inWhave' tio't hat! time yet to
RTant th netfeMary lieehses. In fact
the forms needed to secure permission
to make aneb purchases were not ready
for htnnaaea until ywitertlhy.''
It wnt-h. fit ieaat Uodav before
any HdbqIuIb .'flrutfstoTe' br other' s-
tablishment will fet a Urease for the ther of the scheme," and Governor
permitted falent lirtoor, s", due to1 McCarthy 'a representative for the Ter
the 'ttbseaca of p. . Qooke, chairman i ritory, Iri securing proper regulations of
of th Okhn 'titiuor board, fhtrn ran ' the iiermitted importation of liipior for
be no meeting until after he re urns
from ttiwnit In -float Ifaturdny. Carloa
ItUg, ewretary'of the bbard, savs that
the twenty' fonr hours' 'notice will be
requirect to call a meeting. Whether
the licenses ran be issued at once will
have to ba decided also.
Bara Are Tp
i''n tbfc tleahtime aale, transporting,
iinpbrtiilg rtr alvfng awaV bf liquor
will be -a violation of the Hawaii Pro
hibition -ArV, arrbohgh' physicians atid
drugstores can continue to use alcohol
ror enaipounOlnr thedieine, for which
they are of required to aecure a
license. But the-alCohoV must be in
a irierli kri'eonipo(in3' hhd to Ihipnrt it
a pif harftb 1 aeeBrod under the
requlations prOmnljrate.l 'by the Gov
ernor. 1 r." !' f ' i' ,.'
On the eVa a- tbh.Uion net j
Kunif; iniu rurri, .or qurni.on was
; .1 . . . .
.Ta" !V:?!l.bH
sold until 'the new drills of licenses
are issued.
Dealers in this eommoditv seemed to
have taken it that their business was
not affected by the new prohibition
as the kind of nteohol thev sell
not usually considered as an in-
toxirating liquor. This is because j
such grades as wood and denatured
alcohol are often used for the purposes
specified. However, there are grades
of prairi alcohol largely ued for me
rhanical purposes, such as for wireless
I 'unls and chemical mrrtnrea, which
can be made IntA'a pawible drink with
out danger of instant suicidal results.
vVTut Jb a Drink?
In an offhand decision, Assistant At
torney Oenernl Harry Irwin has ruled
that the transporting, importing and
sale of the various grades of alcohol
come within the meaning of the pro
hibition act, "if such alcohol can be
drunk so it will make tbe drinker
drunk before it will kill bim aa a
quick poison." He explains, though,
that the ' distinction regarding the
various grades and posslbje use qf tbe
nlcohol jwilj 'have to' D determined
"by th facts rather than the law"
in ench ease.
t follows that. In view of fbia ruling,
h!jh scboolti, tajlege,.oY other institu
tioi.s buying fctain"alcrihol for client
ical tests nnd uses will have to follow
the territorisl regulations and secure
permits fdr the in rc base or Importh
' tion of (he quani)y Heeded. It may
follow also t hat 't iris Hawaii Prohibi
tion Art will-stop the manufacture of
atedhrN frttm' rtolaseea, ' f 't the Puia
Uttpttr mill o ' hflaiii, althodgh need
pately for Wetfhaeal ifai'PosM, unless
denhforM atf tt ehuld' trot possibly be
used ni' I bevragi ' 1
Kkfy Ii Affeetad7
IncirJittitalri'CTstrict Attorney Huber
nniit'fft annfh'er hff.tln.iof ileMsinn- Ihn!
! He beffevefl "thail the nrfval ttutliorttits
1 will liaie to follow the Governor's
' regulations a he aecure permits through
I t he lirenM IritpecUr ta Import grain
ilk-phut lor use h) the wireless plants.
ThAuandVdf 'ga noils' of the liquor are
required to supply tuese wireless plants
6hJtl."e Vorloua Islttns, It ts said.
' 'The "law .mlijiei' ho exceptions,"
said both Attofhevs Htlher and Irwin
when asked whether the navy would
have to get the importation permits.! session dining the period of traus
As one of tliem poiuted out, it is doubt I puliation.
Granting of joint jurisdiction to the
territorial courts and officials in the
enforcement of the Hawaii Prohibition
Act, which went into effect at mid
night last niefht, by congressional
amendment of the new law, has been
recoihmended to Attorney Oenernl
Oregofy is the best way violations may
b checked and violators prosecuted
had punished.
Thia recommendation has been msdo
to Attorney (leneral Gregory by Dis
trict Attorney 8. C. Huber and Marshal
J. J. Rmiddy upon he suggestion of
Assistant Attorney General Harry Ir
win. The reco'mmendation is the fed
efal Authorities' answer to the attor
ney general's qnery as to how the pro
hibition law is to be enforced in Ha
waii. Unless some such action is taken
and taken" almost immediately, the fed
eral authorities and others believe the
United States marshal's staff will have
to he increased to a force of twenty
v or thirty deputies, In order to cope
with the situation as it stands today,
and provide the needed machinery
trietly to enforce the new low.
If some such action is taken the
several district and circuit courts of
the Islands and approximately "0 i
lieeinen will be as much responsible for
the enforcement of the federal prol.i
bltion law as they are for the enforce
meat of the territorial litpipr statutes.
Method Ii Easy
Amendment of the Hawaii Prohibi
tion Act, no equal jiirisiftetion will be
gfven to all territorial courts and ofti
eials with that of federal authority,
will be a simple and effective expedi
ent. Alt that will be necessary, it is
aaid, is for congress to add a short
section to the prohibition act, express
ly stating' that this jurisdiction has
beenx'teiided jointly to the Territory
with that nOw imposed in the federal
courts and onicials.
Hope is expressed that Attorney
General Oregory win realize the need
j t.;k .............. .... 1. 1. rn
VI linn O limip'l'l"1"', " " ' ' ,n ,u,ij
informed of the local conditions and
ht,w the w6rk of a large territorial staff
of policemen must be enntinued, or
their services retdace.l with .federal
bffirers, and that he will at once ask
congress to pass the necessary amend
ment. Beveral benefits will be derived from
the passage of the joint jurisdiction
amendmear, in the opinion or Assist-
ant Attorney Oerteral Irwin ''the fa
i sacramental, mechanical, mediciiiul and
stvnnne: purposes.
(!hief of the benefits will be thn
great saving of expenses to the fed
eral government, and to witnesses, de
fendants, and all other litignnts in
volved in any liquor cases, he says.
For instance, he points out, by this
joint jurisdiction arrangement a de
fendant arrested in Kailuu, Kona,
could be tried in the Kailua district
or circuit court, without the necessity
of being brought to Honolulu with all
the necessary witnesses fend arresting
officers,, as Will tie Otherwise reojuiriJd,
a the law stands.
"Besides," Attoruey Irwin aaya,
"The present requirements of the law
will U.hke persons on the outside Is
lands backward about giving informa
tion regarding violations, us they
not want to become mixed up in
a CUliri HCIIOIl WHICH winini I " C"
"''Also;'' be .ays, "the Island poll
a court aetiou wnicn won hi require
will have an interest iu arresting vio
latori of the law so long as the de
feudants caa be prosecuted in the
courts of their district, which they
,. L,.ru:.s uo.ol.l it'll knip HO I CI 11 u nit
i tn,v ii,;.,., iH tt ,niter for federul
. attention. "
In addition, Attorney Irwin asserts
that under the present legul require
ments of the prohibition act Ihe Cult
ed Btates marshal's force, now con
sisting of only two deputies, will bu
entirely inadequate to control vinln
tios of the law.
It is he who estimates that it would
require at least twenty five or thirty
deputy marshals to provide a force t"
replace the services of the territorial
police, unless the services of these to
lice iu enfuv'ing the law as expressly
required by statute.
Huber Trust Folic
While District Attorney Huber be
lieves the assistant attorney general
has struck the right solution of the
probleni of the federal snthorities in
gaining the necessary machinery to en
force the prohibition law, he exprc-se l
confidence yesterday that the territori
al police could be depended upon to
arrest all violators of the prohibition
act iu the meantime while the aineud
ment to the act is being secured froin
But us has been pointed out lief ire),
all violator of the act arrested now
run be prosecuted in the territorial
courts only for the sale or alcoholic
liquor. With this the cuse, violators,
charged with iliegul transportation, im
porting, exporting or giving awav of
liquor -will have to be prosecuted iu
the' federal court, until cougress.
amends the Hawaii Prohibition At.
ful if a shipping company could be
induced to accept the alcohol fur trans
portation to these Islands, nnless an
order for transportation had been "f
cured through the necessary channels.
The order for transportation ntithor
ir.es and requests the movement of all
tumors imported, and is the "scrap
of paper" which chauffeurs, transfer
men, rnilrouds and shipping companies
must have under the new regulations
o save them from arrest for knowing
ly transporting liquor upon the prima,
facie evidence of liquor ill their pos-
xml | txt


text:
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This hotel location map for the Gipsy-Hill-Hotel has been provided as a guide to help show the approximate location of the hotel in Exeter. Visit the accommodation links below to check Gipsy-Hill-Hotel availability and more information including photographs, address and links to hotel reviews.
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text:
January 8, 2013 in Activism
by sleepingnebula
This content was reported as spam by our users, click here to view. Display Article
she has disappeared off of youtube a year ago.
please watch her passionate, eye-opening, genuine videos.
there's a war on for your mind, and alex jones is a part of that war, but he's not exactly on your side.
if you'll submit your own mind to alex's words, and not try to see anything else, then you are just as closed-minded as those who he calls sheeple.
for those who don't like this post, move along to another portion of this site. i do not care if you disagree with this post.
i only care to open the minds of those who are willing to listen to this missing girl's message.
thank you.
p.s. http://www.youtube.com/watch?v=DT1TLRrIcHw
Tags: alex, illuminati, jones, nwo, zionist
workinclasshero said on January 9, 2013
You have been reported for offensive content. Go to another site and spread your disinfo. BTW the girl in the video was a crackhead, not very reliable buck' o brown.
pig love said on January 8, 2013
You're an idiot, try again. That guy doesn't know jack about the card game, it wasn't made in 95′. History Now is alive and well my friend, you are too gullible.
sleepingnebula said on January 10, 2013
where can i see where she is?
You must be logged in to post a comment.
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text:
Birchmont match play underway with close matches, rallies and super shots
In the opening day of match play Wednesday at The Birchmont, perhaps no one had a sweeter shot in the men's championship than Brandon Myers.
The Motley native and New Mexico State junior eagled the par-4 16th hole at Bemidji Town & Country Club on a 70-yard chip shot to defeat Ben Cahill of Grand Rapids 4 and 2.
"I thought it was going in right when I hit it," Myers said. "The ball sailed right over the flag and hit the back of the green, then spinned back about 20 feet to the front of the green and dropped in the hole. That's a great way to win a match."
The win moved Myers into the men's championship round of 16, which continues today.
Myers draws Mike Christensen of Grand Rapids.
"I've really got my work cut out for me, I knew I'm either going to play a 16-year-old who's playing on fire or a former pro," said Myers before turning to a joking tone. "Yep, just a former pro - no big deal."
Christensen moved on by defeating 16-year-old Tyler Rustad of Moorhead 3 and 2. He was 2-under par when the match ended on the 16th hole and
Christensen is coming off a stroke-play tournament championship at the Nemadji (Superior, Wis.) Invitational. He was a former two-time state high school champion at Grand Rapids, a collegiate player at Duke University and professional golfer.
Myers, who is an intern this summer at the Legacy Courses at Cragun's in Brainerd, has played in the last four Birchmonts and is looking to advance past the second round for the first time.
He lost in the second round last year to Jeff Wiltse of Bemidji.
Wiltse was the 2007 qualifying medalist and he was unable to replicate the success he found at the Birchmont last season.
Bemidji High School grad Chris Curb defeated Wiltse 5 and 4 in a battle of Bemidji. After falling behind with a bogey on the third hole, Curb birdied the fourth and fifth holes to take a lead he never relinquished.
It was a strong showing for Curb. He finished the front nine at one-under and was even when the match ended on the 14th hole.
"Today I was really able to make my putts and get up and down," Curb said. "I was a little surprised to win the way I did. I was thinking this was going to be a really close match - Wiltse is a very good player."
Birchmont men's medalist Jesse Nelson showed his blistering five-under score of 67 in Tuesday's qualifying round was no fluke.
The Bemidji State student drew 1993 Birchmont men's champion Chad Skarperud and outlasted the former champ for a 1-up victory.
Nelson was in control for most of the match until a wild finish. Skarperud rallied from a 3-down deficit to take a 1-up lead when Nelson bogeyed the 16th. But Nelson bounced back to move the match all square with a birdie on the 17th and won the match when Skarperud double-bogeyed the 18th.
"It was really a battle today and he's really a very good player," Nelson said. "I don't even know what I shot (in stroke play), I was just trying so hard to focus on the match.
Also winning in a close 1-up match was Thomas Strandemo of Fargo, N.D over Daryl Schomer of St. Cloud. He rallied from a 2-down deficit after five holes and took the lead for good when Schomer bogeyed the eighth hole.
Others moving on to Wednesday's round of 16 were: Neil Rude, Devin Hexner, Matt Skarperud, Tim Skarperud, Tom Jenkins, Justin Cross, Brad Anderson, David Mooney, Thomas Strandemo, Josh Bergrud, Johnny Larson and Paul Daman.
Larson, the 2007 champion from Springfield, will go up against Daman of Bemidji after advancing past Todd Hillier of Hoople, N.D. 2 and 1.
Larson took control of his match on the back nine. He pulled the match all square with an eagle on the par-5 10th hole and went ahead for good with a birdie on the 11th.
He was three-under par when the match ended on the 17th hole.
Daman, a fourth grade teacher at Northern Elementary School, led his match wire-to-wire. Foss tried to rally with an eagle on the 15th hole, but was unable to birdie the 16th to keep the match alive.
Cross turned in good performance Wednesday and defeated Russ Newman 4 and 3. He was three-under par when the match ended on the 15th hole. Both had eagles in the match: Newman on the par-5 fourth hole and Cross on the par-5 15th to win the match.
Ben Moen of Bemidji could not muster the same magic he had at last season's Birchmont when he reached the quarterfinal round.
Jenkins, a three-time Bemidji Town & Country Club Vandersluis champion, beat Moen 5 and 4. Jenkins was one-under par when the round ended and he advances to face Cross Thursday.


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Minivan collides with bison on County Road 143
Ed Smith, owner of Eagle Feather Security, said one of his employees, Ray Roberts, was lucky not to be seriously injured after his Chevy Venture minivan collided with two buffalo at County Road 143 near the Waboose Lake Trail last week.
"He's very lucky; he said a buffalo went over the roof," Smith said. "I towed it back for him to Pine Point; it's a mess," he said of the van.
One of the buffalo was seriously injured and had to be put down by a tribal police officer who responded to the accident, Smith said.
"It's a real danger," he said. "They just come out across the road and there's no way you can avoid them."
The White Earth Police Department confirmed that the accident occurred.
County Road 143 is a paved road that runs east-west through the northern part of the Tamarac Wildlife Refuge.
Refuge officials are urging the public to take extra caution while recreating or driving through the refuge.
"There continues be domestic bison on Tamarac Refuge," Visitor Services and Environmental Education Manager Kelly Blackledge Kelly Black ledge said in a news release. "Their behavior is unpredictable and they can be difficult to see at night crossing refuge roads."
Some 15-20 domestic bison escaped earlier this month from a private pasture adjacent to the refuge.
"Every effort is being made to round up the animals," Blackledge said. "However, the forested and river landscape, along with limited access, is making it difficult. As with any domestic livestock, it is the responsibility of the owner to remove the animals."
She said refuge staff is coordinating with the landowner to allow access and provide for public safety.
"The decision was made by the landowner (Dan Bergstrom) to cull the animals and work with local meat processing lockers," Blackledge said.
"This is an on-going effort," she added. "If you see any bison on the refuge, please report it to Tamarac staff right away by calling 218-847-2641."


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American Teacher Community Screening in Bowling Green
American Teacher Partner Organizations
Tuesday, March 27, 2012 from 6:00 PM to 8:00 PM (CDT)
Event Details
You are invited to the premiere of "American Teacher," a documentary that looks at education today through the eyes and experiences of America's teachers.
Narrated by Academy Award-winner Matt Damon
- Free Admission (Reservation Required)
- Light Refreshments
- Limited Seating
"American Teacher" raises important questions about America's teachers. It should spark a much-needed conversation.
–U.S. Secretary of Education Arne Duncan
When & Where
Bowling Green Junior High School
900 Campbell Lane
Bowling Green,
Tuesday, March 27, 2012 from 6:00 PM to 8:00 PM (CDT)
Add to my calendar
American Teacher Partner Organizations
American Teacher is presented by
- 16th District PTA
- Council for Better Education
- Heart of Kentucky United Way
- Jefferson County Public Schools
- Jefferson County Teachers Association
- Kentucky Association of School Administrators
- Kentucky Association of School Councils
- Kentucky Association of School Superintendents
- Kentucky Chamber of Commerce
- Kentucky Education Association
- Kentucky Educational Television
- Kentucky PTA
- Kentucky School Boards Association
- Metro United Way
- Morehead State University
- Prichard Committee for Academic Excellence
- Rowan County Schools
- United Way of Southern Kentucky
- United Way of Central Kentucky
- United Way of the Bluegrass
- United Way of the Ohio Valley
*Though these organizations are not endorsing the film, we believe it will get us all thinking more about education and what we can do to help. The event is aimed at continuing the conversation about education and raising awareness of issues important to our community.


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You Tube
November 10, 2008
Gainesville, Fl: The Gators911Truth action gets edgy on the corner of the University of Florida campus. Gators911 recriuted 4 more UF students to the group in the past few weeks.
The Obama win for President has significantly nulled accusations that the 9/11 Truth movement is using the issue as a political hammer to bash Bush and the Republicans.
But now, 9/11 Truth must work harder to hold the Obama team accountable to follow through with Bush's broken promise to guarantee a criminal investigation of 9/11.
VIDEO: https://www.youtube.com/watch?v=xZ9kQcnIcl4


text:
B.Swim Vogue Pucker-Up Buttercup Swim Bottom L53VO
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text:
|24 Jun 2012
Windows 7 home 64bit
Computer not starting
I have no idea what I should do, I think I may need my windows disk but I don't know
I don't know what it trying to say since my min pin stepped on the screen, it's been working fine but this is what showed up when I tried starting up
|My System Specs
|24 Jun 2012
MS Windows 7 Home Premium 64-bit SP1
Sounds like something may have broke in the laptop... i'm not to familar with laptops but i could be your software.. i do see repairing..... something can't make it out, i'd have at it with a new windows disc.. if not the only soulution i would have is to open it up and see if anythings broken or loose /:
|My System Specs
|24 Jun 2012
The LCD display is defective. You can use an external display or replace the LCD itself, which is not too difficult to do.
|My System Specs
|25 Jun 2012
min pin = Miniature Doberman Pincher maybe????
|My System Specs
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Monday May 9, 2011 11:31 am
Paula Abdul and Simon Cowell Together Again on X Factor
Posted by K.C. Morgan Categories: Music, Prime Time, Reality, FOX, Gossip,
Paula Abdul will join Simon Cowell at the judging table… but not on American Idol. The two former judges are going to reunite on X Factor USA, which will also run on FOX.
Abdul joins Cheryl Cole and Antonio "LA" Reid at the table. X Factor USA looks for singing talent, soloists and group acts, aged 12 and above.
Read More | Yahoo! News UK
Gallery: Paula Abdul and Simon Cowell Together Again on X Factor
- Related Tags:
- fox, gossip, judges, music, paula abdul, reality, simon cowell, singers, singing, x factor, x factor usa
This is pathetic i think most Americans will not swallow this so fast.
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We have had the the Herb Schildt discussion before. Some people like him some dont.
We have had the the Herb Schildt discussion before. Some people like him some dont.
Mr. C: Author and Instructor
Schildt is less on the theories, more on the code. I use his books as more of a reference, and learn the theories from the courses I'm taking.
The C++ complete reference and MFC from the ground up are probably the two best references I've read on each respective subject.
I'm in AB right now.. and It's real easy for me (I knew C++ real well before taking it) but people in my class have a harder time, so for anybody out there I'd recommend having a good grasp on C++ before beginning. My teacher is strong on theory but weak in C++ (the only language she's "mastered" is Pascal) I recommended the Schildt book to her for this reason, and it seems to be helping her and the rest of the class out a lot.


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Long Pond Natural Area
Open to the Public
Things To Do
Loons, river otter and more View All
Plan Your Visit
Get Directions
Remote and Undeveloped
The Conservancy manages 1,500 feet of shoreline around this remote, undeveloped 99-acre pond. Long Pond is surrounded by a northern hardwood forest and includes habitat for such birds as the common loon and mammals like the river otter and bobcat.
767 acres
Long Pond Natural Area includes a Vermont Fish and Wildlife fishing access, paddock Hill Trail and unmarked logging roads. There are no facilities.
Please read our Preserve Visitation Guidelines.
What to See: Plants
Long Pond is surrounded by a northern hardwood forest composed of sugar maple, red maple, yellow birch, American beech, balsam fir, white and red spruce and white ash. The understory includes hophornbeam, striped maple and a few old yellow birches. The most important ecological feature is a mature northern white cedar swamp on either side of Sawmill Brook at the south end of the pond. The dense canopy of cedar is broken occasionally by large white spruce. There are a number of sedges found in the area including sweet gale, leatherleaf and speckled adler.
What to See: Animals
Birds include common loon, great blue heron, mallard and black duck, red-tailed hawk, bald eagle, osprey, American kestrel, pileated woodpecker, hermit thrush, purple finch and black-throated green warbler. You'll also find such mammals as mink, snowshore hare, red-backed vole, long-tailed weasel, river otter, bobcat, porcupine and moose.
Directions for the trail:
From Greensboro, take Cemetery Ridge Rd. pas the post office onto Garvin Hill Rd., for about 3 miles. At the Y junction, bear left on Hill Rd. In about 0.5 mile, bear right onto Hillcrest Rd. Go 0.75 mile and turn left on Tamarack Ranch Rd. There is an old converted schoolhouse on the right-hand corner. Tamarack Ranch Rd. becomes a rough woods road requiring a high-clearance vehicle. Drive in as far as you feel comfortable, avoiding right-hand turns. Park on the roadside and walk on until you reach the Conservancy trail sign-in box. The Paddock Hill Trail begins here, uphill to your right. For the trail to the pond's north shore, walk past the sign-in box (on your right) for a few yards, turning left at a signpost with markers.
Directions to the Pond's west shore:
To access the cedar swamp and pond, from the center of Greensboro, take Wilson St. north for 0.1 miles. Bear right at the Greensboro Town Hall onto Laredon Ave./Baker Hill Rd./Town Highway #8. You will see Long Pond Road on your right after 4.5 miles. Park along Town Highway #8 and walk down Long Pond Road. Canoes can be launched at the road's end, but the put-in is a long carry.
Have you been to this preserve? Are you thinking of visiting? See what others are saying about their experiences and add your comments below.
Add Your Comments
Time for you to join the discussion. Tell us about your experience at this preserve. What plants and animals did you see? When did you go? You can help others plan their visit when you share your thoughts. And thank you for visiting one of our nature preserves!comments powered by Disqus
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text: Guys, When I purchased my Galaxy Tab one of the major items I found missing was a 'Zoom function' for the Camera App. Not only that but the photo's although good for a 3MP sensor were not that brilliant. Having done a bit of research I've found the 'perfect' answer in the form of using a third-party App by the name of Vignette. Part of Vignette is an ability to use a very clever 'crop tool' which acts as a live zoom feature. It is absolutely brilliant. What is even more amazing is when you do zoom in (crop) and take a shot the photo is still very good. OK it's nothing like a true Optical Zoom but providing you hold your hand steady you will be able to get a decent enough focus for a save photo. Not only that but when taking a normal shot and comparing the same shot with the Tab's dedicated Camera App the quality is miles different. It is so much Sharper and the colours more natural. If this is not enough to interest you let me then tell you of the other tricks up it's sleeve..... The Tab's Camera App has a limited amount of Settings you can apply whereas Vignette has a 'Mammoth' amount of settings and effects for you to choose from. The list is way too much to explain here but you can do all manner of photo shooting. One neat trick is an ability to take several shots and have them shown as one picture. By altering some of the settings you can also save a custom ND Filter setting which for those who are photo skilled will know make by far the best Black & White photography. Finally ~ I am in no way connected with Vignette. I have no part in it's PR, Marketing or Development. The sole reason for stating these facts is purely to let you all know if you take your photography with interest then look no further than Vignette. You can obtain Vignette on The Market here. There is also a Demo version available here.